The design and synthesis of alanine-rich a-helical peptides constrained by an s,s-tetrazine photochemical trigger: A fragment union approach

Article abstract of DOI:10.1021/jo402680v

The design and synthesis of alanine-rich a- helical peptides constrained in a partially unfolded state by incorporation of the S,S-tetrazine phototrigger has been achieved, permitting, upon photochemical release, observation by 2D-IR spectroscopy of the subnanosecond conformational dynamics that govern the early steps associated with a-helix formation. Solid-phase peptide synthesis was employed to elaborate the requisite fragments, with full peptide construction via solution-phase fragment condensation. The fragment union tactic was also employed to construct 13C?18O isotopically edited amides to permit direct observation of conformational motion at or near specific peptide bonds.

Full text of DOI:10.1021/jo402680v

Article
pubs.acs.org/joc
The Design and Synthesis of Alanine-Rich α‑Helical Peptides
Constrained by an S,S‑Tetrazine Photochemical Trigger: A Fragment
Union Approach
Joel R. Courter, Mohannad Abdo, Stephen P. Brown, Matthew J. Tucker,† Robin M. Hochstrasser,‡
and Amos B. Smith, III*
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
S
* Supporting Information
ABSTRACT: The design and synthesis of alanine-rich α-
helical peptides constrained in a partially unfolded state by
incorporation of the S,S-tetrazine phototrigger has been
achieved, permitting, upon photochemical release, observation
by 2D-IR spectroscopy of the subnanosecond conformational
dynamics that govern the early steps associated with α-helix
formation. Solid-phase peptide synthesis was employed to
elaborate the requisite fragments, with full peptide construction via solution-phase fragment condensation. The fragment union
18
tactic was also employed to construct 13C O isotopically edited amides to permit direct observation of conformational motion
at or near specific peptide bonds.
Previously developed phototriggers include non-native
disulfide bonds, pioneered by Hochstrasser and DeGrado,20,21
and the methoxybenzoin-based systems developed by Chan and
co-workers.22−25 However, these triggers have limitations,
including rapid recombination of the initial photoproducts,20,26
the inherent chemical reactivity of the derived photoproducts,
and, in the case of methoxybenzoin-based systems, the
introduction of obtrusive non-native chemical appendages as
a result of the phototrigger structure. Criteria for the ideal
phototrigger, as previously defined,18 include (1) photo-
chemical fragmentation on a time scale shorter than the
conformational changes under observation, (2) a reasonably
high photochemical yield, (3) inert products with negligible
side reactions along the photochemical pathway, (4) function-
ality permitting ready incorporation within peptides and
proteins, (5) sufficient stability to permit synthetic manipu-
lations and purification both in solution and on solid supports,
and (6) photochemical cleavage at light frequencies compatible
with the amide backbone and amino acid side chains.
To meet these criteria, we introduced the S,S-tetrazine
chromophore (Figure 1A).27−29 Importantly, the photo-
products comprise inert and unobtrusive thiocyanates and
nitrogen; moreover, the S,S-tetrazine phototrigger can be
inscribed readily between two cysteine residues to constrain a
peptide within a narrow distribution of conformers.28 We also
described UV-pump transient 2D-IR-probe experiments to
monitor the structural relaxation of a 24 residue alanine-rich
peptide containing a kink induced by the S,S-tetrazine
phototrigger (Figure 1B).30 Upon release of the constraint,
INTRODUCTION
■
Peptides and proteins comprise dynamic entities that, in many
cases, exploit conformational changes and/or transient
molecular interactions to achieve their biological function.1−5
Understanding the early kinetic events that govern conforma-
tional dynamics in peptides, and in turn proteins, is critical for
the development and refinement of models that relate dynamic
structure to the observed biological function.6−8 Structural
snapshots of such biomolecules can be achieved at atomic
resolution by X-ray crystallography and nuclear magnetic
resonance measurements. However, these static structures do
not provide information on transiently sampled nonequilibrium
structures. Structural change on the millisecond time scale can
be defined by nuclear magnetic resonance spectroscopy,9 while
advances in time-resolved X-ray crystallography10−14 permit
temporal resolution on subnanosecond time scales within a
crystal, although this approach remains technically challenging.
Two-dimensional infrared (2D-IR) spectroscopy, developed
and pioneered by Hochstrasser and colleagues,15−17 in
conjunction with ultrafast photochemical release (i.e., trigger-
ing),5,18,19 provides an attractive alternative method to initiate
and subsequently observe the earliest kinetic processes
associated with conformational change (i.e., protein folding
and misfolding). For example, incorporation of a phototrigger
to confine a peptide/protein within a narrow distribution of
conformers, followed by ultrafast photochemical release of the
geometric constraint, would permit precise temporal control. In
particular, the peptide conformation would evolve, with some
initial spatial coherence, toward a different distribution,
ultimately reaching the native equilibrium distribution of
peptide/protein conformations.
Received: December 3, 2013
Published: December 20, 2013
© 2013 American Chemical Society
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wherein the introduced cross-link(s) typically aim to introduce
minimal changes to the ground-state conformation of the
peptide,32 we require the S,S-tetrazine “staple” to introduce a
well-defined local perturbation to the peptide conformation. Of
interest, the S,S-tetrazine staple can be rapidly released upon
photolysis.
A variety of (i, i + 2/4) spacings between the cysteine
residues inscribing the S,S-tetrazine system were initially
investigated by molecular dynamics (MD) simulations, with
distance constraints between the sulfur atoms employed to
model the phototrigger.30 The resultant constrained MD
simulations predicted that the S,S-Tet-AKA peptide would
assume a kinked α-helical conformation wherein the S,S-
tetrazine moiety would disrupt the helical structure between
Cys10 and Cys12 (cf. Figure 1B, Topt = 0 ps).30 Our synthetic
targets thus became S,S-Tet-AKA and isotopically edited
versions thereof (Figure 2).
Figure 1. (A) Photolysis of the S,S-tetrazine phototrigger can be
achieved with laser pulses at various wavelengths that afford two
thiocyanates and molecular nitrogen in less than 50 ps. (B) Schematic
representation of S,S-tetrazine phototriggering applied to S,S-Tet-
AKA. Photolysis of the S,S-tetrazine (shown in space-filling
representation) removes the structural constraint to furnish the bis-
SCN peptide photoproduct. The 2D-IR measurements at various time
delays following the photolysis pulse (Topt) directly monitor the
conformational relaxation within the central CAC motif (the main
chain of the peptide in the CAC region is shown in sticks).
initiated by a subpicosecond pulse of UV irradiation (λ = 355
nm), the formation of a single α-helix turn was directly
observed by 2D-IR spectroscopy without “unzipping” of nearby
helix turns (vide infra).
Figure 2. The AKA peptide (top) was employed for the design of an
S,S-tetrazine-constrained synthetic target, S,S-Tet-AKA (bottom).
By constraining the center of an α-helix with an S,S-tetrazine
moiety, we envisioned a perturbation not dissimilar to that
induced by a proline residue.33 To estimate the potential
helicity of the designed target peptides relative to the original
AKA peptide (Scheme 1), model peptides AKAA11P and
AKAA10C/A12C were constructed via solid-phase peptide syn-
Herein we describe the design and requisite synthesis of a
constrained 24-residue alanine-rich α-helical peptide possessing
the S,S-tetrazine phototrigger (S,S-Tet-AKA; cf. Figure 2) by
means of a solution-phase fragment coupling tactic. The
30
18
synthesis of 13C O edited isotopomers is also described.
