Photoswitchable elements within a peptide backbone-ultrafast spectroscopy of thioxylated amides

Article abstract of DOI:10.1021/jp045151t

A series of thioxo compounds, thioacetamide, N-methylthioacetamide, a cyclic thioxoamide [(S)-5-thioxopyrrolidine-2-carboxylic acid ethyl ester], two thioxylated dipeptides (Ala-Ψ[CS-NH]-Ala and Phe-Ψ[CS-NH]-Ala) and a thioxylated dodecapeptide (Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu-Arg-Gln-His- Ψ[CS-NH]-Nle-Asp-Ser-Ser-Thr-Ser-Ala-Ala, or [thioxo-His¹²]-S- peptide; Nle = norleucine) are investigated by ultrafast spectroscopy in the visible and near UV. The different molecules show very similar absorption dynamics featuring a rise of a strong visible absorption band on the subpicosecond and picosecond time scale. The decay of the visible absorption occurs within 150-600 ps. The observations are interpreted by the ultrafast formation of triplet states and their decay on the subnanosecond time scale. Comparison with published ER experiments on N-methylthioacetamide indicates that the cis-trans isomerization around the thioxopeptide bond is terminated within less than 1 ns.

Full text of DOI:10.1021/jp045151t

4770
J. Phys. Chem. B 2005, 109, 4770-4775
Photoswitchable Elements within a Peptide BackbonesUltrafast Spectroscopy of
Thioxylated Amides
†
†
†
,†
‡
‡
H. Satzger, C. Root, P. Gilch, W. Zinth,* D. Wildemann, and G. Fischer
BioMolekulare Optik, Department f u¨ r Physik, Ludwig-Maximilians-UniVersit a¨ t M u¨ nchen, Oettingenstrasse 67,
D-80538 M u¨ nchen, Germany, and Max Planck Research Unit for Enzymology of Protein Folding,
Weinbergweg 22, D-06120 Halle/Saale, Germany
ReceiVed: October 25, 2004; In Final Form: December 23, 2004
A series of thioxo compounds, thioacetamide, N-methylthioacetamide, a cyclic thioxoamide [(S)-5-
thioxopyrrolidine-2-carboxylic acid ethyl ester], two thioxylated dipeptides (Ala-Ψ[CS-NH]-Ala and Phe-
Ψ[CS-NH]-Ala) and a thioxylated dodecapeptide (Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu-Arg-Gln-His-Ψ[CS-
1
2
NH]-Nle-Asp-Ser-Ser-Thr-Ser-Ala-Ala, or [thioxo-His ]-S-peptide; Nle ) norleucine) are investigated by
ultrafast spectroscopy in the visible and near UV. The different molecules show very similar absorption
dynamics featuring a rise of a strong visible absorption band on the subpicosecond and picosecond time
scale. The decay of the visible absorption occurs within 150-600 ps. The observations are interpreted by the
ultrafast formation of triplet states and their decay on the subnanosecond time scale. Comparison with published
IR experiments on N-methylthioacetamide indicates that the cis-trans isomerization around the thioxopeptide
bond is terminated within less than 1 ns.
1
. Introduction
or protein chain opens up a wide range of important applications,
two major difficulties result: The dye molecules impose distinct
Protein folding is one of the most important still unresolved
changes of the local or global protein structure. Even if their
geometry is optimized, they still may deviate from the size and
structure of the replaced amino acid residue. In addition, the
different chemical properties may impose problems with bio-
compatibility. To prevent these difficulties, isosteric thioxopep-
tides have been used that allow predictable structural changes
by photoswitching. Peptide bond thioxylation is characterized
by the replacement of the carbonyl oxygen at one or more
defined peptide bonds by a sulfur atom. Generally, thioxopep-
tides showed minimum deviation from the biological properties
processes in the cell. Folding bridges the gap between the
information held in the genetic sequence and protein structure.
