Switch peptide via Staudinger reaction

Article abstract of DOI:10.1021/ol802268e

(Chemical Equation Presented) A new transformation based on the Staudinger reaction is described, and its application in the design of a novel switch element to control peptide folding is demonstrated. We found that the azide switch is activated rapidly in water to promote acyl transfer using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) via the Staudinger reaction. Our findings expand the repertoire of uses of the Staudinger reaction in chemical biology and the number of available triggers for use in switch peptides.

Full text of DOI:10.1021/ol802268e

ORGANIC
LETTERS
2008
Vol. 10, No. 22
5243-5246
Switch Peptide via Staudinger Reaction
Natalia Nepomniaschiy,† Valerie Grimminger,‡ Aviv Cohen,† Saviana DiGiovanni,‡
Hilal A. Lashuel,*,‡ and Ashraf Brik*,†
Department of Chemistry, Ben-Gurion UniVersity of the NegeV, P.O. Box 653,
Beer SheVa 84105, Israel, and Brain Mind Institute, Ecole Polytechnique Federale de
Lausanne (EPFL), 1015 Lausanne, Switzerland
hilal.lashuel@epfl.ch; abrik@bgu.ac.il
Received September 29, 2008
ABSTRACT
A new transformation based on the Staudinger reaction is described, and its application in the design of a novel switch element to control
peptide folding is demonstrated. We found that the azide switch is activated rapidly in water to promote acyl transfer using
tris(2-carboxyethyl)phosphine hydrochloride (TCEP) via the Staudinger reaction. Our findings expand the repertoire of uses of the Staudinger
reaction in chemical biology and the number of available triggers for use in switch peptides.
The Staudinger reaction, discovered nearly a century ago,
occurs between a phosphine and an azide to form an aza-
ylide.1 This transformation has been exploited in several
reactions of high synthetic importance wherein the aza-ylide
intermediate, with its highly nucleophilic nitrogen atom,
serves to trap various electrophiles.2 Staudinger ligation is
one example of such reactions, in which an ester moiety is
placed within a phosphine structure to capture the nucleo-
philic aza-ylide by intramolecular cyclization, leading to a
stable amide bond.3
While the aza-ylide intermediate is known to be stable in
organic solvents, it tends to hydrolyze rapidly in aqueous
media to furnish the primary amine and the phosphine oxide
products. In the traceless Staudinger ligation, however, the
reduction of the azide to amine is a competing side reaction,
thus reversing the capture step and leading to two peptide
fragments.4 We envisioned that placing an electrophile and
an azide rather than the former and phosphine within the
same molecule would allow for acyl transfer and amide
formation that can be triggered upon selective azide reduction.
One possible application of this modified Staudinger
reaction would be in designing new switch elements for use
in “switch peptides” wherein a switch element, based on
† Ben-Gurion University of the Negev.
‡ Ecole Polytechnique Federale de Lausanne.
(1) Staudinger, H.; Meyer, J. HelV. Chim. Acta 1919, 2, 635–646.
(2) (a) Gololobov, Y. G.; Kasukhin, L. F. Tetrahedron 1992, 48, 1353.
(b) Nyffeler, P. T.; Liang, C. H.; Koeller, K. M.; Wong, C. H. J. Am. Chem.
Soc. 2002, 124, 10773–10778. (c) Kurosawa, W.; Kan, T.; Fukuyama, T.
J. Am. Chem. Soc. 2003, 125, 8112–8113.
(4) (a) Soellner, M. B.; Dickson, K. A.; Nilsson, B. L.; Raines, R. T.
J. Am. Chem. Soc. 2003, 125, 11790–11791. (b) Lin, F. L.; Hoyt, H. M.;
Van Halbeek, H.; Bergman, R. G.; Bertozzi, C. R. J. Am. Chem. Soc. 2005,
127, 2686–2695. (c) Tam, A.; Soellner, M. B.; Raines, R. T. J. Am. Chem.
Soc. 2007, 129, 11421–11430.
(3) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007–2010.
10.1021/ol802268e CCC: $40.75
Published on Web 10/30/2008
2008 American Chemical Society

