Design, synthesis, and photochemical validation of peptide linchpins containing the S,S-tetrazine phototrigger

Article abstract of DOI:10.1021/ol301490h

The design, solid-phase synthesis, and photochemical validation of diverse peptide linchpins, containing the S,S-tetrazine phototrigger, have been achieved. Steady state irradiation or femtosecond laser pulses confirm their rapid photofragmentation. Attac

Full text of DOI:10.1021/ol301490h

ORGANIC
LETTERS
2012
Vol. 14, No. 13
3518–3521
Design, Synthesis, and Photochemical
Validation of Peptide Linchpins
Containing the S,S-Tetrazine Phototrigger
Mohannad Abdo, Stephen P. Brown, Joel R. Courter, Matthew J. Tucker,
Robin M. Hochstrasser,* and Amos B. Smith III*
Department of Chemistry, University of Pennsylvania, Philadelphia,
Pennsylvania 19104, United States
smithab@sas.upenn.edu; hochstra@sas.upenn.edu
Received May 30, 2012
ABSTRACT
The design, solid-phase synthesis, and photochemical validation of diverse peptide linchpins, containing the S,S-tetrazine phototrigger, have
been achieved. Steady state irradiation or femtosecond laser pulses confirm their rapid photofragmentation. Attachment of peptides to the C- and
N-termini will provide access to diverse constrained peptide constructs that hold the promise of providing information about early peptide/protein
conformational dynamics upon photochemical release.
Understanding the early kinetic events that govern
conformational dynamics in peptides and proteins provides
an avenue for the development of models of folding and, in
turn, the misfolding of proteins.1 To explore these early
events, ultrafast photochemical triggers are required to
initiate conformational transitions that occur on the pico-
to nanosecond time scale.2 With the appropriate design,
incorporation of a phototriggering moiety would confine a
peptide/protein construct within a narrow distribution
of conformers. Photolysis would then permit release of
the geometric constraint with precise temporal control
for interrogation with now available state-of-the-art spec-
troscopic techniques, such as two-dimensional infrared
spectroscopy (2D-IR),3 to monitor the conformational
evolution on the pico- to nanosecond time scale, as the
peptide/protein relaxes to a new equilibrium conformation.4
The first example of a photochemical tactic to explore
the dynamics of peptide and protein folding was intro-
duced by Hochtrasser and DeGrado, exploiting the photo-
lysis of a disulfide bond.5 While effective in releasing a
peptide system, homolysis of the disulfide bond required
high energy (λ = 270 nm). The photochemical event
revealed two reactive sulfur-centered radicals that could
either undergo rapid geminate recombination or react with
the protein side chains. In related work, Chan and co-
workers developed 30-methoxy and 30,50-dimethoxy-
benzoins that are both water-soluble and undergo rapid
photolysis with a time constant of 5 ps.6 The analytical
methods employed however did not permit temporal
resolution of the conformational dynamics that occur
on the ultrafast time scale, but instead focused on global
(1) (a) Sosnick, T. R.; Barrick, D. Curr. Opin. Struct. Biol. 2011, 21,
12–24. (b) Chung, H. S.; McHale, K.; Louis, J. M.; Eaton, W. A. Science
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Science 2011, 334, 517–520.
(2) (a) Lu., H. S. M.; Volk, M.; Kholodenko, Y.; Gooding, E.;
Hochstrasser, R. M. J. Am. Chem. Soc. 1997, 119, 7173–7180. (b) Volk,
M. Eur. J. Org. Chem. 2001, 14, 2605–2621.
(3) Kim, Y. S.; Hochstrasser, R. M. J. Phys. Chem. B 2009, 113,
8231–8251.