Its detailed understandingsthe possibility to predict protein
structure and function from a genetic sequenceswould open
unprecedented applications in biotechnology and medicine (for
a recent review on the topic see ref 1). On the way to an
understanding of folding processes, experiments are undertaken
to study structural dynamics occurring in small proteins or
oligopeptides. These experiments should yield experimental
information to test and to improve simulation and prediction
methods. Dynamic experiments on short time scales rely on the
possibility to induce structural changes at a well-defined starting
time by an external triggering event. Most interesting for
investigations of the rapid processes in protein folding are optical
techniques, where structural dynamics can be induced by the
absorption of an ultrashort light pulse and where the structural
changes are followed by optical or infrared spectroscopy. Two
different molecular approaches are currently undertaken: (i) The
trigger molecule, for example, an azobenzene, is attached to
5,6
of the parent oxopeptide structures: In this way the absorption
spectrum of the peptide group is red-shifted from a range <230
nm to ∼260 nm for the ππ* transition. Light absorption at this
wavelength induces trans to cis photoisomerization without
photodecomposition. In short thioxopeptides, the absorption of
the cis isomer is slightly red-shifted (5 nm) as compared to the
trans form. In the dark, reequilibration was characterized by a
3
relaxation time of about 10 s, a time by far long enough to
study the functional modifications caused by the isomer-induced
7
2
backbone rearrangement. Illumination around 300 nm induces
side chains or the termini of a peptide chain. Light absorption
the accelerated re-formation of the trans isomer.
and isomerization of the dye then induces a local strain on the
amino acid chain, causing a modification of the secondary
structure. (ii) Another more direct trigger process occurs when
the switched moiety is part of the polypeptide backbone. In this
case, a direct switching of secondary structure elements of the
protein may result. It has been shown recently that the
incorporation of modified azobenzene molecules in cyclic
peptides allowed the structure of cyclic peptides to switch
reversibly between two families of configurations.3 While the
incorporation of photoswitchable dye molecules in a peptide
Oligopeptide derivatives of thioamides are small molecules
containing the thioxylated peptide bond and allow one to study
the basic photophysical properties of a biologically relevant
moiety. The photochemistry of one of the simplest thioaceta-
mides that can undergo photoisomerizationsN-methylthioac-
etamide, or NMTAAshas recently been studied by Helbing et
4
al., employing femtosecond transient absorption and IR spec-
,4
troscopy. In these experiments the spectroscopic changes
occurring upon ππ* excitation of trans-NMTAA have been
monitored. Transient IR spectra indicate that the formation of
the cis isomer and the recovery of the ground-state trans form
has biphasic kinetics. Time constants of 8.5 and 250 ps have
been determinded for the two phases. The ratio between cis
*
Corresponding author: tel +49 89 21809201; fax +49 89 21809202;
e-mail zinth@physik.uni-muenchen.de.
†
‡
Ludwig-Maximilians-Universit a¨ t M u¨ nchen.
Max Planck Research Unit for Enzymology of Protein Folding.
1
0.1021/jp045151t CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/17/2005

Photoswitchable Elements within a Peptide Backbone
J. Phys. Chem. B, Vol. 109, No. 10, 2005 4771
formation and trans recovery is about 30% for either phase.
Surprisingly, only the 250-ps component recurs in the transient
absorption experiment in the visible region. This experiment
on the other hand features kinetics with a time constant of ca.
to arrive at an optical density of ∼1 OD at the excitation
wavelength of 265 nm (this corresponds to concentrations of
1-3 mM). The sample was exchanged between subsequent laser
shots by pumping through a flow cell (fused silica windows,
optical path length 0.5 mm) by a peristaltic pump. Care was
taken to avoid accumulation of the cis product by utilizing
sufficient sample amounts (∼5 mL).
1
ps, which might have been missed in the IR experiment due
to a lower time resolution. Setting these details of the photo-
kinetics aside, these results suggest that the incorporation of a
thioxoamide group in a peptide should allow for the photoin-
duction of conformational changes on the 100 ps time scale.
Here, we will present femtosecond transient absorption studies
on three linear peptides where one peptide bond has been
replaced by its thioxo congener. These experiments will show
that the trans-cis isomerization of thioxopeptides proceeds in
a similar fashion as in NMTAA. Furthermore, we looked at
other thioxoamides in an attempt to shine light on the mecha-
nism of the isomerization. Of particular interest is a cyclic
thioxoamide where the isomerization pathway is blocked.