Going forward, the general fragment coupling protocol
developed to construct S,S-Tet-AKA can now be applied to
peptides and proteins of greater complexity to initiate
conformational changes that occur on subnanosecond time
scales.
Scheme 1. Synthesis of the AKA Variants AKAA11P and
AKAA10C/A12C
RESULTS AND DISCUSSION
■
Design and Synthesis. We initiated studies to construct
peptides containing the S,S-tetrazine within the 24-residue
alanine-rich AKA peptide (Figure 2), a system introduced by
Baldwin and colleagues31 to adopt in the ground state an α-
helical structure that has subsequently often been employed in
folding and relaxation studies. We reasoned that introduction of
the S,S-tetrazine moiety within the middle of a 24-residue
peptide would permit both the N- and C-terminal segments to
maintain an α-helical conformation, while the amino acids
between the cysteine residues would remain nonhelical because
of the geometric constraints enforced by incorporation of the
S,S-tetrazine phototrigger (Figure 1B). Upon photochemical
release of the constraint imposed by the S,S-tetrazine
phototrigger, the terminal α-helices would direct the central
kinked residues to equilibrate rapidly to the preferred extended
α-helical conformation. In contrast to peptide “stapling”,
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thesis (SPPS) employing N-methylindole aminomethyl resin.
The proline mutant AKAA11P was specifically constructed to
mimic the S,S-tetrazine-constrained peptide before photolysis
(i.e., S,S-Tet-AKA), while the double cysteine mutant
AKAA10C/A12C was synthesized to mimic the bis-thiocyanate
peptide photoproduct.
Scheme 2. SPPS of Tetrapeptide 3 and Steady-State
Photolysis of 3 To Afford Bis-Thiocyante Tetrapeptide 4
The far-UV CD spectra of the S,S-Tet-AKA (prepared as
described below), AKAA11P, and AKAA10C/A12C peptides were
recorded; an absorption band at 222 nm, diagnostic of helical
structure, was observed for the native AKA peptide (Figure
3).31 The fractional helicity [θ222] of the native AKA was
nate peptide 4, thus confirming that the proximal basic Lys side
chain does not perturb the steady-state photolysis of the
electron-deficient S,S-tetrazine. The disappearance of the 13C
NMR signals for the tetrazine ring (δc ≈ 170 ppm) with the
concomitant appearance of the SCN resonances at δc ≈ 113
ppm27 proved to be particularly diagnostic for monitoring the
S,S-tetrazine photolysis (see the Supporting Information).
Having achieved the successful construction of a peptide
model system and validated the photochemical fragmentation,
we turned to the synthesis of S,S-Tet-AKA (Scheme 3). The
solid-phase conditions employed earlier to incorporate the S,S-
tetrazine component in the model peptide27,29 unfortunately
proved ineffective when the fully constructed peptide backbone
remained immobilized on the solid support. A number of
reaction modifications, such as including denaturants, elevating
Figure 3. Far-UV CD spectra of AKAA11P in phosphate buffer (dark
blue) and TFE (magenta), AKAA10C/A12C in TFE (red), and the final
synthetic target S,S-Tet-AKA in TFE (green).
determined to be 0.72 in aqueous phosphate buffer (data not
shown). The band at 222 nm nearly disappeared upon
incorporation of the proline residue within AKAA11P, suggesting
that the fractional helicity was significantly reduced (ca. 0.05).33
To ensure that a desirable degree of fractional helicity can be
achieved for a kinked AKA-type peptide system, trifluoroetha-
nol (TFE), which is known to maximize the peptide helicity
and increase the solubility,34 was employed to enhance the
helicity of AKAA11P. Pleasingly, a value of 0.34 was observed for
the AKAA11P peptide in TFE solution. A slight increase in the
fractional helicity to 0.49 was observed for the AKAA10C/A12C
peptide in TFE. These results were highly encouraging,
suggesting that following photolysis of the S,S-tetrazine-
constrained AKA peptide (i.e., S,S-Tet-AKA), a more helical
conformation should evolve, as suggested by the helicity of the
analogous AKAA10C/A12C peptide indicated by the CD spectra.
Turning to the synthesis of the 24-residue target peptide S,S-
Tet-AKA, we recently introduced an SPPS synthetic protocol
to incorporate the S,S-tetrazine phototrigger within short tri-,
tetra-, and peptapeptide linchpins.29 The compatibility of a
lysine in the i + 1 position relative to the S,S-tetrazine, however,
had not been explored. To validate the compatibly of a
protected Lys side chain under the conditions required to insert
the S,S-tetrazine, tetrapeptide 3 was constructed via SPPS, with
both the lysine side chain bearing the standard tert-butyl
carbamate protecting group and with the cysteine side-chain
thiols masked with the 4-monomethoxytrityl (Mmt) protecting
groups (Scheme 2). The synthesis proceeded without incident.
Equally important, we demonstrated that tetrapeptide 3
underwent steady-state photolysis to yield solely bis-thiocya-
Scheme 3. Attempted Synthesis of the S,S-Tet-AKA Peptide
on the N-Methylindole Solid Support
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the reaction temperature, or employing more polar solvent
systems failed to improve the situation. An alternative synthetic
strategy was thus explored wherein orthogonally protected Cys
residues35 [i.e., thio-tert-butyl (St‑Bu) at Cys10 and Mmt at
Cys12] were incorporated during SPPS in an attempt first to
introduce the S,Cl-tetrazine (to give 6 in Scheme 3) and then
to remove the second thiol protecting group with concomitant
formation of the S,S-tetrazine moiety. This tactic also proved
ineffective. Equally unsuccessful was our tactic employed to
replace the disulfide bond in the peptide hormone oxytocin
with the S,S-tetrazine,27 which called for treatment of the
AKAA10C/A12C peptide in an aqueous 0.1 M ammonium
bicarbonate solution with dichlorotetrazine (2), as this
approach also failed to furnish the desired S,S-Tet-AKA
peptide. Presumably the immobilized peptide has a preference
to adopt the α-helical structure on the solid support, a
conformational bias that could not be sufficiently perturbed to
incorporate the S,S-tetrazine phototrigger.
Undeterred, we turned to a fragment condensation strategy
carried out in solution to construct the S,S-Tet-AKA peptide.
Early studies in our laboratory had demonstrated that the S,S-
tetrazine moiety is stable toward amide bond coupling
protocols.29 Initially a model system comprising a two-fragment
union tactic was explored. To this end, S,S-tetrazine peptide 8,
which was readily constructed on the Wang resin with the Lys
side chain reprotected as the tert-butyl carbamate, furnished
coupling partner 9 (Scheme 4A). The requite dodecapeptide
coupling partner 10, corresponding to the C-terminal peptide
fragment, was next prepared via a standard SPPS protocol. With
fragments 9 and 10 in hand, our previously developed fragment
union tactic utilizing HBTU and ethyl 2-cyano-2-
(hydroxyimino)acetate (oxyma)29,36 was employed to achieve
union at the Cys12−Ala13 amide bond; the yield was 53%
(Scheme 4B). Subsequent treatment with TFA furnished
peptide 11 in nearly quantitative yield. Importantly, peptide
11 corresponds to residues 7−24 of the full synthetic target S,S-
Tet-AKA.
