Tetrazine phototriggers: Probes for peptide dynamics

Article abstract of DOI:10.1002/anie.201000500

(Presented Chemical Equation) Surrender your secrets, peptides! Of several 3, 6-disubstituted tetrazines examined as phototriggers for the investigation of peptide dynamics, the most useful candidates were disulfenyl tetrazines. The thiocyanate fragments resulting from photol-ysis (see scheme) were detected by ultrafast infrared spectroscopy. Chromophore insertion into oxytocin and photofragmentation to trigger peptide relaxation were both demonstrated.

Full text of DOI:10.1002/anie.201000500

Communications
DOI: 10.1002/anie.201000500
Phototriggering
Tetrazine Phototriggers: Probes for Peptide Dynamics**
Matthew J. Tucker, Joel R. Courter, Jianxin Chen, Onur Atasoylu,
Amos B. Smith, III,* and Robin M. Hochstrasser*
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Ultrafast photochemical triggers hold the promise of provid-
ing information on the dynamics of peptide and protein
folding.[1] Prior to photolysis, bonding to the trigger constrains
the peptide to have a narrow structure distribution. Photo-
chemical triggering releases the constraints and thus permits
the molecule to evolve to a different equilibrium distribution.
The structure evolution, even when ultrafast, can be followed
by infrared probe or two-dimensional infrared spectroscopy.
Fast phototriggering can thus reveal early kinetic events in
protein dynamics by providing a means to explore the free-
energy landscape of folding and misfolding. Several photo-
triggers have been developed for this purpose,[1,2] but there
remain significant challenges. For example, disulfide bonds in
peptides can be used as a phototriggers. Deep UV light severs
the disulfide bond and releases the structural constraints; such
experiments have been carried out in short helical pepti-
des,[1a,b] cyclic peptides,[1d] and b hairpins.[1c] Although disul-
fide photolysis offers the capability of initiating ultrafast
structure equilibration, limitations preclude the broad gen-
Scheme 1. Photofragmentation of s-tetrazines.
parent s-tetrazine, close to unity in the vapor phase.[6] The
quantum yields and light-induced pathways are environ-
mentally sensitive,[7] and it is well-established that the photo-
reaction of s-tetrazine occurs on the ground state following
internal conversion.[8]
With these observations as background, we report herein
the potential of incorporating the s-tetrazine structural motif
in peptides as an ultrafast photochemical trigger to initiate
early events in peptide/protein folding. Although the overall
photochemical yield of substituted s-tetrazines has been
suggested to be lower in the condensed phase than in the
gas phase,[5,9] the variation of yield with substitution has not
been examined systematically. Our initial investigations
therefore focused on the condensed-phase photophysical
properties of the parent s-tetrazine along with dimethyl and
diphenyl 3,6-substituted s-tetrazines as model compounds to
guide the development of amino acid based systems.
The photochemical quantum yield for this series of 3,6-
disubstituted tetrazines was measured in the condensed phase
by nanosecond flash photolysis; the bleach signal of the
parent molecules was observed at around 542 nm after a 1 ms
delay, when all photochemical processes were complete. The
photolysis yield per shot was determined from the ratio of
DOD, the optical density of the bleach signal, and OD(0), the
optical density before irradiation at 532 nm. The mechanism
of the photodissociation of simple tetrazines involves a
barrier crossing on the electronic ground state.[8] The parent
molecule, s-tetrazine (R = H), dissociates on the subnano-
second timescale with a high photolysis yield (ca. 90%). As
substituents are attached to the tetrazine, the number of
modes accessible after internal conversion is greatly
increased, and the photolysis yield is decreased as a result
of vibrational-energy redistribution. For example, in the case
of 3,6-dimethyl-s-tetrazine (R = Me), the yield is decreased to
18%. This trend continues for 3,6-diphenyl-s-tetrazine (R =
Ph), for which the yield is further decreased to 1–2% owing to
the involvement of the ring modes of the phenyl groups in the
vibrational-energy distribution after internal conversion.
Because of this overall reduction in the yield by substitution,
photodissociation reactions with this mechanism are not
useful for triggering relaxation in peptide systems.