Data Analysis. Qualitative information on the number and
the time scales of the involved kinetic processes was obtained
by a data visualization technique based on the logarithmic
10
differentiation of absorption changes (LDAC). In addition, by
singular value decomposition, the number of involved kinetic
components was also estimated. The thereby obtained informa-
tion was used to derive starting values for a global least-squares
fitting algorithm (Levenberg-Marquart) with sums of expo-
nentials (convoluted with the instrument response function,
which is assumed to have Gaussian shape; for additional
information on global data analysis see, for example, refs 11
and 12).
2
. Materials and Methods
Synthesis of Thioxylated Compounds. Thioacetamide was
obtained from Fluka (Germany). It was used without further
purification. Synthesis of thioxylated peptides was done ac-
Spectroscopy. Stationary ultraviolet and visible absorption
spectra were recorded on a Lambda 19 (Perkin-Elmer) spec-
trophotometer. For the femtosecond absorption experiments a
homemade Ti-sapphire laser chirped pulse amplifier (CPA)
system was used, which delivers pulses at a wavelength of 800
nm and a repetition rate of 1 kHz. The excitation pulses at λ )
13
cording to Wildemann et al. N-Methylthioacetamide and (S)-
5
-thioxopyrrolidine-2-carboxylic acid ethyl ester were synthe-
sized according to the following procedure (all chemicals were
purchased from Fluka, Germany): 500 mg (6.8 mmol) of
N-methylacetamide and 200 mg (1.3 mmol) of (S)-5-oxopyr-
rolidine-2-carboxylic acid ethyl ester were each dissolved in
2
(
65 nm (energy ∼ 0.5 µJ) are generated by frequency tripling
frequency doubling to 400 nm and subsequent mixing with
the 800 nm fundamental pulse) of the laser output or by
frequency doubling of light pulses at 530 nm generated by a
1
00 mL of tetrahydrofuran. After addition of 2 equiv of
Lawesson reagent, the mixture was stirred for 2 h at 0 °C. The
solvent was evaporated and the crude products were purified
by reverse-phase high-performance liquid chromatography (RP-
HPLC). Product identity was verified by electrospray ionization
8
non-collinear optical parametric generator (NOPA). The excita-
tion pulses were focused onto the sample cell at a diameter of
ca. 0.2 mm. The absorption changes generated by the excitation
process are recorded by a femtosecond white light continuum
generated from a small part of the fundamental at 800 nm
(ESI)massspectrometry.N-Methylthioacetamideand(S)-5-thioxopyr-
rolidine-2-carboxylic acid ethyl ester were obtained in yields
of 78% and 65%.
9
focused onto a CaF2 plate. The relative (linear) polarization of
the white light probing and the excitation pulses was at the
magic angle. In most experiments probing was performed
between 340 and 750 nm. The temporal resolution of the pump-
probe experiment was deduced from the transient absorption
changes of the dye p-terphenyl. A fit of this rise, performed
under the assumption that p-terphenyl responds instantaneously,
yields an instrumental response time in the range of 200 fs.
The optical density of the sample solutions was adjusted to be
3
. Results
The common motif of all investigated molecules is the
thioxoamide group (see structures in Figure 1a). Three of them
contain amino acid residues. Thioactamide (TAA) is the smallest
of the investigated set. Due to the symmetric substitution of
the nitrogen, no cis-trans isomers exist. In the cyclic thioxoa-
mide CTA the five-membered ring restricts the thioxoamide
bond angle ω to about 0°, thus preventing photoisomerization.
The linear thioxo compounds of this study are known to show
reversible photoisomerization about the C-N bond.7 The
stationary UV/vis absorption spectra of the investigated samples
are similar. The exact band positions are given in Figure 1b.