With construction of the truncated peptide 11 validated, we
turned to a three-fragment condensation tactic to generate the
target peptide possessing the full N-terminal sequence. As the
N-terminus of our previously reported S,S-tetrazine tripeptide
linchpin, employed via solution-phase fragment coupling, was
most conveniently protected as the Boc carbamate,29 we sought
a semiorthogonal protecting group to mask the lysine side
chains. The benzyloxycarbonyl (Cbz) protecting group was
selected and proved to be compatible with the S,S-tetrazine
insertion conditions employed to construct tetrapeptide 13
(Scheme 5A). Importantly, the Cbz protecting groups were
Scheme 5. (A) Evaluation of Benzyloxycarbonyl (Cbz) as an
Alternative Protecting Group for the Lysine Side Chain and
Validation of the Deprotection Conditions; (B) The S,S-
Tetrazine Ring of 15 Is Partially Reduced to the Dihydro-
S,S-Tetrazine System 16 upon Exposure to Hydrogenolysis
Conditions
Scheme 4. (A) Preparation of Lys-Protected S,S-Tetrazine
Hexapeptide 9; (B) Union of S,S-Tetrazine Hexapeptide 9 to
the C-Terminal Peptide Fragment 10 of the S,S-Tet-AKA
Target Peptide, with the Corresponding Residue Numbers
Shown, To Furnish 18-Mer 11
retained during removal of 13 from the resin by treatment with
TFA/TIPSH/H2O (95:2.5:2.5) for 2.5−5 h. However, removal
of the Cbz protecting group from the Lys side chain to furnish
peptide 14 required more strenuous conditions, namely,
treatment with TFA for 24 h.
Turning next to the orthogonal Cbz-removal tactic employ-
ing hydrogenation, we were surprised to observe rapid loss of
the orange color characteristic of the S,S-tetrazine chromo-
phore. However, when the colorless solution was permitted to
stand exposed to air, the solution slowly regained the original
orange color. This observation suggested that hydrogenation
had led to semi-reduction of the S,S-tetrazine ring. To explore
this scenario, we turned to our previously described amino acid-
based S,S-tetrazine 15 (Scheme 5B).27 Similar exposure of 15
to the hydrogenation conditions led to loss of the characteristic
orange color with formation of dihydro-S,S-tetrazine 16 in
nearly quantitative yield (1H and 13C NMR). In an effort to
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distinguish the tautomer obtained upon semihydrogenation,
density functional theory (DFT) calculations at the RB3PW91/
6-31+G(d,p) level were performed using Gaussian 0937 to
determine the frequencies and intensities of the IR transitions
corresponding to the 1,2- and 1,4-dihydro-S,S-terazine
tautomers. A correction factor based on the ratio of known
experimental carbonyl frequencies to those derived from the
DFT calculations was applied to the frequencies. The best
agreement between the calculated and experimental infrared
transitions suggested the formation of 1,2-dihydro-S,S-tetrazine
16. Importantly, exposure of 16 to air over a 24 h period
demonstrated that the dihydro-S,S-tetrazine 16 does indeed
oxidize to give S,S-tetrazine 15.
model (vide supra). For the proposed transient 2D-IR
experiments, this fractional helicity was required in order to
observe conformational reorganization to the final α-helical
structure upon photolysis of the S,S-tetrazine constraint.
Photochemical Studies: Kinetics and Conformational
Response to Photorelease. The synthetic S,S-Tet-AKA
peptide was first subjected to steady-state photolysis (λ = 355
nm) to afford the (SCN)2-AKA peptide (see Figure 1B) in
TFE. We were delighted to find that the fractional helicity of
(SCN)2-AKA was 0.50, representing roughly a 0.07 increase in
the helical content upon release of the S,S-tetrazine photo-
trigger. In view of the fact that only a single α-helix turn is
formed following the S,S-tetrazine photolysis, the observed
modest increase in helicity was expected. Transient 2D-IR
measurements reported elsewhere30 also confirmed the
formation of the single α-helix turn following photolysis with
a time constant of ∼100 ps. To ensure that photolysis of the
S,S-tetrazine linker was complete, formation of the SCN
functionality was confirmed by FT-IR spectroscopy through
observation of the SCN stretching band.30
With the use of the Cbz protecting group validated, the
synthesis of the target S,S-Tet-AKA peptide employing a three-
fragment union tactic was undertaken (Scheme 6). The
Scheme 6. The Successful Three-Fragment Union Approach
To Synthesize S,S-Tet-AKA
The tripeptide model system 2129 (Figure 4 inset) was also
employed to provide insights into the photochemistry of the
Figure 4. Time-dependent spectrum of the SCN transient absorption
observed upon photolysis (355 nm excitation) of S,S-tetrazine
tripeptide 21 to furnish bis-SCN tripeptide 22, measured at 2163
cm−1 from 0 to 100 ps. The raw data points are black squares outlined
in blue, while the exponential fit to data, with a time constant τ = 56
ps, is shown by the green curve.
kinked region. Upon photolysis of 21, the yield of 22 measured
by nanosecond transient absorption was 28%, slightly larger
than the 20% previously observed for the photolysis of
noncyclic S,S-tetrazine peptide 15.28 The increase in photo-
chemical yield is likely related to the conformational strain
exerted on the S,S-tetrazine system when the Cys residues are
in an i, i + 2 relationship within cyclic peptide 21. The time
scale of SCN formation upon flash photolysis of 21 was
determined by femtosecond transient spectroscopy27,28 by
measuring the time dependence of the increase of the SCN
absorption band intensity of the peptide photoproduct 22
centered at 2163 cm−1 (Figure 4). The time constant for SCN
formation was determined to be ∼56 ps, again slightly shorter
than that previously reported for the acyclic system 15. On the
basis of these experiments, the time required for formation of
the final photoproduct 22 is expected to be on the order of tens
of picoseconds, while the initial time scale for S,S-tetrazine ring
cleavage of acyclic 15 was determined by prior experiments to
be ∼10 ps.28
appropriate linchpin 17 was available from our model studies,29
while the requisite C- and N-terminal peptide fragments 18 and
20 were constructed via SPPS employing the N-methylindole
and 2-chlorotrityl resins, respectively. The free amine of peptide
18 was first coupled to S,S-tetrazine linchpin 27, followed by
the removal of the Boc protecting group, to furnish peptide 19
in 43% yield for the two steps. Peptide 19 in turn was coupled
to the N-terminal peptide fragment 20, followed by treatment
with TFA, to complete the construction of the peptide S,S-Tet-
AKA.30 The final union and deprotection proceeded in 25%
yield after HPLC purification.