protection of the R-amino group, is placed adjacent to an
ester bond (electrophile) to temporally disrupt peptide
secondary structure and/or function (Scheme 1A).5 Upon
native amide bond.6 By studying the Staudinger reaction,
we recognized that replacing the -NH2 of depsipeptide by
an azide could allow for amide formation that can be
triggered upon selective azide reduction (Scheme 1B, path
b). However, a mechanism in which the aza-yilde nucleo-
philic amine is attacking the ester carbonyl should not be
excluded (Scheme 1B, path a).
Scheme 1. (A) General Concept of Switch Peptides; (B)
Principle of Switch Peptide via Staudinger Reaction
To examine the effectiveness of the reduction of the
R-azido group to the amine functionality at the N-terminus,
we prepared peptide 1 (N3-SLYRAG) as a model system
using standard Fmoc solid-phase peptide synthesis (SPPS)
and azido-glycine as the N-terminal amino acid. Although
various methods are known for the conversion of azide to
amine in organic solvents, this transformation has not been
fully exploited and developed under physiological-like
conditions. To search for such conditions, we examined
several potential azide reducing agents (e.g., borohydride
reagents and propanedithiol) suitable for peptidic systems
in water.7
However, none of these reagents gave satisfactory results,
as the reactions were sluggish and gave low yields (∼15%),
despite extending the reaction time for 10 h. Inspired by the
known ability of substituted phosphines to reduce an azide,
we tested the water-soluble phosphine TCEP.8 We found that
TCEP is an excellent azide-reducing reagent of azido-peptide
in water. Reduction of 1 to give peptide 2 (H2N-SLYRAG)
was completed within 6 min using TCEP (10 equiv) in 200
mM phosphate buffer, pH 7.5 (see Supporting Information).
The reduction with TCEP was dependent on the pH of the
reaction mixture, increasing at a high pH, which is consistent
with the pK value of 7.6 of TCEP.9 Moreover, the reaction
rate increased with higher amounts of TCEP, 10 equiv of
which gave the best results with t1/2 of ∼60 s.
induction of acyl transfer (Soff to Son state), via specific
triggers, the peptide regains its native backbone, secondary
structure, and function(s). These findings combined with
recent success in the application of orthogonal triggering
strategies to induce and/or reverse secondary structure
transitions and peptide self-assembly have created greater
interest in the development of new switch elements that could
introduce greater flexibility and specificity.5 The desired
switch should be stable under physiological conditions, can
efficiently be introduced into peptide systems, is amenable
to rapid activation by specific and selective triggers under
mild conditions, and can be operated orthogonally with other
switch elements. Here we report on a new transformation
based on the Staudinger reaction and its application to the
development of a new switch element based on the azido
ester motif (Scheme 1B), which fulfills all of the criteria
mentioned above.
Encouraged by these results, we designed several model
peptide systems (3-5) that included the depsipeptide unit
with the azido group to examine its utility as a switch element
based on the intramolecular O-N acyl transfer. The synthesis
of the model peptide with the azide functionality was carried
out fully on solid support (see Supporting Information).
Having these precursors at hand, we then focused on the
O-N acyl transfer step using the TCEP reduction conditions.
Thus, peptide 3 was dissolved in 200 mM phosphate buffer,
pH 7.5 followed by the addition of TCEP (10 equiv). The
progress of the reaction was followed using HPLC and mass
spectrometry by monitoring the appearance of the product,
which showed a decrease of 26 mass units upon reduction
of the azide product (Figure 2). Under these conditions, the
reaction was successfully completed within 7 min with t1/2
of ∼80 s, thus the azide switch fulfils an important criterion
of the desired switch element. Authentic samples of the
amine intermediate 6 and the product 7 were prepared for
Activation of switch elements, based on the intramolecular
O-N acyl transfer in situ, regardless of the nature of the
trigger (e.g., chemical, enzymatic, photolytic) occurs through
regeneration of the primary amine followed by O-N acyl
transfer via a five-membered ring intermediate to yield the
(5) (a) Sohma, Y.; Yoshiya, T.; Taniguchi, A.; Kimura, T.; Hayashi,
Y.; Kiso, Y. Biopolymers 2007, 88, 253–262. (b) Tuchscherer, G.;
Chandravarkar, A.; Camus, M.-S.; Berard, J.; Murat, K.; Schmid, A.; Mimna,
R.; Lashuel, H. A.; Mutter, M. Biopolymers 2007, 88, 239–252. (c) Santos,
S. D.; Chandravarkar, A.; Mandal, B.; Mimna, R.; Murat, K.; Sauce`de, L.;
Tella, P.; Tuchscherer, G.; Mutter, M. J. Am. Chem. Soc. 2005, 127, 11888–
11889. (d) Taniguchi, A.; Sohma, Y.; Kimura, M.; Okada, T.; Ikeda, K.;
Hayashi, Y.; Kimura, T.; Hirota, S.; Matsuzaki, K.; Kiso, Y. J. Am. Chem.
Soc. 2006, 128, 696–699.
(6) This step is quite reminiscent of the S- to N-acyl transfer intrinsic
to native chemical ligation: Dawson, P. E.; Muir, T. W.; Clarklewis, I.;
Kent, S. B. H. Science 1994, 266, 776–779.
(7) (a) Rolla, F. J. Org. Chem. 1982, 47, 4327–4329. (b) Firouzabadi,
H.; Adibi, A.; Zeynizadeh, B. Synth. Commun. 1998, 28, 1257–1273.
(8) Faucher, A.-M.; Grand-Maˆıtre, C. Synth. Commun. 2003, 33, 3503–
3511.
(9) Cline, D. J.; Redding, S. E.; Brohawn, S. G.; Psathas, J. N.;
Schneider, J. P.; Thorpe, C. Biochemistry 2004, 43, 15195–15203.
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Org. Lett., Vol. 10, No. 22, 2008