(5) (a) Volk, M.; Kholodenko, Y.; Lu, H. S. M.; Gooding, E. A.;
DeGrado, W. F.; Hochstrasser, R. M. J. Phys. Chem. B 1997, 101, 8607–
8616. (b) Lu, H. S. M.; Volk, M.; Kholodenko, Y.; Gooding, E.;
Hochstrasser, R. M.; DeGrado, W. F. J. Am. Chem. Soc. 1997, 119,
7173–7180.
(4) (a) Remorino, A. Hochstrasser, R. M. Acc. Chem. Res. 2012,
in press, doi: 10.1021/ar3000025. (b) Kolano, C.; Helbing, J.; Kozinski,
M.; Sander, W.; Hamm, P. Nature 2006, 444, 469–472. (c) Kolano, C.;
Helbing, J.; Bucher, G.; Sander, W.; Hamm, P. J. Phys. Chem. B 2007,
111, 11297–11302.
(6) (a) Rock, R. S.; Chan, S. I. J. Am. Chem. Soc. 1998, 120, 10766–
10767. (b) Hansen, K. C.; Rock, R. S.; Larsen, R. W.; Chan, S. I. J. Am.
Chem. Soc. 2000, 122, 11567–15568. (c) Chen, R. P.-Y.; Huang, J. J.-T.;
Chen, H.-L; Jan, H.; Velusamy, M.; Lee, C.-T; Fann, W.; Larsen, R. W.;
Chan, S. I. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7305–7310.
r
10.1021/ol301490h
Published on Web 06/26/2012
2012 American Chemical Society

changes (ms) of the peptide conformation. Photochemical
switches have also been employed, for example azoben-
zenes, to enable fast, reversible photoisomerization to
permit conformational reorganization.7 This constraint
however significantly limited the accessible conforma-
tional space available to the peptide or protein.
Recently, we introduced and validated the feasibility of
incorporating the S,S-tetrazine phototrigger system be-
tween two cysteine residues.8 Photolysis of the S,S-tetrazine
construct proceeded rapidly with a ps time constant to
furnish thiocyanate photoproducts, in conjunction with loss
of dinitrogen (Figure 1).
Standard Fmoc-based Solid-Phase Peptide Synthesis
(SPPS; Figure 2), with the requisite cysteine side chains
protected as para-monomethoxytrityl (Mmt) thioethers,
was employed to construct the target peptide linchpins
employing either N-methyl indole aminomethyl or Wang
resins. Selective removal of the Mmt cysteine side-chain
protecting groups, followed by insertion of the S,S-tetra-
zine ring by treatment with 3,6-dichlorotetrazine under
mildly basic conditions, and then removal of the peptide
from the resin successfully furnished tripeptides4aꢀc, with
an overall yield of 15ꢀ30%. Pleasingly, steady-state irra-
diation of linchpins 4a and c at a wavelength of 355 nm led
solely to the formation of the anticipated bis-thiocyanate
peptide photoproducts 5a and b. The photolysis was
monitored by LC-MS and 1H, 13C NMR. The rapid
disappearance of the 13C signals for the tetrazine ring
carbons (δc ≈ 170 ppm), with concomitant appearance
of the SCN resonance at δc ≈ 113 ppm proved particularly
diagnostic [see Supporting Information (SI)].
Figure 1. The photolysis of S,S-tetrazine (ꢀ)-1, under a variety
of solvents and light sources, yields thiocyanate (ꢀ)-2 with loss
of dinitrogen.
The S,S-tetrazine scaffold possesses a number of advan-
tages when compared to other nonreversible trigger sys-
tems: (1) flash photolysis can be conducted with near-UV
(λ = 355 or 410 nm) laser pulses, which would not lead to
damage of the peptide; (2) acceptable photolysis yields are
observed; and (3) the thiocyanate photoproducts are rela-
tively inert and provide a potentially useful IR probe. In
addition to identifying the S,S-tetrazine phototrigger as a
suitable candidate, we successfully replaced the native di-
sulfide bond of the peptide oxytocin with the tetrazine
moiety as a model system.9 Subsequent photophysical ana-
lysis of the derived cyclic peptide revealed that the peptide
backbone and the side chains do not interfere with the
photofragmentation reaction, highlighting the biocompat-
ibility of the S,S-tetrazine phototrigger. With these experi-
ments as background, we next sought to broaden the scope
of the S,S-tetrazine trigger with the development of a general
protocol to incorporate the trigger within a peptide system
having two cysteine residues as desired along a peptide chain.