Synthetic S,S-Tet-AKA was characterized by HPLC−MS and
MALDI-TOF MS. Pleasingly, the far-UV CD spectra of S,S-
Tet-AKA in TFE (Figure 3) indicated the fractional helicity to
be 0.43, similar to that of the proline-kinked mutant AKAA11P
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To monitor the picosecond structural transitions within the
amide backbone by 2D-IR upon photochemical release of S,S-
Tet-AKA, the amide-I stretching modes were differentiated by
employing carbonyl isotopic editing. Specifically, we incorpo-
AKAA17*/A18*) do not support conformational changes, we
have proposed a model for this conformational transition
wherein the dominant motion of the peptide is predominantly
through rotation about the ψ dihedral angle.30
18
rated the 13C O double label at defined positions along the
peptide backbone.38−42 Our modular fragment coupling
strategy was well-suited to prepare these peptides, as we had
CONCLUSION
■
We report here the design and validation of a general synthetic
protocol that permits incorporation of the S,S-tetrazine
photochemical trigger into complex peptides. Photochemical
release and monitoring by 2D-IR spectroscopy permits atomic-
level definition of the microscopic motions that specific
peptide/protein structural components explore along con-
formational reorganization paths to reach a final equilibrium
structure. The specific photochemical trigger, S,S-tetrazine, was
introduced into the alanine-rich α-helical AKA peptide to
disrupt the extended helical conformation, similar to a
helix−“proline turn”−helix. The bias to assume an extended
α-helix conformation, however, initially hindered efforts to
incorporate the S,S-tetrazine phototrigger directly employing
our previously developed protocols. To overcome this
challenge, the separate peptide fragments were constructed
through SPPS, and a fragment union tactic was executed in
solution to furnish the relatively large 24-residue S,S-Tet-AKA
peptide system, which pleasingly adopts a well-defined initial
conformational distribution. The initial helix−turn−helix
conformation with the minor S,S-tetrazine perturbation within
the middle of the sequence permitted 2D-IR observation of the
microscopic structural transitions following ultrafast photo-
chemical release. A detailed account of the conformational
dynamics observed for the S,S-Tet-AKA peptide is now
available.30 Pleasingly, the synthetic methods developed herein
hold the promise of wide applicability for the construction of
diverse peptides and proteins possessing the ultrafast photo-
chemical trigger S,S-tetrazine to define the early events in
peptide/protein conformation reorganization.
18
previously reported a 13C O isotopically edited S,S-tetrazine
linchpin29 that we utilized here to prepare labeled peptide S,S-
Tet-AKAC10*/A11* (Figure 5). In addition, to monitor the
Figure 5. Isotopically edited peptides S,S-Tet-AKAC10*/A11*, S,S-Tet-
AKAA5*/A6*, and S,S-Tet-AKAA17*/A18* were synthesized to resolve the
amide-I stretching modes from the remaining backbone stretching
18
modes. The positions of the 13C O-labeled amides in the synthetic
peptides are shown as blue circles.
EXPERIMENTAL SECTION
backbone dynamics distal to the S,S-tetrazine phototrigger, two
■
adjacent units of 13C O-Ala43 were incorporated within two
18
General Experimental Methods. Organic solvents used for
reactions and washes were of reagent grade and degassed by purging
with nitrogen prior to use. Di-tert-butyl dicarbonate (Boc2O),
diisopropylethylamine (DIPEA), acetic anhydride, N-methylimidazole,
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium (HBTU), 1-
(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT), ethyl 2-
cyano-2-(hydroxyimino)acetate (oxyma), Fmoc-Ala-OH, Fmoc-Cys-
(Mmt), Fmoc-Asp(Ot-Bu)-OH, Fmoc-Gly-OH, Fmoc-Pro-OH,
Fmoc-Ser-OH, Fmoc-Trp(N-Boc)-OH, Fmoc-Val-OH, Fmoc-Ala-
OH (1-13C), L-cysteine (1-13C), and 18O water were purchased from
commercial vendors and used as received. Wang resin (LL) and N-
methylindole aminomethyl resins for peptide synthesis were obtained
from Novabiochem.
Solid-phase syntheses were carried out in peptide synthesis reaction
vessels (25 or 50 mL) with coarse-porosity fritted glass supports and
Teflon stopcocks. The reaction vessels had 14/20 ground glass joints
that allowed the resin to be kept under a nitrogen atmosphere while
removing the monomethoxytrityl groups from cysteine. Photolysis
experiments were performed in a Srinivasan−Griffin photoreactor.
Microwave irradiation was employed using 2−5 mL sealed microwave
reaction vessels, and an IR temperature sensor was used to control the
temperature.
S,S-tetrazine peptides, the first pair within the N-terminal
peptide fragment, leading to the synthesis of S,S-Tet-
AKAA5*/A6*, and the second pair within the C-terminal peptide
fragment, furnishing S,S-Tet-AKAA17*/A18*. Standard SPPS
18
utilizing 13C O-Ala at the requisite positions, followed by
the fragment union tactic, was employed.
Upon evaluation of S,S-Tet-AKAA5*/A6* and S,S-Tet-
AKAA17*/A18* by standard 2D-IR methods, the coupling
between these dipoles indicated that the peptides have an α-
helical structure in both the N- and C-terminal domains,
respectively.44,45 Subsequent evaluation of the N- or C-
terminal-labeled residues by transient 2D-IR probe spectrosco-
py revealed no changes in the angle between the dipoles
following S,S-tetrazine photolysis, suggesting that these regions
of the peptide do not undergo conformational changes. As
previously reported, only in the case of S,S-Tet-AKAC10*/A11*
,
where the residues are in the domain of the peptide that is
inscribed by the S,S-tetrazine constraint, did we observe
significant changes in the conformation of the peptide.30
Equally important and as described in detail elsewhere,30 the ϕ
and ψ angles could be extracted from the transient 2D IR
A chloranil test was used to examine the presence of free amines on
resin. A test tube containing a small amount of resin (10−12 beads)
known to have a free primary amine was used as a reference along with
another test tube containing a small amount of the same resin after
acylation. Each resin was first washed with DMF (3 × 1 mL) and then
to each in DMF (1 mL) was added 1 drop of 2% (w/w) acetaldehyde/
18
spectra of the 13C O amides of Cys10 and Ala11 to reveal
directly the rate of relaxation to the full helical structure. As the
distal residues (cf. S,S-Tet-AKAA5*/A6* and S,S-Tet-
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DMF, immediately followed by 1 drop of 2% (w/w) 2,3,5,6-
tetrachloro-1,4-benzoquinone (p-chloranil)/DMF and allowed to
stand at room temperature for 5 min. The reaction solution was
then removed by syringe and washed with DMF. The free amine resin
(control) turned a deep-blue color while the acylated resin remained
unchanged, suggesting that the amine had been completely acylated.
The alizarin−cyanuric chloride test was used to test for alcohols on
resin. Two resins in separate test tubes, one Wang resin and the other
acylated Wang resin, were used. Each was washed with DMF (3 × 1
mL), and then a cocktail of N-methylmorpholine (1 mL), cyanuric
chloride (5 mg), and DMF (3 mL) was added to each tube and heated
at 70 °C for 20 min. The resin beads were washed with DMF (3 × 1
mL). Next, a solution of N-methylmorpholine (1 mL), alizarin (5 mg),
and DMF (3 mL) was added, and after 5 min the resin beads were
washed with DMF. The Wang resin turned dark red, while the acylated
resin remained colorless.
Resin washing was conducted with the indicated solvent and was
allowed to contact the resin for 30 s during each wash. The solvent was
pushed through the frit using an “air push” apparatus made from a 15
mL disposable syringe and a 14/20 septum, or nitrogen gas was used
in cases of an inert atmosphere requirement.
Preparative-scale reversed-phase chromatography was conducted
using binary HPLC pumps and a UV−vis dual-wavelength detector.
The separations were achieved with a Waters XBridge Prep BEH 130
C18 5 μm OBD 19 mm × 100 mm column. The eluent was
acetonitrile (HPLC grade) and Millipore water with 0.05% formic acid
buffer, unless otherwise noted. Linear gradient elutions with a flow rate
of 15 mL/min were employed, and specific gradients for each
compound are noted.
