A perfluoroaryl-cysteine SNAr chemistry approach to unprotected peptide stapling

Article abstract of DOI:10.1021/ja400119t

We report the discovery of a facile transformation between perfluoroaromatic molecules and a cysteine thiolate, which is arylated at room temperature. This new approach enabled us to selectively modify cysteine residues in unprotected peptides, providing access to variants containing rigid perfluoroaromatic staples. This stapling modification performed on a peptide sequence designed to bind the C-terminal domain of an HIV-1 capsid assembly polyprotein (C-CA) showed enhancement in binding, cell permeability, and proteolytic stability properties, as compared to the unstapled analog. Importantly, chemical stability of the formed staples allowed us to use this motif in the native chemical ligation-mediated synthesis of a small protein affibody that is capable of binding the human epidermal growth factor 2 receptor.

Full text of DOI:10.1021/ja400119t

Communication
pubs.acs.org/JACS
A Perfluoroaryl-Cysteine SNAr Chemistry Approach to Unprotected
Peptide Stapling
Alexander M. Spokoyny,† Yekui Zou,† Jingjing J. Ling,† Hongtao Yu,‡ Yu-Shan Lin,‡
and Bradley L. Pentelute*,†
†Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
‡Department of Chemistry, Tufts University, Medford, Massachusetts 02155, United States
S
* Supporting Information
ABSTRACT: We report the discovery of a facile
transformation between perfluoroaromatic molecules and
a cysteine thiolate, which is arylated at room temperature.
This new approach enabled us to selectively modify
cysteine residues in unprotected peptides, providing access
to variants containing rigid perfluoroaromatic staples. This
stapling modification performed on a peptide sequence
designed to bind the C-terminal domain of an HIV-1
capsid assembly polyprotein (C-CA) showed enhance-
ment in binding, cell permeability, and proteolytic stability
properties, as compared to the unstapled analog.
Importantly, chemical stability of the formed staples
allowed us to use this motif in the native chemical
Figure 1. (A) SNAr reaction between thiolate and a perfluoroaromatic
species. (B) Model reactions carried out between protected cysteine
derivative 1 and two commercially available perfluoroaromatic reagents
2a and 2b. In both cases the reaction yields are >95% as determined by
in situ 19F NMR spectroscopy (see SI).
ligation-mediated synthesis of a small protein affibody that
is capable of binding the human epidermal growth factor 2
receptor.
ysteine derivatization of peptides and proteins has evolved
unprotected peptides. We further show how this chemistry can
be applied toward peptide stapling rendering a new class of
bioactive peptides and small proteins.
We focused on determining optimal conditions for coupling
cysteine thiols and perfluorinated substrates using a model
system featuring protected amino acid precursors. Reacting
protected cysteine 1 with hexafluorobenzene 2a in the presence
of base in polar organic solvents (see SI) for 4.5 h led to
substantial formation of the disubstituted product 3a (Figure 1,
see SI for characterization details).
C
into a powerful research and technological tool for
postsynthetic modification of these biomolecules.1 There are
now several methods available to modify cysteine, which
include alkylation, conjugate addition, oxidation, and reduc-
tion.2 With the majority of reports on alkylation chemistry (vide
supra), methods for mild arylation of cysteine are extremely
rare.1,2 This is surprising, since it has been known for a long
time that naturally occurring enzymes (e.g., glutathione S-
transferase) are capable of selectively arylating glutathione via
nucleophilic aromatic substitution (SNAr).3
Recently, Davis and co-workers4 utilized Mukaiyama’s
reagent to form an arylated cysteine intermediate that
underwent conversion to dehydroalanine. This suggests that
it is possible to arylate cysteine in the context of bioconjugation
using sufficiently activated reagents.1 To probe arylation of
cysteines under mild conditions, we evaluated perfluoroar-
omatic molecules. These species are known to exhibit high
reactivity for SNAr reactions under nonforcing conditions,5 are
chemically robust and commercially available, and possess a
convenient 19F NMR handle that can enable characterization of
the products screened in situ up to midmicromolar concen-
trations. Schubert recently suggested this system satisfies many
of the requirements of a “click” reaction, making it potentially