The thioxo group has a pronounced UV absorption band around
270 nm. In NMTAA this transition is slightly blue-shifted (260
nm) as compared to the other compounds (∼268 nm). The
thioxyl-
ated dodecapeptide ([thioxoHis ]-S-peptide) mimics a polypep-
tide chain where the thioxopeptide moiety is the linker between
two long peptide segments, because the peptide bond between
the amino acid residues His and Nle (norleucine) was
thioxylated. When the UV absorption bands of NMTAA and
the thioxo peptides are excited, pronounced and long-lasting
1
OD at the excitation wavelength of 265 nm, so that the
excitation energy is almost completely absorbed halfway through
the optical path length. After the transient absorption of the
sample solution was measured, the experiment was repeated
with the neat solvent. In the wavelength region above 500 nm,
signal contributions of the solvated electron could be observed
both in ethanol and in water. Scaling the transient absorption
of the solvent by a factor of 0.5 and subtracting this signal from
the transient absorption of the sample solution could nicely
remove the signal contributions of the solvent. Delay time zero
for the individual probe wavelengths was determined by a
Sellmeier fit to the (chirped) delay time zeroes of the solvent
signal. The data shown have been corrected for the chirp of the
probe light and signal contributions from the solvent. The
complete set of data was recorded over a longer period of time.
Therefore, the excitation conditions and accordingly the signal
magnitudes for different samples varied and should not be
compared.
,14
1
2
1
2
13
7
,14
changes of the absorption spectrum occur.
The original
absorption band decreases and a new absorption band centered
around 280 nm grows in. The difference absorption spectrum
for Phe-Ψ[CS-NH]-Ala is shown in Figure 1c. The absorption
change is caused by the trans to cis photoisomerization around
the thioxo peptide bond of Phe-Ψ[CS-NH]-Ala resulting in 19%
Samples were dissolved in absolute ethanol except for the
12
[
thioxoHis ]-S-peptide, which was dissolved in aqueous phos-
phate buffer (2.8 mM, pH 7). The concentrations were adjusted

4772 J. Phys. Chem. B, Vol. 109, No. 10, 2005
Satzger et al.
in the absorption spectrum on the time scale of ∼0.5-5 ps,
which leads to a spectrum with a pronounced absorption band
centered in the visible region. This band peaks around 540 nm
for all samples except for TAA and CTA, for which it is
centered around 470 nm. These spectral features decay with
time constants of some 100 ps. Numerical values were retrieved
by a global fitting procedure that employs a sum of exponentials
convoluted with the experimental response. The retrieved decay-
associated spectra and time constants support the qualitative
picture of the kinetic behavior (data are compiled in Table 1).
TABLE 1: Compilation of Time Constants and Absorbance
a
Maxima Obtained in the Global Analysis of Transient
Absorption Experiments on the Thioxo Compounds
compound
τ
1
, ps
τ
2
, ps
3
τ , ps
TAA
NMTAA
CTA
Ala-Ψ[CS-NH]-Ala
Phe-Ψ[CS-NH]-Ala
0.4 (600, -) 3.9 (470, -) 500 (470, +)
0.8 (580, -) 3.8 (500, -) 280 (540, +)
0.3 (500, -) 2.4 (460, -) 670 (470, +)
0.3 (530, -)
0.2 (530, -)
180 (540, +)
120 (530, +)
350 (530, +)
12
[thioxoHis ]-S-peptide 0.8 (540, -)
a
Absorbance maxima in nanometers are given in parentheses; -
indicates a rise, + indicates a decay.
For all compounds studied, a delayed rise of the transient
absorption in the visible is retrieved. For TAA, NMTAA, and
CTA this rise is noticeably biphasic with time constants of 0.3-
0
.8 ps (∼0.5 ps in the following) and 2.4-3.9 ps (∼4 ps in the
following). The spectra associated with the two time constants
have approximately equal amplitude but differ in shape. The
∼
0.5 ps spectra represent an absorption rise that peaks at longer
wavelength (500-600 nm) than the ∼4 ps spectra (460-500
nm). For the peptides the rise can be described single-
exponentially with one time constant ranging from 0.2 to 0.8
ps. Notably, the decay-associated spectrum of this one rise
component has its peak around 550 nm, that is, in between the
peaks of the spectra associated with the two time constants
retrieved for TAA, NMTAA, and CTA. The decay of the
transient absorption in the visible can be described single-
exponentially for all compounds. The time constants of this
decay range from 120 ps (Phe-Ψ[CS-NH]-Ala) to 670 ps (CTA)
(
see Table 1). The differences between the compounds are
Figure 1. (a) Molecular structures of thioacetamide (TAA), N-
methylthioacetamide (NMTAA), and the cyclic thioxoamide (S)-5-
thioxopyrrolidine-2-carboxylic acid ethyl ester (CTA). (b) Absorption
spectra of TAA, CTA, NMTAA, and the investigated peptides Ala-
clearly visible, when the transient data are spectrally integrated
over the transient absorption band in the visible region (Figure
3
is faster for the peptide Phe-Ψ[CS-NH]-Ala (120 ps) than for
NMTAA (280 ps); the cyclic compound CTA (670 ps) shows
a very similar time constant as TAA (500 ps).