General Resin Loading Procedures. Wang Resin. The resin
(0.22 mmol) was placed in a peptide synthesis vessel and swelled with
CH2Cl2 (10 mL) for 1 h. The solvent was drained, and a premixed
solution of Fmoc-AA-OH (0.88 mmol, 4.0 equiv), MSNT (0.88 mmol,
4.0 equiv), and methylimidazole (0.66 mmol, 3.0 equiv) dissolved in
CH2Cl2 (5 mL) was added to the resin. The contents were gently
rocked for 2 h and then drained, and the resin was washed with
CH2Cl2 (3 × 5 mL). The procedure was repeated and the resin carried
on to the next step.
N-Methylindole AM Resin. N-Methylindole resin (0.22 mmol) was
placed in a peptide synthesis vessel and swelled with CH2Cl2 (10 mL)
for 1 h. The solvent was drained, and the resin was then treated with a
solution of 20% piperidine/DMF (2 × 7 mL), allowing each treatment
to contact the resin for 10 min. The resin was then washed with DMF
(3 × 5 mL), and a premixed solution of Fmoc-AA-OH (1.1 mmol, 5
equiv), HBTU (1.1 mmol, 5 equiv), oxyma (1.1 mmol, 5 equiv), and
DIPEA (2.2 mmol, 10 equiv) dissolved in DMF (5 mL) was added to
the resin. The contents were rocked gently for 1 h and then drained,
and the resin was washed with DMF (3 × 5 mL). The coupling
procedure was repeated and the resin carried on to the next step.
2-Chlorotrityl Chloride Resin. 2-Chlorotrityl chloride resin (0.22
mmol) was placed in a peptide synthesis vessel and swelled with
CH2Cl2 (10 mL) for 1 h. The solvent was drained, and then a
premixed solution of Fmoc-AA-OH (0.26 mmol, 1.2 equiv) and
DIPEA (0.88 mmol, 4 equiv) dissolved in CH2Cl2 (5 mL) was added
to the resin. The contents were rocked gently for 1 h and then drained,
and the resin was washed with DMF (3 × 5 mL). The coupling
procedure was repeated and the resin carried on to the next step.
General Procedures for Solid-Phase Peptide Synthesis. The
resin-bound Fmoc-amino acid (0.22 mmol) was washed with DMF (3
× 5 mL) and then treated with a 20% piperidine/DMF solution (2 × 7
mL), allowing each treatment to contact the resin for 10 min. The
resin was washed with DMF (3 × 5 mL), and then a premixed solution
of Fmoc-protected amino acid (1.1 mmol, 5.0 equiv), HBTU (1.1
mmol, 5.0 equiv), oxyma (1.1 mmol, 5.0 equiv), and DIPEA (2.2
mmol, 10.0 equiv) dissolved in DMF (5 mL) was added to the resin.
The contents were rocked gently for 1 h and then drained, and the
resin was washed with DMF (3 × 5 mL). The Fmoc deprotection
procedure was repeated, followed by the coupling of the amino acid
sequence to synthesize the desired peptide. The N-terminus was
protected as the Boc- or Ac-derivative by first employing the Fmoc
deprotection procedure and washing the resin with DMF (3 × 5 mL).
A premixed solution of Boc-anhydride (2.2 mmol, 10 equiv)/acetic
anhydride (2.2 mmol, 10 equiv) and DIPEA (4.4 mmol, 20 equiv) in
DMF (5 mL) was added to the resin. The contents were rocked gently
for 1 h and then drained, and the resin was sequentially washed with
DMF (3 × 5 mL) and CH2Cl2 (3 × 5 mL).
S,S-Tetrazine Tethering during SPPS. The resin-bound peptide
(0.22 mmol) containing two S-Mmt-protected Cys groups was swelled
with CH2Cl2 (10 mL) for 1 h. The peptide synthesis vessel was then
sealed with a septum, vented to a mineral-oil-filled bubbler, and
attached to a two-neck collection flask. The solvent was drained, and a
premixed solution of CH2Cl2/TIPSH/TFA (92.5:5.0:2.5 peptides on
the Wang resin; 95:4:1 for loaded N-methylindole AM resin) was
added to the resin, after which a stream of nitrogen was introduced
through the collection flask and bubbled through the resin bed to
agitate the orange reaction mixture for 3 min. The contents were
drained by pushing the liquid contents into the collection flask using a
stream of nitrogen, and the resin was washed with CH2Cl2 (10 mL).
The procedure was repeated four additional times until the reaction
solution became clear, suggesting that the monomethoxytrityl groups
had been completely removed. The resin was washed with 10%
DIPEA/ClCH2CH2Cl (3 × 5 mL). To the resin was added a solution
of dichlorotetrazine (2) (0.24 mmol, 1.1 equiv) in ClCH2CH2Cl (5
mL) followed by neat DIPEA (0.5 mL). The peptide vessel was
wrapped in foil to exclude ambient light, and the contents were
agitated for 18 h. The solvent was drained, and the resin was washed
with CH2Cl2 (3 × 5 mL), MeOH (3 × 5 mL), and CH2Cl2 (3 × 5
mL).
1H NMR (500 MHz), 13C NMR (125 MHz), and 2D NMR spectra
were recorded with either a 5 mm dual inverse probe or a 5 mm DCH
CryoProbe. Analytical LC−MS analyses were conducted on a binary
gradient HPLC system connected to a diode array detector and a mass
spectrometer with electrospray ionization. The HPLC−MS samples
were analyzed as solutions in water or acetonitrile, prepared at 0.15−
0.20 mg/mL concentration. The analytical HPLC−MS analyses were
conducted with an Atlantis C18 column (4.6 mm × 50 mm; 5 μm),
and linear gradients of 0.05% formic acid in acetonitrile and 0.05%
formic acid water were utilized with a flow rate of 2 mL/min. High-
resolution mass spectrometry (HRMS) was performed with a MALDI-
TOF-TOF mass spectrometer using electrospray ionization in positive
or negative mode, depending upon the analyte. All FTIR spectra were
obtained using default parameters. Absorption spectra were taken with
a UV−vis spectrometer, and CD spectra were obtained with a 1 mm
sample holder.
Femtosecond IR Transient Absorption Method. The optical
density (OD) of the samples at the excitation wavelength (355 nm)
ranged from 0.09 to 0.12 in a 400 μm path length CaF2 cell for
femtosecond transient IR experiments. Fourier transform-limited 70 fs
pulses with center frequencies of 2150 cm−1 were used in the
femtosecond IR transient absorption experiments for the samples in
chloroform. The energy of the 355 nm excitation pulse (5 μJ) was
attenuated by an iris. The beam radius of the IR probe pulse was 75%
of the visible pump pulse radius at the sample cell. The beam radius of
the IR probe pulse was 75% of the visible pump pulse radius at the
sample cell. The visible beam was passed through a hole in the center
of the parabolas used to collect the infrared probe pulses, and the
overlap between the pump and probe pulses was achieved by first
using a 200 μm pinhole at the sample holder and then maximizing the
transient absorption signal of a Si wafer. The transient absorption of
the carbonyl stretch of fluorenone was utilized as a standard to
optimize the overlap of the pump beam. The timing between the
pump and probe pulses was varied by using an automated translation
stage with femtosecond time resolution. The transmitted probe pulse
was focused onto the focal plane of a monochromator equipped with a
64-element mercury−cadmium−telluride array detector. The appro-
priate monochromator grating was chosen, comprising 150 lines per
mm groove. The kinetic trace was collected (averaged over 16 scans,
1600 shots per scan) over a 100 ps time range with 1 ps steps.