attractive for bioconjugations (Figure 1A).5c,d Here we report
the discovery of a cysteine perfluoroarylation, which can be
used for mild functionalization of cysteine thiolate moieties in
Disubstitution (1,4) was observed exclusively in this process,
even when hexafluorobenzene was used in excess (10-fold)
with 1. Progress of this reaction was monitored by the in situ
19F NMR spectroscopy, where the starting material and product
exhibited two distinct resonances (δ, 138 and 167, respectively,
see Figure 1 and SI). Importantly, no substitution other than
1,4 was observed under these conditions, highlighting the
unique regioselectivity of this process. The observed reactivity
pattern was previously rationalized by the steric hindrance of
the 2,5-(ortho) C−F sites5a and simultaneous activation of the
4-(para) C−F moiety5bof the aromatic ring by the thioether
moiety formed in the monosubstituted species. The ability of
the sulfur to stabilize the negative charge in the intermediate
Received: January 8, 2013
Published: April 5, 2013
© 2013 American Chemical Society
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species after the first substitution ultimately leads to increased
rate of a second thiolate substitution, thus favoring
disubstituted species. Similar reactivity was observed when
decafluorobiphenyl species 2b was used instead of 2a (see SI
and Figure 1). We evaluated chemoselectivity of this SNAr
process by screening several amino acids, which could
potentially compete with the thiolate as a nucleophile (see
SI). We observed no reactivity when competing reactions
between carboxylate and amine unprotected cysteine analogs of
1 and when lysine and histidine were used under similar
reaction conditions in DMF at room temperature. The unique
regio- and chemoselectivity observed in the reactions between
perfluoroaromatic molecules 2a and 2b and cysteine 1 thus
prompted us to evaluate this chemistry for stapling of
unprotected peptides.
of these variants. We thus synthesized a model peptide 6
(Scheme 1) that is capable of binding the C-terminal domain of
an HIV-1 capsid assembly polyprotein (C-CA).7 In particular,
Cowburn et al. previously showed that stapling this sequence
via olefin metathesis (approach originally developed by Grubbs
and co-workers,6g later elaborated by Verdine et al.6c) could
efficiently stabilize its α-helical conformation, rendering it more
efficient in intracellular uptake and binding (Kd ∼ 1 μM) to C-
CA (Scheme 1, peptide 9).8
Scheme 1. Peptides Studied in the Context of the C-CA
Model
Previous studies established chemical transformations for
side-chain cross-linking in a polypeptide for residues positioned
in an i, i + 4/7 fashion. The resulting peptide structure is
commonly referred to as “stapled” motif.6 Researchers
previously showed that “stapled” peptides exhibit different
chemical and biological properties than their parent-unstapled
surrogates.6 While a number of well-established approaches for
peptide stapling currently exist,6 we envisioned that cysteine
perfluoroarylation may lead to a new class of these species with
new properties owing to the rigidity and lipophilicity of the
perfluoroaromatic linkers. To test the developed protocol (vide
supra) with unprotected peptides, we designed and synthesized
two short peptide sequences 4 and 5 (for 5 see SI) that
contained cysteine residues separated by 3 amino acids (i, i +
4).
By simply incubating unprotected peptide (0.5−2 mM) 4 in
the presence of 50 mM solution of TRIS (tris(hydroxymethyl)-
aminomethane) base in DMF for 4.5 h with either 2a or 2b
substrates, significant conversion of the starting material was
observed by LC-MS (Figure 2 and SI) yielding stapled peptides
4a,b. These same experiments were carried out with peptide 5
(see SI for details on 5 series), and similar outcomes were
observed. Progress of the reaction and the identity of the
products was monitored by LC-MS and in situ 19F NMR
spectroscopy (Figure 2 and SI). In both cases, the observed
conversions of the starting peptide were >90%.
Peptide 6 (Scheme 1A) featuring 14 amino acid residues
with two cysteines positioned in an i, i + 4 fashion was
successfully synthesized via Boc/benzyl in situ neutralization
solid-phase peptide synthesis.9 Peptide 6 was further stapled
with 2a and 2b through the developed protocol on a
multimilligram scale, affording the desired peptides in ∼70%
yield after RP-HPLC purification. Circular dichroism (CD)
spectroscopy was used to probe the effect of stapling on the
conformational features of the peptides (Figure 3A and SI).