Herein we describe the design, synthesis, and photophy-
sical validation of a series of peptide linchpins containing
the S,S-tetrazine phototrigger. In addition, we demon-
strate the feasibility of employing these linchpins in solu-
tion phase fragment coupling, which should permit the
ready construction of more complex, relevant peptide and
protein systems.
Figure 2. SPPS was used to access resin bound tripeptides 3;
selective deprotection, tetrazine insertion, standard cleavage
conditions, and subsequent photolysis yielded peptides 4 and 5
respectively.
With the goal of incorporating the S,S-tetrazine linch-
pins within larger peptide/protein scaffolds, the compat-
ibility of various peptide constituents present during the
solid-phase peptide synthesis, in conjunction with the
effect of proximal side chains on the photochemical
behavior of the S,S-tetrazine phototrigger, was explored.
To this end, tripeptide tetrazines 4dꢀj were prepared
(Figure 3). Although 4k could be successfully constructed
and released from the solid support, the conditions to
release the pbf-protected Arg side chain led to decomposi-
tion, presumably due to the incompatibility of the guani-
dinium functionality with the tetrazine system.10 Indeed,
treatment of simple S,S-tetrazine (ꢀ)-1 with basic guani-
dine led to similar decomposition. Importantly, tripeptide
linchpins 4dꢀi underwent clean steady-state photo-
lysis,11 demonstrating that the amino side chains do not
(7) (a) Bredenbeck., J.; Helbing, J.; Sieg, A.; Schrader, T.; Zinth, W.;
Renner, C.; Behrendt, R.; Moroder, L.; Wachtveitl, J.; Hamm, P. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 6452–6457. (b) Bredenbeck, J.;
Helbing, J.; Kumita, J. R.; Woolley, G. A.; Hamm, P. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 2379–2384. (c) Ihalainen, J. A.; Bredenbeck, J.;
Pfister, R.; Helbing, J.; Chi, L.; Van Stokkum, I. H. M.; Woolley, G. A.;
Hamm, P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 5383–5388.
(8) Tucker, M. J.; Abdo, M.; Courter, J. R.; Chen, J. X.; Smith, A. B.;
Hochstrasser, R. M. J. Photochem. Photobiol. A 2012, 234, 156–163.
(9) Tucker, M. J.; Courter, J. R.; Chen, J. X.; Atasoylu, O.; Smith,
A. B.; Hochstrasser, R. M. Angew. Chem., Int. Ed. 2010, 49, 3612–3616.
(10) S,S-Tetrazine 1 was stable to guanidine hydrochloride; however
basic guanidine resulted in a complex mixture. For further details refer
to Supporting Information.
(11) 4dꢀi were photolyzed under standard conditions to give single
compounds by 1H NMR.
Org. Lett., Vol. 14, No. 13, 2012
3519

Figure 4. Isotopically labeled S,S-tetrazine containing tripep-
tide units with doubly labeled carbonyls highlighted in yellow
and singly labeled in green.
Figure 3. Tetrazine tripeptides synthesized in 7ꢀ30% overall
yield with the exception of 4k which undergoes decomposition
upon purification.
perturb the photolysis of the electron-deficient S,S-tetrazine
chromophore.
To monitor structural transitions on a fast time scale
employing 2D-IR, differentiation of the amide I IR
stretching modes of the diverse peptide carbonyls would
be required.12 To this end, tripeptides 4l,m and 7 were
prepared (Figure 4), wherein the alanine and/or cysteine
amino acid carbonyls were labeled with 13C or correspond-
ing 13C/18O, the latter prepared byH218O exchange13 of the
13C-amino acid (see SI). Tripeptides 4l,m were then pre-
pared according to the previously developed solid-phase
conditions employing the isotopically labeled amino acids.