). There is no obvious trend in the decay behavior. The decay
1
2
Ψ[CS-NH]-Ala, Phe-Ψ[CS-NH]-Ala, and [thioxoHis ]-S-peptide. (c)
Absorption change induced by cw illumination of the peptide Phe-Ψ-
[CS-NH]-Ala.
The experimental results on NMTAA presented here are in
line with the UV/vis femtosecond transients reported in ref 4.
In both studies a rather intense excited-state absorption peaking
around 350 nm is observed immediately after photoexcitation.
With the decay of this peak, a new species with an absorption
maximum at ∼540 nm grows in. While we have determined
two time constants of 0.8 and 3.8 ps for this transient, ref 4
gives one constant of 1.1 ps. Treating the rise in our data with
just one exponential yields a time constant of 2 ps, in rather
good agreement with ref 4. We note, however, that we find a
pronounced biphasic character of the rise for NMTAA. In both
studies, the decay of the 540 nm feature is found to occur with
very similar time constants of 260 ps (ref 4) and 280 ps (this
study).
7
cis isomer in the photoequilibrium. In the dark the original
absorption spectrum of Phe-Ψ[CS-NH]-Ala is recovered with
a time constant of ∼2000 s (sodium phosphate buffer, pH 7.0,
7
2
83 K). The long lifetime of the cis isomer at this condition is
equivalent to a large rotational barrier of the potential energy
surface in the electronic ground state.
Excitation of the thioxo compounds with ∼200 fs laser pulses
centered at 265 nm results in spectrotemporal behavior that is
qualitatively alike for all compounds. Exemplary results depicted
in Figure 2 for N-methylthioacetamide NMTAA and Phe-Ψ-
[
CS-NH]-Ala illustrate this similarity. Immediately after excita-
tion there is a spectrally broad absorption increase. As can be
seen from the difference spectra recorded at distinct delay times
tD (Figure 2, second row), this absorption increase is large in
the UV at 350 nm, but it extends throughout the whole visible
region. Apparently this absorption represents the initially
populated ππ* state. Subsequently there is a pronounced change
4
. Discussion
The experimental results may be summarized as follows:
After the population of the excited electronic state, there is a

Photoswitchable Elements within a Peptide Backbone
J. Phys. Chem. B, Vol. 109, No. 10, 2005 4773
Figure 2. Femtosecond transient absorption data for NMTAA (left) and Phe-Ψ[CS-NH]-Ala (right). The first row gives an overview, the second
row shows transient spectra at selected delay times, and the third row depicts decay-associated spectra obtained from a global analysis. Note that
in the first row the time scale is linear from -1 to 1 ps and logarithmic thereafter.
Figure 3. Temporal behavior of the spectral integral of the transient absorption peaking around 500 nm. Note that the time axis is linear from -1
ps to 1 ps and logarithmic thereafter. Data for TAA, NMTAA, and CTA are compiled in the graph on the left; those for the thioxylated peptides
on the right.
rise of a strong visible absorption band centered around 550
nm on the (sub)picosecond time scale. While for TAA,
NMTAA, and CTA this rise is distinctly biphasic, a single
exponential suffices to describe this rise for the linear thioxo-
peptides. The spectra associated with the single-exponential rise
resemble the “average” of the two rise spectra of the

4774 J. Phys. Chem. B, Vol. 109, No. 10, 2005
Satzger et al.
thioxoamides. The decay of this absorption band occurs on the
100 ps-1 ns time domain. Aside from differences in the
observed time constants, the different samples show very similar
behavior. It can safely be stated that the spectrotemporal
response of the investigated compounds is determined by their
common “chromophore”, namely, the thioxoamide group. The
interpretation of the experimental findings therefore has to focus
on the (electronic) properties of this group.