765
dx.doi.org/10.1021/jo402680v | J. Org. Chem. 2014, 79, 759−768

The Journal of Organic Chemistry
Article
Synthesis and Isolation of Peptides. AKA Peptide. This
previously reported peptide31 was prepared from 0.050 mmol of
loaded methylindole AM resin following the general procedures. The
final product was purified by reversed-phase HPLC (gradient 5−35%
organic over 10 min) to give 13.7 mg (13% yield) of a white
amorphous powder after lyophilization. HRMS (ES) m/z 2093.1575
[(M + Na)+; calcd for C91H155N29O26Na, 2093.1596]; MALDI-TOF
m/z 2071.014 [(M + H)+; calcd for C92H156N29O26, 2071.1776].
AKAA11P Peptide. The AKAA11P peptide was prepared from 0.050
mmol of loaded methylindole AM resin utilizing the general
procedures and purified by reversed-phase chromatography (gradient
5−35% organic over 10 min) to afford 27.8 mg (27% yield) of
AKAA11P as a white amorphous powder after lyophilization. HRMS
(ES) m/z 2097.1933 [(M + H)+; calcd for C93H158N29O26,
2097.1980]; MALDI-TOF m/z 2097.123 [(M + H)+; calcd for
C93H158N29O26, 2097.1980].
5.5 mg (80% yield) of 9 as a red-orange amorphous powder after
lyophilization. HRMS (ES) m/z 808.2853 [(M + Na)+; calcd for
C30H47N11O10NaS2, 808.2847].
Peptide Building Block 10. The peptide sequence comprising the
N-terminal fragment of the target peptide was assembled from 0.10
mmol of loaded methylindole AM resin employing the general
procedures, cleaved from the resin to retain the N-terminal Fmoc
protecting group, and purified by reversed-phase chromatography
(gradient 5−35% organic over 10 min) to give 65.4 mg (54% yield) of
10 as a white amorphous powder after lyophilization. HRMS (ES) m/z
1206.6654 [(M + H)+; calcd for C57H88N15O14, 1206.6635]. Next, to
install the requisite N(ε)-Boc protecting groups, a 25 mL round-
bottom flask was charged with the above peptide (30.0 mg, 0.025
mmol) and NaHCO3 (10.5 mg, 0.125 mmol, 5 equiv), which were
then dissolved in water (2.0 mL). A premixed solution of Boc2O (16.4
mg, 0.075 mmol, 3 equiv) dissolved in 1,4-dioxane (2.0 mL) was
added, and the reaction solution was heated to 45 °C with stirring.
After 2 h, completion of the reaction was observed by LC−MS, and
the solvent was evaporated under reduced pressure. The crude
reaction mixture was dissolved in DMF (2.0 mL), and then added
dropwise by syringe with stirring to a 25 mL round-bottom flask
charged with 10% piperidine/DMF (2.0 mL). Once the addition was
completed, the reaction solution was allowed to stir at room
temperature for 10 min and then concentrated under reduced
pressure. The resulting residue was dissolved in 1:1 acetonitrile/
water and then purified by reversed-phase chromatography (gradient
5−60% organic over 10 min) to give 19.8 mg (67% yield) of 10 as an
amorphous powder after lyophilization. HRMS (ES) m/z 1184.6713
[(M + H)+; calcd for C52H94N15O16, 1184.6998].
Peptide 11. To a 10 mL teardrop flask containing peptide 10 (3.3
mg, 0.0028 mmol) in DMF (1 mL) was added dropwise a premixed
solution of 9 (4.4 mg, 0.0056 mmol, 2.0 equiv), oxyma (0.80 mg,
0.0056 mmol, 2.0 equiv), HBTU (2.1 mg, 0.0056 mmol, 2.0 equiv),
and DIPEA (2.3 μL, 0.014 mmol, 5 equiv) in DMF (1.0 mL). After 3 h
of stirring, completion of the reaction was observed by LC−MS, and
the solution was concentrated under reduced pressure. The resulting
residue was taken into 1:1 acetonitrile/water and then purified by
reversed-phase chromatography (gradient 5−70% organic over 10
min) to give 2.9 mg (53% yield) of the protected peptide intermediate
as a light-orange amorphous powder after lyophilization. HRMS (ES)
m/z 1973.9673 [(M + Na)+; calcd for C82H38N26O25NaS2,
1973.9666]; MALDI-TOF m/z 1973.761 [(M + Na)+; calcd for
C82H38N26O25NaS2, 1973.9666]. Next, a 10 mL teardrop flask charged
with the protected peptide intermediate (2.9 mg, 0.0015 mmol) and a
solution of 1:3 TFA/CH3CN (2 mL) was added in one portion. After
2 h of stirring, completion of the reaction was observed by LC−MS,
and the solution was concentrated under reduced pressure to afford,
without any purification, 2.5 mg of peptide 11 as a light-orange
amorphous powder. HRMS (ES) m/z 1651.8291 [(M + H)+; calcd for
C67H114N26O19S2, 1651.8273]; MALDI-TOF m/z 1651.355 [(M +
H)+; calcd for C67H114N26O19S2, 1651.8273].
AKAA10C/A12C Peptide. The AKAA10C/A12C peptide was prepared
from 0.036 mmol of loaded methylindole AM resin employing the
general procedures and purified by reversed-phase chromatography
(gradient 5−35% organic over 10 min) to give 10.5 mg (15% yield) of
AKAA10C/A12C as a white amorphous powder after lyophilization.
HRMS (ES) m/z 2157.1082 [(M
+
Na)+; calcd for
C91H155N29O26NaS2, 2157.1037]; MALDI-TOF m/z 2157.315 [(M
+ Na)+; calcd for C91H155N29O26NaS2, 2157.1037].
S,S-Tetrazine Tetrapeptide 3. Tetrapeptide 3 was synthesized from
0.22 mmol of loaded methylindole AM resin employing the general
procedures and purified by reversed-phase chromatography (gradient
5−45% organic over 12 min) to give 12.2 mg (10% yield) of 3 as an
orange amorphous powder after lyophilization. All of the resonances
for 3 were assigned using 2D NMR (COSY, HMBC, and HSQC): 1H
NMR (500 MHz, DMSO-d6) δ 8.49 (d, Ala-3-NH, J = 8.5 Hz, 1H),
8.48 (d, Cys-4-NH, J = 7.0 Hz, 1H), 8.19 (d, Lys-1-NH, J = 8.0 Hz,
1H), 8.01 (q, Me-NH, J = 4.5 Hz, 1H), 7.84 (d, Cys-2-NH, J = 6.5 Hz,
1H), 4.71 (dt, Cys-2-Hα, J = 6.5, 3.5 Hz, 1H), 4.68 (ddd, Cys-4-Hα, J
= 6.5, 4.5, 2.0 Hz, 1H), 4.50 (dd, Cys-4-Hβ, J = 15.0, 4.5 Hz, 1H), 4.46
(dd, Cys-2-Hβ, J = 15.0, 2.5 Hz, 1H), 4.26 (dq, Lys-1-Hα, J = 8.0, 4.5
Hz, 1H), 4.14 (dq, Ala-3-Hα, J = 8.5, 7.0 Hz, 1H), 3.45 (dd, Cys-4-Hβ,
J = 14.5, 2.0 Hz, 1H), 3.34 (dd, Cys-2-Hβ, J = 14.5, 4.0 Hz, 1H), 2.76
(t, −CH2−NH2, J = 7.5 Hz, 2H), 2.64 (d, CH3−NH, J = 4.5 Hz, 3H),
1.88 (s, CH3−CO, 3H), 1.54−1.47 (m, 4H), 1.36−1.28 (m, 2H), 1.04
(d, Ala-3-CH3, J = 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ
171.4, 171.2, 170.3, 169.6, 168.9, 166.9, 52.5, 51.3, 51.0, 47.6, 38.8,
32.4, 31.1, 30.3, 26.7, 26.0, 22.6, 22.5, 19.8; HRMS (ES) m/z 557.2072
[(M + H)+; calcd for C20H33N10O5S2, 557.2077].