A modification to this protocol was required to construct
tripeptide 7, as the 18O enrichment procedure proved
incompatible with the para-monomethoxytrityl sulfur-
protected cysteine. Pleasingly employing the thio-tert-butyl
sulfur protected amino acid during the 18O exchange14
followed by solid-phase peptide synthesis proved effective.
Sequential removal of the cysteine protecting groups with
tributylphospine and TFA prior to insertion of the tetrazine
moiety permitted preparation of tripeptide 7.
Figure 5. Infrared spectra (1550ꢀ1770 cmꢀ1) of 4a (red), 4l
(green), and 7 (black) recorded in CD3OD.
Following the successful preparation of the 4aꢀm series
of tetrazine tripeptide linchpins, designated as (i, iþ2)
with regard to separation of Cys residues, we next sought
to study larger macrocyclic congeners that inscribe the
tetrazine ring. Standard Fmoc-based solid-phase peptide
synthesis was employed to construct the (i, iþ3) and (i, iþ4)
target peptides 9a,b and 9c respectively (Figure 6). With
these model linchpins in hand, the photofragmentation
reaction was again investigated. Nanosecond flash photo-
lysis15 experiments revealed a dependence of the photo-
chemical yield on the size of the macrocyclic peptide con-
strained by the S,S-tetrazine. Peptide 4c, with an (i, iþ2)
relationship between the Cys residues, afford the highest
photoproduct yield upon flash photolysis (>25%). The
(i, iþ3) relationship in 9b led to a decrease in photoproduct
to>15%; a further decrease to ∼10% wasobserved for the
(i, iþ4) peptide 9c. The observed photochemical yield
dependence is likely related to the conformational strain
exerted by the peptides on the S,S-tetrazine ring. Impor-
tantly the photochemical yields are sufficient that popula-
tion changes are observable by 2D IR, while adequate
intact linchpin is maintained to enable spectroscopic data
to be accumulated over an extended period of time.
IR analysis of the isotopically labeled tripeptides revealed
that the 13C/18O and 13C amino acid amide carbonyl
stretching bands are shifted by ∼60 and ∼30 cmꢀ1, respec-
tively, with the doubly labeled peak appearing at ∼1590 cmꢀ1
,
well separated from the main amide I band (Figure 5). The
stretching frequency of the singly labeled carbonyl, however,
remained somewhat buried in the main band.
(12) (a) Torres, J.; Adams, P. D.; Arkin, I. T. J. Mol. Biol. 2000, 300,
677–685. (b) Fang, C.; Wang, J.; Charnley, A. K.; Barber-Armstrong,
W.; Smith, A. B.; Decatur, S. M.; Hochstrasser, R. M. Chem. Phys. Lett.
2003, 382, 586–592. (c) Fang, C.; Wang, J.; Kim, Y. S.; Charnley, A. K.;
Barber-Armstrong, W.; Smith, A. B.; Decatur, S. M.; Hochstrasser,
R. M. J. Phys. Chem. B 2004, 108, 10415–10427. (d) Fang, C.;
Hochstrasser, R. M. J. Phys. Chem. B 2005, 109, 18652–18663.
(13) Marecek, B.; Song, B.; Brewer, S.; Belyea, J.; Dyer, R. B.;
Raleigh, D. P. Org. Lett. 2007, 9, 4935–4937.
(14) Gormer, K.; Waldmann, H.; Triola, G. J. Org. Chem. 2010, 75,
1811–1813.
(15) Papp, S.; Vanderkooi, J. M.; Owen, C. S.; Holtom, G. R.;
Phillips, C. M. Biophys. J. 1990, 50, 177–186.