4
Helbing et al. have performed quantum chemical calculations
on the CASSCF level addressing the character of the excited
states. In line with spectroscopic results, they have identified
four excited states in the energetic range of interest. Two of
these states have singlet multiplicity and nπ* and ππ* orbital
character. The other two carry triplet multiplicity. As only the
1
singlet ππ* state shares a substantial transition moment with
Figure 4. Scheme illustrating the relative energetic ordering of the
the ground state, it is the state initially populated. In their
calculations Helbing et al. focus on structural distortions of the
thioxoamide, which bring the different electronic states in
energetic vicinity so that transitions between them can take
place. Accordingly, important coordinates are the CdS bond
elongation and the pyramidalization of the thiocarbonyl carbon.
The torsion of the central thioxopeptide bondsthe coordinate
that connects the trans and the cis isomershas virtually no
influence on the energies of the excited states. This is in line
with our finding that the “locked” thioxoamide CTA has very
similar photokinetics as those undergoing unperturbed cis-trans
isomerization. The data from the locked molecule CTA support
the interpretation that the isomerization occurs during the
deactivation of the electronically excited states but does not
strongly promote this decay. The calculations of Helbing et al.
also point to the fact that very similar deactivation patterns are
expected if the reaction proceeds in states with triplet multiplic-
ity. It seems very likely that indeed most of the observed kinetics
are due to processes associated with triplet states.
excited states of the thioxo compounds and possible deactivation paths
1
following the excitation of the ππ* state. The ordering of the singlet
states is derived from the spectroscopic results.25 In analogy to aromatic
thiocarbonyls, an inversion of the nπ* and ππ* states in the triplet
manifold is assumed.
3
3
manifold, that is, a nπ* to ππ* transition. For the thioxoamides
TAA, NMTAA, and CTA we associate the 3-4 ps component
with this transition. For the linear thioxopeptides within this
reasoning this IC process is somewhat faster so that kinetically
it cannot be distinguished from the ISC. The final recovery of
the electronic ground state, either in trans or in cis form, occurs
then by another ISC process on the 100 ps-1 ns time scale.
This above assignment requires an inversion of the triplet states,
3
3
that is, the ππ* has to lie below the nπ* state. To our
knowledge there are no spectroscopic data on the energetics of
the triplet state of thioxoamides. Yet there are results on aromatic
thiocarbonyls21 stating that in polar solvents such an inversion
occurs. An inversion for the thioxoamides is thus at least not
unlikely.
It is well-known that, in molecules with close-lying ππ* and
nπ* states, intersystem crossing can be very efficient since the
spin flip can be accompanied by a change of the orbital character
On the basis of the assumption that triplet states play an
essential role in the excited-state deactivation, one can speculate
on the role of the spin state for the recovery of the ground state
and the biphasic formation of the cis isomer reported by Helbing
15 16
of the state (El-Sayed’s rule ). For example, in aromatic
ketones, intersystem crossing times of well below 10 ps have
been measured.1 Recently, some of the present authors have
reported an intersystem crossing time of 1 ps for xanthone,
4
et al. (cf. Figure 4). According to El-Sayed’s rules, a transition
7,18
3
from the nπ* state to the ground state should be faster than
3
from ππ*. Thus, one might envision a branching during the
1
9
3
3
which is an aromatic ketone with a very rigid structure. This
decay of the nπ* state: both the ππ* state would be populated
and the ground state would be recovered. Upon this ISC process
a large amount of energy (2-3 eV) has to be transferred into
nuclear degrees of freedom, leading to a strong vibrational
excitation of the ground state. Such a vibrational excitation is
known to relax in the 10 ps time domain (see, for example,
refs 22-24). This reaction scheme can be used to explain the
fast component in the isomerization process observed in the IR
experiments: Isomerization may occur either directly during
the ISC process or from the vibrationally excited ground state.