Steady-State Photolysis of Tetrapeptide 3. A solution of 3 in
CD3OD (∼5 mg/mL) was photolyzed under steady-state irradition for
4 h to afford bis-SCN peptide 4. 1H NMR (500 MHz, CD3OD) δ 4.72
(dd, J = 8.5, 4.5 Hz, 1H), 4.66 (dd, J = 7.5, 5.0 Hz, 1H), 4.34 (q, J =
7.0 Hz, 1H), 4.30 (dd, J = 8.0, 5.5 Hz, 1H), 3.64 (dd, J = 14.0, 4.5 Hz,
1H), 3.54 (dd, J = 14.0, 5.0 Hz, 1H), 3.37 (dd, J = 14.0, 9.0 Hz, 1H),
3.36 (dd, J = 14.0, 7.5 Hz, 1H), 2.99−2.94 (m, 2H), 2.77 (br s, 3H),
2.05−2.01 (overlapping m, 2H), 2.04 (overlapping s, 3H), 1.73−1.68
(m, 2H), 1.44 (d, J = 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ
172.7, 172.5, 170.3, 169.1, 169.0, 113.6, 113.5, 53.3, 52.8, 52.7, 49.5,
39.14, 35.6, 31.5, 27.1, 26.3, 23.0, 22.8, 18.8; HRMS (ES) m/z
529.2007 [(M + H)+; calcd for C20H33N8O5S2, 529.2015].
S,S-Tetrazine Peptide 8. Peptide 8 was prepared from 0.13 mmol of
loaded Wang resin employing the general procedures and purified by
reversed-phase chromatography (gradient 5−30% organic over 12
min) to give 17.8 mg (20% yield) of 8 as an orange amorphous
powder after lyophilization. HRMS (ES) m/z 686.2522 [(M + H)+;
calcd for C25H40N11O8S2, 686.2503].
Peptide 9. A 25 mL round-bottom flask was charged with 8 (6.0
mg, 0.0088 mmol) and NaHCO3 (1.8 mg, 0.022 mmol, 2.5 equiv),
which were then dissolved in water (2.0 mL). A premixed solution of
Boc2O (2.9 mg, 0.013 mmol, 1.5 equiv) dissolved in 1,4-dioxane (1.0
mL) was added, and the reaction solution was heated to 45 °C with
stirring. After 30 min, completion of the reaction was observed by
LC−MS, and the reaction solution was directly purified by reversed-
phase chromatography (gradient 5−50% organic over 10 min) to give
S,S-Tetrazine Tetrapeptide 13. The Cbz-protected peptide 13 was
synthesized from 0.11 mmol of loaded Wang resin employing the
general procedures and purified by reversed-phase chromatography
(gradient 10−80% organic over 10 min) to give 11.9 mg (16% yield)
of an orange amorphous powder after lyophilization. HRMS (ES) m/z
700.1962 [(M + Na)+; calcd for C27H35N9O8NaS2, 700.1948]. The 1H
NMR spectrum (DMSO-d6) indicated the presence of the diagnostic
β-Cys resonances for peptides contining the S,S-tetrazine phototrigger;
however, multiple conformers were observed for this protected
peptide intermediate (see the Supporting Information).
S,S-Tetrazine Peptide 14. To a 10 mL teardrop flask charged with
13 (2.0 mg, 0.0030 mmol) was added a solution of 19:1 TFA/TIPSH
(3 mL). After 2 h of stirring, completion of the reaction was observed
by LC−MS, and the solution was concentrated under reduced
pressure. The resulting residue was taken into water (1 mL) and then
purified by reversed-phase chromatography (gradient 5%−45%
organic over 10 min) to give 1.5 mg (90% yield) of 14 as an orange
amorphous powder after lyophilization. 1H NMR (500 MHz, DMSO-
d6) δ 8.44 (d, J = 6.5 Hz, 1H), 8.48 (d, J = 8.5 Hz, 1H), 8.18 (d, J = 8.0
766
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The Journal of Organic Chemistry
Article
Hz, 1H), 7.83 (d, J = 7 Hz, 1H), 7.74 (br s, 1H), 6.51 (s, 2H), 4.79 (br
m, 1H), 4.74 (dt, J = 6.5, 3.5 Hz, 1H), 4.54 (m, 2H), 4.25 (ddd, J =
8.6, 4.8, 3.5 Hz, 1H), 4.17 (ddd, J = 7.6, 6.4, 1.0 Hz, 1H), 3.52 (dd, J =
14.9, 1.8 Hz, 1H), 3.37 (dd, J = 15.2, 3.5 Hz, 1H), 2.76 (br m, 2H),
1.88 (s, 3H), 1.63 (br m, 1H), 1.52 (br m, 4H), 1.37−1.31 (br m, 3H),
1.00 (d, J = 6.5 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ 171.1,
170.8, 170.6, 170.4, 169.4, 166.8, 52.4, 51.6, 50.9, 47.4, 38.6, 32.3, 30.9,
30.3, 26.6, 22.5, 22.3, 19.7 (note: one peak overlapped with the solvent
signal); HRMS (ES) m/z 544.1777 [(M + H)+; calcd for
C19H30N9O6S2, 544.1760].
mg, 0.021 mmol, 1.1 equiv), HBTU (8.0 mg, 0.021 mmol, 1.1 equiv),
and DIPEA (8.4 μL, 0.048 mmol, 2.5 equiv) in DMF (1.5 mL). After 8
h of stirring, the reaction solution was concentrated under reduced
pressure. To the resulting residue was added neat TFA (4 mL). After
24 h of stirring, the solution was concentrated under reduced pressure,
taken into 1:1 acetonitrile/water, and then purified by reversed-phase
chromatography (gradient 5−35% organic over 10 min; the formic
acid buffer for the mobile phase was replaced with 0.05% TFA) to
provide 10.5 mg (25% yield) of S,S-Tet-AKA as an amorphous
powder after lyophilization. HRMS (ES) m/z 2213.1260 [(M + H)+;
calcd for C93H154N33O26S2, 2213.1184]; MALDI-TOF m/z 2213.598
[(M + H)+; calcd for C93H154N33O26S2, 2213.1184].