A variety of heteroatom-substituted 3,6-tetrazines were
evaluated in an effort to identify substituents that would lead
to increased photoproduct yields. Pleasingly, acceptable
photolysis yields of 24% at 355 nm and 12% at 410 nm
were observed for 3,6-bisthiobenzyl-s-tetrazine (R = SBn)
upon flash photolysis. The sulfenyl substitution causes the
appearance of an additional band centered at 410 nm, which
has been attributed to either a separate n!p* transition[10] or
charge transfer involving sulfur. This band is particularly
useful because it presents the opportunity to initiate photol-
ysis with a frequency-doubled Ti:sapphire laser pulse at
approximately 400 nm that is not absorbed by a peptide or
ꢀ
erality of this technique. Homolytic S S bond scission reveals
two reactive radicals that can undergo geminate recombina-
tion, as well as reactions with protein side chains. Moreover,
the UV excitation required to dissociate the disulfide bond
also excites the peptide backbone. Another example is
azobenzene, which undergoes fast, reversible photoisomeri-
zation. When azobenzene is designed into a peptide, a light
pulse can be used to cause the system to shift reversibly
between significantly different equilibrium configurations.[3]
To optimize the knowledge gained on the early events in
peptide/protein folding and unfolding, the triggering process
should 1) be initiated on the picosecond timescale from a
narrow structure distribution near equilibrium and be faster
than the early events in conformational reorganization, such
as single-bond rotations, hydrogen-bond formation, or helix
nucleation;[4] 2) have a high photochemical yield; 3) produce
inert products with negligible side reactions along the
photolysis pathways; 4) be biocompatible for ease of incor-
poration into peptides and proteins; and 5) be photochemi-
cally accessible to nondamaging light-pulse frequencies. s-
Tetrazine and its congeners are a class of compounds that
fulfill most of these requirements. For example, tetrazine
photoproducts are relatively inert, unobtrusive nitriles and
molecular nitrogen (Scheme 1).[5] Furthermore, the n!p*
transition at 532 nm permits excitation in the visible region of
the spectrum with a photoproduct yield, in the case of the
[*] Dr. M. J. Tucker, J. R. Courter, Dr. J. Chen, O. Atasoylu,
Prof. A. B. Smith, III, Prof. R. M. Hochstrasser
Department of Chemistry, University of Pennsylvania
231 S. 34th Street, Philadelphia, PA 19104-6323 (USA)
E-mail: smithab@sas.upenn.edu
hochstra@sas.upenn.edu
[**] This research was supported by the NIH; instrumentation was
developed at the Research Resource (NIH P41RR001348 and
RR001348S).
Supporting information for this article, including detailed synthetic
procedures, full characterization data for all new compounds, and a
description of the methods employed to obtain the photophysical
data, is available on the WWW under http://dx.doi.org/10.1002/
anie.201000500.
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Communications
protein. Although the heavy-atom effect,[11] whereby the
photochemical yield of the tetrazine fragmentation reaction:
a photoproduct yield of 11% was observed upon flash
photolysis of (ꢀ)-3a. A linear dependence of the photolysis
yield on the irradiation intensity was found at lower energies
(< 10 mJ), whereas the yield saturates at higher energies as a
result of two photon-absorption processes that were not
studied in detail.
We next sought to verify that the photofragmentation
produced the anticipated thiocyanate product (ꢀ)-4b
(Scheme 3). The steady-state photolysis was conducted with
sulfur atoms slow down the energy flow out of the tetrazine
ring, may offer an explanation for the effect of the sulfur
atoms on the yield, it is considered more likely that new
photochemical pathways are opened up by sulfur substitution.
An absorption band observed in the transient spectrum of 3,6-
bisthiobenzyl-s-tetrazine at around 510 nm for up to 1 ms, with
a lifetime dependent on the oxygen concentration, is attrib-
uted to the triplet–triplet absorption.[12] Yuasa and Fukuzumi
observed intersystem crossing in 3,6-diphenyl-s-tetrazine
derivatives and demonstrated that the triplet state is photo-
stable.[13] The photoproducts in the present examples are
therefore believed to be formed through the singlet manifold,
with triplet formation being a nonproductive pathway. The
fluorescence lifetime for 3,6-bisthiobenzyl-s-tetrazine was
determined to be 585 ps, which suggests photolysis on this
timescale. Having identified a favorable tetrazine scaffold, the
next step in designing a trigger suitable for incorporation
within a peptide was to develop an amino acid based tetrazine
derivative in which the tetrazine trigger was incorporated
between two cysteine groups.