While the CD spectra of 6 and 6b (minima at 208 and 222 nm)
showed an α-helical motif, the spectrum of 6a revealed an
unusual behavior featuring a maxima at 222 nm and a minima
at 208 nm (Figure 3A).6a We hypothesized that the C6F4 linker
may be responsible for this behavior and thus performed
control CD spectroscopic measurements on 3a and 3b. Indeed,
in the case of 3a we observed a well-defined CD spectrum
exhibiting absorbance maxima at 222 nm. For 3b, a similar
spectrum but with a decreased molar residual ellipticity (MRE)
value centered at ∼226 nm was observed (Figure 3B). While
the nature of the signals in the CD spectra of 3a and 3b is yet
to be determined, we were able to semiquantitatively estimate
the helical content of 6, 6a,b, assuming that MRE values arising
from the introduction of the perfluorinated staple do not
change significantly between 3a/3b and 6a/6b, respectively.
We observed that while species 6 exhibited ∼16% α-helical
structure, stapling with 2a significantly enhances the α-helical
content of the stapled peptide 6a up to 53%. For 6b, stapling
was done with a longer linker (2b) and the α-helical content
changed to 36%. This modest change is not surprising since the
length of 2b is significantly longer than the distance between
two Cys residues positioned at i, i + 4 in a model α-helix. These
observations were further corroborated from the molecular
dynamics simulations of peptides 6 and 6a,b (see SI).
We then decided to evaluate our stapling protocol on more
sophisticated peptide sequences to understand how perfluor-
oaromatic bridges affect the chemical and biological properties
Figure 2. (A) Perfluoroarylation of cysteine residues in an unprotected
peptide 4. (B) LC-MS traces of the crude reaction mixtures (214 nm,
UV) and mass spectra (inset) of the stapled peptides 4a (bottom) and
4b (top).
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differences, where the stapled peptides 6a,b were identified as
better binders than 6. Significantly, observed binding trend
correlates with the α-helicity of the peptides, which is
consistent with the work of Cowburn and co-workers.8 We
further investigated whether these stapled peptides possess
enhanced cellular interaction properties due to the combination
of altered secondary structure and enhanced hydrophobicity
provided by a perfluorinated staple moiety.10 To evaluate these
properties we synthesized FITC-based dye-labeled variants of
6a,b referred to as 7a,b (Scheme 1). Stapling of the
unprotected FITC-labeled peptide 7′ (SI) occurred smoothly
and was not inhibited by the presence of a thiourea moiety (in
contrast to the case of the olefin metathesis approach).6c Upon
incubation of these peptides at 5 μM concentrations with
HEK293T cells for 4 h, we observed significant uptake of the
stapled peptides 7a,b (Figure 3E) localized in the endosomes
(green punctate fluorescence) and in the cytosol (green
fluorescent haze). Importantly, no fluorescent positive cells
were observed for the unstapled variant 7 via confocal
microscopy, and the control experiment with 9 (peptide analog
stapled via olefin metathesis, Scheme 1) suggested that 7b
qualitatively exhibits similar uptake properties. Additional
experiments with flow cytometry revealed a similar trend in
peptide uptakes (Figure 3D and SI). Further control experi-
ments with nonstapled peptide variants 8a and 8b (Scheme 1B)
featuring perfluoraromatic units installed only on one cysteine
residue suggest that stapling is an essential requirement
rendering these peptides cell permeable.
Finally, we evaluated whether the developed stapling
chemistry is compatible with the conditions used for the total
chemical synthesis of proteins. We chose to install a staple on
the C-terminal helix of a previously engineered affibody that has
∼1 nM affinity for the human epidermal growth factor 2 (HER-
2) protein receptor.11 Affibodies 10 and 10a were prepared via
consecutive native chemical ligation12 from three fragments,
where the C-terminal peptide was stapled using the developed
protocol (Figure 4A and SI). Upon examination of the CD
spectra of the synthesized affibodies 10 and 10a we observed a
well-defined α-helical motif (Figure 4B), suggesting the
introduction of the perfluoroaryl staple did not alter the overall
protein topology (note, that the contributions of the
perfluoroaryl moieties in these CD spectra still persists, vide
supra). Notably, temperature-dependent melting properties of
10a were not compromised from the introduction of the staple.
Furthermore, upon evaluating binding properties of these
affibodies (Figure 4C) we observed similar binding of the
stapled affibody as compared to the alkylated congener 10.
While the concept of utilizing stapled peptides in the protein
total synthesis was originally described by Verdine and
Kennedy in their recent patent application,13 to the best of
our knowledge, this is the first example demonstrating
incorporation of stapled peptide motif within the structure of
a larger protein scaffold without significantly altering its
functional properties.