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Org. Lett., Vol. 14, No. 13, 2012

C-terminus was then coupled to phenylalanine benzyl-
amide employing standard Boc-based peptide solution-
phase synthesis to furnish 13. Following removal of the
Boc protecting group (cf. 14), the N-terminus was then
coupled to N-R-Boc-N-ε-Cbz lysine to furnish 15 (Figure 8).
Overall the yields were good (ca. 43% over 4 steps).
Figure 6. The S,S-tetrazine incorporated at different positions
along the peptide backbone to furnish 9a,b (i, iþ3) and 9c (i, iþ4)
macrocylcic peptides.
Building upon the successful synthesis and development
of the (i, iþ2) peptide linchpins, we focused attention next
on constructing larger peptide constructs containing the
S,S-tetrazine with an (i, iþ2) linchpin relationship between
the Cys residues. The solid-phase conditions employed
earlier to incorporate the S,S-tetrazine chromophore un-
fortunately proved unworkable when g8 amino acid pep-
tides were immobilized on the solid support, despite
employing a wide variety of reaction paradigms, such as
including denaturants, elevated temperatures, and using
3,6-dibromotetrazine16 (Figure 7). We also explored early
incorporation of the tetrazine unit 4b; repeated iterative
treatment with piperidine employed in the solid-phase
peptide synthesis tactic however led to decomposition of
the S,S-tetrazine.17,18 We, thus, chose to develop and
deploy the S,S-tetrazine linchpins employing a fragment
union tactic, whereby readily prepared peptide residues
would be attached to the C- and N-termini of the tetrazine
linchpins to access more complex peptides and proteins.
Figure 8. Use of a chromophore containing (i, iþ2) peptide
linchpin in a solution phase fragment condensation approach.
In summary, an effective synthetic protocol has been
designed and validated to access a variety of peptide
linchpins containing the S,S-tetrazine phototrigger. With
the appropriate Boc protected tetrazine linchpins available
from the corresponding (i, iþ2), (i, iþ3), and (i, iþ4) peptide
linchpins, access to a wide variety of peptide or protein
scaffolds that contain the S,S-tetrazine photochemical trig-
ger should be feasible. These constrained peptides with an
(i, iþ2), (i, iþ3), and (i, iþ4) relationship between the Cys
residues present an avenue for irreversibly constraining a
variety of common secondary peptide structural motifs, and
ultimately the tertiary and quaternary structures of more
complex proteins. Importantly, a variety of spectroscopic
methods, such as IR probe and 2D IR spectroscopy, are
now available to track conformational transitions on the
pico- to nanosecond time scale, as the designed peptides, at
some known displacement from the equilibrium state, relax
to new equilibrium conformations upon the triggering event.
Acknowledgment. Financial support was provided by
the NIH GM 12694 and the RLBL facility grant (NIH P41
RR 001348). The authors also thank Drs. George Furst
and Rakesh Kohli at the University of Pennsylvania for
assistance in obtaining NMR and high-resolution mass
spectra, respectively.
Figure 7. Attempted synthesis of lengthy peptides containing the
S,S-tetrazine on solid support.
To prepare a tetrazine linchpin suitable for solution
phase fragment union, the N-terminus of tripeptide 4a
inscribing the S,S-tetrazine was protected as the tert-
butyl carbamate. With peptide linchpin 12 in hand, the
Supporting Information Available. Details of the initial
photophysical studies, and the preparation and spectro-
scopic characterization for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(16) For synthesis of 3,6-dibromotetrazine refer to Supporting
Information.
(17) Alternative protocols to remove the Fmoc protecting group
including morpholine, triaminoethylamine, DBU, and TBAF were
unsuccessful.
(18) Evidence for displacement of bis-(alkylthio)-tetrazine: Panek,
J. S.; Zhu, B. Tetrahedron Lett. 1996, 45, 8151–8154.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 13, 2012
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