Scheme 3. Photolysis of (+)-3b.
l = 254 nm owing to the higher absorbance of (+)-3b at this
1
Previous synthetic studies demonstrated that the reaction
of electrophilic dichloro-s-tetrazine (1)[14] with thiolates
favors the symmetrical bissubstituted derivatives.[15] The key
to reproducibly high yields of tetrazine (ꢀ)-3a (Scheme 2)
was the exclusion of ambient light. This reaction can also be
conducted under aqueous conditions by using 1,4-dioxane as a
cosolvent to provide (+)-3b. The introduction of the amino
acid based substituents did not significantly perturb the
wavelength. The photolysis of (+)-3b was monitored by H
and 13C NMR spectroscopy (Figure 1), which indicated quan-
titative conversion into (ꢀ)-4b, as exemplified by the
disappearance of the 13C signal for the tetrazine ring with
the concomitant appearance of the SCN resonance at dC =
113 ppm.[16]
Figure 1. 13C NMR (d=200–100 ppm) spectroscopic monitoring of the
steady-state photolysis of (+)-3b (20 mgmLꢀ1, CD3OD, quartz NMR
tube). Top: 13C NMR spectrum of the starting material (+)-3b;
bottom: 13C NMR spectrum after irradiation for 4 h (complete con-
version into (ꢀ)-4b).
The fluorescence of tetrazine (ꢀ)-3a decays mainly within
a lifetime of 851 ps (with a few-percent component of 1.67 ns).
To further investigate the timescale of photolysis, we per-
formed femtosecond transient IR measurements; the results
demonstrated that the ester carbonyl modes of (ꢀ)-3a are
sensitive to photolysis. The transient difference spectrum
obtained after a 1 ns time delay was similar to the difference
spectrum obtained after complete photolysis from the sta-
tionary spectrum of (ꢀ)-4b (Figure 2). The IR transient
absorption peaks at 1765 and 1745 cmꢀ1 arise from the ester
absorption after photolysis of the molecule, whereas the
bleach signals at 1730 and 1755 cmꢀ1 are due to the removal of
parent molecules. These results demonstrate that the photol-
ysis occurs within 1 ns.
The thiocyanate vibrational mode of the photoproduct
was monitored at different delays between the optical pump
Scheme 2. Synthesis of cysteine-derived tetrazines and X-ray crystal
structure of (ꢀ)-3a.
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Figure 2. Carbonyl ester region of the femtosecond IR transient
absorption spectrum of (ꢀ)-3a in CD3OD (blue), and stationary FTIR
spectrum of the photolysis product AcHN-Cys(SCN)-COOMe (red).
Figure 3. Time-dependence spectra of the SCN transient absorption
following the photolysis of (ꢀ)-3a (triangles: raw data; red line: fit)
and AcNH-1,6-S,S-Tet-oxytocin (squares: raw data; blue line: fit) in
CHCl3/MeOH (95:5 v/v). Inset: SCN region of the femtosecond IR
transient absorption spectrum of (ꢀ)-3a at a delay of 1 ns (black), and
FTIR absorption band of the SCN stretching frequency after steady-
state irradiation of (ꢀ)-3a at 400 nm (red). The asymmetry of the
transient absorption peak is not physically meaningful, but is caused
by the detector.
and infrared probe. The transient absorption spectrum used to
monitor the photolysis of (ꢀ)-3a in the SCN region contained
one peak at 2163 cmꢀ1 at 1 ns (inset of Figure 3, black curve).
The peak corresponds to the 2163 cmꢀ1 band observed in the
FTIR spectrum of the compound after irradiation at 400 nm
(inset of Figure 3, red curve). The thiocyanate signal was
estimated from the number of photons and the photochemical
yield of 166 mOD on the assumption that two identical
thiocyanates with similar stationary lineshapes formed upon
photolysis. The observed thiocyanate signal corresponded to
120 mOD at the 2163 cmꢀ1 peak at a time delay of 1 ns for the
photolysis of (ꢀ)-3a. However, the predicted and measured
integrated absorption are in close agreement, which suggests
that indeed two thiocyanates are formed. The thiocyanate
transient at the peak maximum was fit to an exponential
growth (Figure 3), and the time constant was determined to
be 177 ps for (ꢀ)-3a. The rate constant corresponds to a rate
of conversion that is fast relative to the S1 lifetime, which
suggests that the dissociation to produce the SCN groups
occurs from a highly excited state.
purification,[17] then provided the target peptide: AcNH-1,6-
S,S-Tet-oxytocin.
Detailed NMR spectroscopic studies support the location
of the tetrazine ring between the two cysteine side chains.