Figure 3. (A) CD spectra of 6 and 6a,b and (B) of 3a,b. (C) Biacore
binding sensograms of 6 and 6a,b with immobilized C-CA
(adsorption/desorption time, 300 s). (D) Flow cytometry data for
FITC-based peptides incubated with HEK293T cells (25 μM;
fluorescence response is averaged for each sample and normalized to
7b). (E) Z-stack accumulated fluorescent confocal microscopy images
(DNA, blue; cell membrane, red; peptides, green (FITC)) of the
HEK293T cells treated with peptides 7, 7a,b (5 μM), unstapled
controls 8a and 8b and NYAD-2, 9.
To test whether the perfluoroaryl cross-links affect the
proteolytic stability of the modified variants, we compared the
distributions of cleaved products of 6 and 6a,b by LC-MS after
treatment with protease. From these experiments we observed
enhanced proteolitic stabilities of stapled 6a,b compared to
unstapled peptide 6 when these species were incubated with
trypsin or chymotrypsin (see SI). This is consistent with the
observed conformational changes in stapled peptides 6a,b
rendering these structures less prone to amide-bond cleavage
by proteases. A drastic difference was observed when proteinase
K was incubated with these peptides. As such, no Leu-Leu
cleavage was observed for 6a,b (in contrast to 6) upon
prolonged incubation of these peptides in the presence of
proteinase K (3 h, see SI), suggesting that the perfluoroaryl
staple is capable of protecting the amide bonds positioned
between the two Cys residues (see SI). We then evaluated and
compared the binding affinities of peptides 6 and 6a,b using
surface-plasmon resonance assay enabled by the Biacore
system. C-CA protein domain was immobilized on the Au-
based chip via covalent attachment chemistry, and peptide
analytes were evaluated at 5 μM concentrations (Figure 3C and
SI). From these experiments we observed binding affinity
In conclusion, we demonstrated a new and mild synthetic
platform for cysteine arylation. The developed method operates
at room temperature in polar organic solvents and shows
excellent selectivity and functional group tolerance. As a result,
we were able to utilize this approach toward stapling
unprotected peptides, thereby positively altering their biological
properties. We further demonstrated incorporation of a
perfluoroaryl staple within the small trihelical affibody protein.
We believe this approach may expand the toolkit of available
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(GM101762 for A.M.S.), MIT start-up fund for B.L.P., and
Tufts start-up fund for Y.-S.L. J.J.L. is a recipient of a National
Science Scholarship (Ph.D.) from A*STAR, Singapore. FACS
core facility (MIT) is supported by the National Cancer
Institute (P30-CA14051). The authors thank Ms. Amy
Rabideau, Dr. Xiaoli Liao, Mr. Mark Simon, and Mr. Rocco
Policarpo for technical assistance, Ms. Wendy Salmon (White-
head Institute) for help with confocal microscopy, and Prof. R.
John Collier (Harvard) for contributing select laboratory
equipment used in this work. The content of this manuscript
is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes
of Health.
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chemical transformations to alter the properties of biomole-
cules that contain a thiol. In addition, our approach is
complementary in several aspects to the olefin metathesis
mediated peptide stapling. The chemistry reported here
operates on unprotected peptides, does not necessitate the
use of a metal-based catalyst, and poses no requirement for
expensive non-natural amino acid precursors.6c Furthermore, in
contrast to bromoalkyl/benzyl chemistry that can produce
overalkylated species, this platform is selective toward cysteines
and provides staples featuring lipophilic perfluorinated
moieties.6f Efforts to establish in-depth structural understanding
of the described stapled system, elucidate precise mechanism
for the observed cellular uptake, and broaden the synthetic
scope of the cysteine arylation are currently under active
investigation in our laboratories.
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ASSOCIATED CONTENT
* Supporting Information
Full experimental and characterization details. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Larsson, B.; Hoiden-Guthenberg, I.; Widstrom, C.; Carlsson, J.;
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AUTHOR INFORMATION
Corresponding Author
blp@mit.edu
■
(12) Kent, S. B. H. Chem. Soc. Rev. 2009, 38, 338.
(13) Verdine, G. L.; Kennedy, E. J. U.S. Patent Application
20110144306, Filed July 23, 2009.
Notes
The authors declare no competing financial interests. Patent
application covering the content of this manuscript was filed by
the MIT-TLO on Sept 23, 2012.
ACKNOWLEDGMENTS
■
We thank Prof. Stephen L. Buchwald (S.L.B.) for his
encouragement and support. This work was partially funded
by the National Institutes of Health (GM46059 for S.L.B.),
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