In the first case, the manifestation of the cis ground-state
spectrum could be delayed by the vibrational cooling time. In
the second case, isomerization proceeds in parallel with the 10
ps cooling process. Both interpretations could explain why the
10 ps component found in the time-resolved IR-experiments is
not detected by UV/vis spectroscopy. The part of the population
1
1
ps time constant has been assigned to a transition from a ππ*
3
to a nπ* state. That process is followed by a slower internal
conversion within the triplet manifold populating the lowest
3
triplet state, the ππ* state. This process proceeds with a time
constant of 10 ps. The behavior of the visible absorption of the
thioxoamide compounds parallels that of xanthone. For the
thioxoamides (TAA, NMTAA, and CTAA), biphasic rise
behavior was observed. The spectral signatures of the rise of
the peptides indicate that for them the two time constants are
so close that they are not distinguished. Thus, by analogy we
assign the time constants of a few hundred femtoseconds to an
3
intersystem crossing (ISC) that populates the nπ* state (cf.
Figure 4). This is even faster than the value reported for
xanthone. The fast ISC process in xanthone is assumed to be
due to the oxygen in the carbonyl group. For the thioxoamides
the sulfur is responsible for the fast ISC. Thus the acceleration
of ISC in the thioxoamides is not surprising since spin-orbit
coupling, which mediates the ISC, is higher by a factor of 2 in
sulfur as compared to oxygen.20 This ISC process should then
be followed by an internal conversion (IC) process in the triplet
3
that has arrived in the ππ* state is “trapped”, since from here
ISC to the ground state is not El-Sayed-allowed. In our
considerations we focused on spin effects in order to obtain a
consistent picture of the observed transients in the thioxoamides.
This does not imply that nuclear motions such as those

Photoswitchable Elements within a Peptide Backbone
J. Phys. Chem. B, Vol. 109, No. 10, 2005 4775
References and Notes
4
pointedout by Helbing et al. do not leave a kinetic imprint and
may be important to explain the observed transients. At the
present level of experimental information, no definite decision
on the molecular mechanisms can be made.
(
1) Dobson, C. M. Nature 2003, 426, 884.
(2) Kumita, J. R.; Smart, O. S.; Woolley, G. A. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 3803.
(3) Sp o¨ rlein, S.; Carstens, H.; Satzger, H.; Renner, C.; Behrendt, R.;
Finally, we turn to the isomerization of the linear thioxopep-
tides. As we have stated above, their kinetics aresbesides the
less pronounced picosecond transientsessentially identical to
those observed in NMTAA. We could not directly detect the
formation of the cis isomer of the peptides, since its spectro-
scopic signature lies too far in the UV for our setup. From
steady-state experiments it is evident that the cis form of the
Moroder, L.; Tavan, P.; Zinth, W.; Wachtveitl, J. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 7998.
(4) Helbing, J.; Bregy, H.; Bredenbeck, J.; Pfister, R.; Hamm, P.; Huber,
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(
5) Sifferlen, T.; Rueping, M.; Gademann, K.; Jaun, B.; Seebach, D.
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(
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7
peptide is formed. For NMTAA the IR experiments have
4
shown that the cis formation is finished within 250 ps, a value
(
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that is also present in the experiments with visible detection.
These kinetics support the standard picture of photoisomeriza-
tion: Since the barrier for isomerization in the electronic ground
state potential surface of the thioxoamides is too high to allow
rapid ground-state isomerization of thermalized molecules at
room temperature, the isomerization has to occur in the excited
electronic state, during the transition to the ground state or
immediately afterward, in the vibrationally hot ground state.
Thus, it is very likely that the processes on the 100 ps time
scale observed here for the peptides terminate the formation of
the cis isomer. This shows that the incorporation of the
thioxoamide group in oligopeptides of 20 amino acid in length,
at least, indeed allows us to induce and to study peptide
dynamics on time scales of >100 ps.
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2
In conclusion, we have investigated a series of thioxo
compounds by visible and near-UV absorption spectroscopy.
We have shown that peptides containing a thioxo peptide bond
show very similar absorption kinetics as thioacetamide and
N-methylthioacetamide. After excitation of the ππ* transition,
we observe absorption transients on the 1 ps and ca. 300 ps
time scale. The initially excited electronic state decays on the
subpicosecond time scale into an intermediate state, most
probably a triplet state. The visible absorption changes recover
with time constants of 120-670 ps. Apparently the cis-trans
isomerization of the investigated thioxo compounds is terminated
within this time.
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