Assignment of the amide NH resonances and calculation of
3
the JNH,Ha values permitted simulation of the peptide back-
bone angles.[18] The position of the tetrazine bridge was
further refined by analysis of the 3J coupling constants
between the well-resolved Cys(Ha) and Cys(Hb) resonances.
This analysis permitted assignment of the tetrazine-bridged
peptide structure as illustrated in Scheme 4. Details of this
analysis and representative peptide structures for the lowest-
energy conformational families are provided in the Support-
ing Information.
Having validated that the tetrazine fragmentation reac-
tion occurs within a few hundred picoseconds upon photol-
ysis, we developed a method to insert the trigger within a
cyclic peptide system. The peptide oxytocin was chosen as a
readily available model system. Initial efforts to reduce the
disulfide of the native peptide with immobilized tris(2-
carboxyethyl)phosphane (TCEP), followed by treatment
under the aqueous conditions for tetrazine insertion, proved
irreproducible. To overcome these limitations, we developed
a strategy for solid-phase peptide synthesis in which the
orthogonal MMT (para-methoxytrityl) protecting group was
used to mask the pivotal thiol groups (Scheme 4). Standard
techniques were used to construct the peptide, with the
N terminus capped as the acetamide. Removal of the Mmt
groups liberated the thiol groups of the Cys residues within
the resin-bound peptide. Subsequent treatment with 1 intro-
duced the tetrazine bridge. The pseudodilution effect induced
by immobilizing the peptide precludes the formation of
tetrazine-bridged peptide dimers. Deprotection and cleavage
from the resin under standard conditions, followed by
Having developed an effective method to insert a
tetrazine trigger within a peptide construct, we next evaluated
the photochemical properties of this system within the
constrained cyclic peptide. The fluorescence decay of
AcNH-1,6-S,S-Tet-oxytocin displays three components: a
fast component at 200 ps (66% of the amplitude) and two
slower components at 1.09 ns (26%) and 4.09 ns (8%). The
large contribution of the 1 ns component is likely to be from
another conformer, as suggested by the NMR spectroscopic
analysis. The rate of formation of the SCN stretching peak
upon photolysis was again measured by femtosecond tran-
sient IR spectroscopy, and the thiocyanate transient was fit to
a single exponential with a time constant of 207 ps for AcNH-
1,6-S,S-Tet-oxytocin (Figure 3). The quantum yield of the
model compound, (ꢀ)-3a, is dependent on the excitation
wavelength. There is a twofold increase in SCN transient
absorption upon excitation at 355 nm as compared to
excitation 400 nm. Clearly, the photochemical transformation
occurs from unrelaxed states following excitation.
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Keywords: IR spectroscopy · peptides · photolysis ·
.
phototriggers · tetrazines
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Scheme 4. Solid-phase synthesis of AcNH-1,6-S,S-Tet-oxytocin. TFA=
trifluoroacetic acid, TIPS=triisopropylsilyl, Trt=triphenylmethyl,
MMT=para-methoxytrityl.
In summary, this study demonstrates that a disulfenyl
tetrazine system can be readily incorporated within a
bisthiolate peptide motif by treatment with 1. The photolysis
rate of this new phototrigger was measured directly by
femtosecond transient absorption to occur on the picosecond
timescale; thus, this method provides rapid access to non-
equilibrium states. As far as we can ascertain, the two SCN
groups are formed at the earliest measured times (ca. 25 ps).
The photochemical yield upon flash photolysis is wavelength-
dependent: 22% at 355 nm. The photolysis can be carried out
to complete conversion by standard photochemical irradi-
ation (l = 256 nm). The principal advantages of the disulfenyl
tetrazine triggering system are that the photolysis can be
initiated with light of 330–400 nm or at lower yield with visible
irradiation, which makes the tetrazine triggering system
biocompatible. Upon incorporation into a cyclic peptide
system, the photolysis rate and photolysis yield of the
tetrazine trigger are similar to those of acyclic versions (e.g.,
(ꢀ)-3a). Thus, the mechanism of dissociation is not influenced
by rigidity of the peptide system or the presence of other side-
[17] Compound (+)-3b was found to be stable to a variety of resin-
cleavage/global-deprotection conditions for up to 12 h, as
monitored by LC–MS.
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13
18O
=
C
chain functionalities. Furthermore, site-selective
[19] C. Fang, R. M. Hochstrasser, J. Phys. Chem. B 2005, 109, 18652.
replacements in the peptide backbone will permit FTIR or
2D IR tracking at the residue level[19] of the resulting peptide
structure.
Received: January 28, 2010
Published online: April 9, 2010
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