Dihydro-S,S-tetrazine 16. A 10 mL teardrop flask equipped with a
stir bar was charged with a solution of 1527 (15.0 mg, 0.035 mmol) in
methanol (5 mL). A spatula tip of 10% palladium on carbon (50% wet
with water) was added to the solution. The heterogeneous mixture was
flushed with hydrogen gas using a balloon and then stirred under a
hydrogen gas environment for 5 min. The mixture was then filtered
through a pad of silica gel, and the filterate was concentrated in vacuo
to afford, without any required purification, 14.9 mg (98% yield) of 16
as a colorless oil. IR (CH2Cl2) 3047 (s), 2985 (s), 2306 (m), 1745 (s),
1679 (s), 1623 (m), 1510 (s) cm−1; 1H NMR (500 MHz, CD3OD) δ
4.69 (dd, J = 8.0, 4.5 Hz, 1H), 3.73 (s, 3H), 3.46 (dd, J = 14.0, 4.5 Hz,
Isotopically Edited Peptides Containing 13C/18O Amides. The
procedures described above were employed to synthesize the S,S-Tet-
AKAC10*/A11*, S,S-Tet-AKAA5*/A6*, and S,S-Tet-AKAA17*/A18* peptides
utilizing either Fmoc-HN-Cys(SSt‑Bu)-13C18O2H or Fmoc-HN-
Ala-13C18O2H, which were prepared according to previously reported
methods.29,43 Mass spectra and LC−MS chromatograms of the
synthetic intermediates are provided in the Supporting Information.
Peptide S,S-Tet-AKAC10*/A11*. HRMS (ES) m/z 2219.1348 [(M +
13C2H154N33 18O2S2, 2219.1336]; MALDI-
12
16
H)+; calcd for
C O
91 24
T O F m / z 2 2 1 9 . 5 2 2 [ ( M
+
H ) +
;
c a l c d f o r
1
1H), 3.22 (dd, J = 14.0, 8.0 Hz, 1H), 1.99 (s, 3H); H NMR (500
12
16
C
13C2H154N33 18O2S2, 2219.1336].
O
MHz, DMSO-d6) δ 8.89 (s, 1H), 4.43 (d, J = 7.5 Hz, 1H), 4.89 (ddd, J
= 9.0, 7.5, 5.0 Hz, 1H), 3.63 (s, 3H), 3.33 (dd, J = 13.5, 5.0 Hz, 1H),
3.06 (dd, J = 13.5, 9.0 Hz, 1H), 1.86 (s, 3H); 13C NMR (125 MHz,
CD3OD) δ 173.5, 172.3, 151.8, 54.0, 53.2, 33.2, 22.5; 13C NMR (125
MHz, DMSO-d6) δ 170.7, 169.3, 149.4, 52.1, 51.5, 31.2, 22.2; HRMS
(ES) m/z 435.1129 [(M + H)+; calcd for C14H23N6O6S2, 435.1121].
To a 10 mL teardrop flask charged with 16 (5.2 mg, 0.012 mmol)
was added methanol (5 mL). The solution was allowed to stand
exposed to air for 8 h, after which the solution, which turned from
colorless to orange, was concentrated in vacuo and then chromato-
graphed on silica gel using 5% methanol/ethyl acetate as the eluent to
give 5.0 mg (97% yield) of 15 as an orange solid. The characterization
data for this material matched that reported previously for 15.27 To
provide evidence as to whether 16 was the 1,2- or 1,4-dihydrotetrazine,
IR analysis aided with simulations were highly suggestive of the 1,2-
dihydrotetrazine (see the Supporting Information).
Peptide 18. Peptide 18, comprising the C-terminal fragment of S,S-
Tet-AKA, was synthesized from 0.25 mmol of loaded methylindole
AM resin according to the general procedures and purified by
reversed-phase chromatography (gradient 5−60% organic over 10
min) to provide 116.1 mg (37% yield) of 18 as a white amorphous
powder after lyophilization. HRMS (ES) m/z 1252.6707 [(M + H)+;
calcd for C58H90N15O16, 1252.6690]; MALDI-TOF m/z 1274.399 [(M
+ Na)+; calcd for C58H89N15O16Na, 1274.6510].
Peptide 19. To a 25 mL teardrop flask containing peptide 18 (53.0
mg, 0.043 mmol) in DMF (3.5 mL) was added dropwise a premixed
solution of 1729 (20.0 mg, 0.043 mmol, 1.0 equiv), oxyma (6.7 mg,
0.047 mmol, 1.1 equiv), HBTU (17.9 mg, 0.047 mmol, 1.1 equiv), and
DIPEA (18.7 μL, 0.11 mmol, 2.5 equiv) in DMF (1.5 mL). After 8 h of
stirring, the reaction solution was concentrated under reduced
pressure. To the resulting residue was added a 1:3 TFA/CH3CN
solution (5 mL). After 2 h of stirring, the reaction solution was directly
purified by reversed-phase chromatography (gradient 10−70% organic
over 10 min) to give 32.8 mg (43% yield) of 19 as an amorphous
powder after lyophilization. HRMS (ES) m/z 1629.7113 [(M + Na)+;
calcd for C69H102N22O19NaS2, 1629.7031]; MALDI-TOF m/z
1629.665 [(M + Na)+; calcd for C69H102N22O19NaS2, 1629.7031].
Peptide 20. Peptide 20, comprising the N-terminal fragment of S,S-
Tet-AKA, was prepared from 0.25 mmol of loaded 2-chlorotrityl
chloride resin following the general procedures and purified by
reversed-phase chromatography (gradient 10−70% organic over 10
min) to afford 109.1 mg (38% yield) of 20 as a white amorphous
powder after lyophilization. HRMS (ES) m/z 1160.5630 [(M + H)+;
calcd for C56H78N11O16, 1160.5628]; MALDI-TOF m/z 1182.469 [(M
+ Na)+; calcd for C56H77N11O16Na, 1182.5448].
91
24
Peptide S,S-Tet-AKAA5*/A6*. HRMS (ES) m/z 2219.1357 [(M +
13C2H154N33 18O2S2, 2219.1336]; MALDI-
12
16
H)+; calcd for
C O
91 24
T O F m / z 2 2 1 9 . 7 8 1 [ ( M
+
H ) +
;
c a l c d f o r
12
16
C
13C2H154N33 18O2S2, 2219.1336].
O
91
24
Peptide S,S-Tet-AKAA17*/A18*. HRMS (ES) m/z 2219.1414 [(M +
13C2H154N33 18O2S2, 2219.1336]; MALDI-
12
16
H)+; calcd for
C O
91 24
T O F m / z 2 2 1 9 . 7 5 6 [ ( M
+
H ) +
;
c a l c d f o r
12
16
C
13C2H154N33 18O2S2, 2219.1336].
O
91
24
ASSOCIATED CONTENT
* Supporting Information
■
S
HPLC−MS and MALDI-TOF-TOF data, a description of the
simulated IR spectra for dihydro-S,S-tetrazine 16, character-
ization data for synthetic intermediates related to the 13C/18O
isotopically labeled peptides, and NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: smithab@sas.upenn.edu.
■
Present Address
†M.J.T.: Department of Chemistry, University of Nevada, Reno,
NV 89557.
Notes
The authors declare no competing financial interest.
‡Robin M. Hochstrasser, Donner Professor of Physical Sciences
at the University of Pennsylvania, passed away on February 27,
2013.
ACKNOWLEDGMENTS
■
Financial support was provided by the NIH (GM 12694) and
an RLBL Facility Grant (NIH P41 RR 001348). The MALDI-
TOF-TOF instrumentation was supported by NSF MRI-
0820996. The authors also thank Drs. George Furst and
Rakesh Kohli at the University of Pennsylvania for assistance in
obtaining NMR and HRMS spectra, respectively.
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