comparison. The intermediate 6 and product 7 have identical
mass but exhibited different retention time with the amine
intermediate eluting earlier on a C18 reverse phase column
(Figure 2). The peak appearing slightly before the product
corresponds to the amine intermediate (6) and elutes with
the same retention time as the authentic sample. The reaction
of the peptide bearing the more sterically hindered amino
acid (e.g., Ala (4) and Val (5), Figure 1) on the ester moiety
Figure 2. Analytical HPLC of the time-course for the Staudinger
reaction of model peptide 3 with a mass of 748.29 Da (calculated
748.36 Da). The MALDI-TOF analysis of peptide 3 also shows a
mass of 722.28 Da corresponding to azide fragmentation, with a
loss of N2 and an addition of two hydrogen atoms, during mass
analysis.10 The product of the O-N acyl transfer reaction 7 and
the amine intermediate 6 shows a mass of 722.24 Da (calculated
722.39 Da).
Figure 1. Model peptides used in this study.
gave slightly slower rates (within 1- to 2-fold) and similar
pH-dependent properties, indicating that steric hindrance does
not affect rate of the reaction dramatically.
In principle, part of the O-N acyl transfer product could
be obtained through an attack of the nucleophilic amine of
the aza-ylide formed between the azide and TCEP, resem-
bling Staudinger ligation (Scheme 1B, path a). Recent studies
by Arndt and co-workers have shown that when similar
peptide systems were exposed to PPh3, in THF or THF/H2O
(4:1) aza-Wittig ring-closure products were obtained in high
yields.11 In these studies, no premature hydrolysis of the
putative iminophosphorane intermediate was observed, i.e.,
amine, even in the presence of 25% v/v water. Our results
show that when TCEP is applied, under physiological-like
conditions, the cyclized product could not be detected by
HPLC or mass spectrometry. This probably occurs as a result
of the instability of the aza-ylide formed with TCEP in water
leading to the primary amine, which subsequently attacks
the ester carbonyl followed by the O-N acyl transfer
(Scheme 1B, path b). Moreover, Raines and co-workers
reported that the use of the water-soluble bis(2-carboxyeth-
yl)thiomethyl phosphine, resembling the structure of TCEP,
to mediate traceless Staudinger ligation in water predomi-
nately afforded the amine byproduct.4c These studies coupled
with our results of the rapid reduction of peptide 1 and the
appearance of the amine intermediate in the conversion of
peptide 3 to 7 support that the O-N acyl transfer product is
obtained primarily through an attack of the reduced primary
amine (Scheme 1B, path b). The rapid reduction of the azide
to the amine intermediate (<1 min) followed by a slower
O-N acyl transfer step (∼6 min) suggests that the latter step
is rate-limiting.
Encouraged by the results obtained with the model
peptides 3-5, we sought to explore the application of our
depsipeptide unit with the azide switch element to control
secondary structure transitions. For this purpose we chose the
C-terminal peptide (9, SALRHYINLITRQRY) of the neu-
ropeptide analogue NPY as a model system.12 This peptide
represents the minimal chain length for retaining significant
binding capacity to NPY receptor Y2 with the R-helical state
as the bioactive conformation. A switch peptide 8 (Figure 1)
was designed to include the isopeptide unit with the azido
functionality at the Ile31-Thr32 position. The synthesis of
peptide 8 with the azide moiety was fully achieved on solid
support, which after cleavage and purification steps gave the
desired peptide in 22% isolated yield (Supporting Information).
It is noteworthy that despite the fact that the esterification in
this case was performed between two sterically hindered amino
acids (Thr and Ile) the synthesis was efficient and low
racemizationlevels(<5%)wereobserved(SupportingInformation).
Initially, we studied the kinetics of the reduction and the acyl
transfer by LCMS. Similar to the model systems, the reduction
step was rapid wherein a complete disappearance of the starting
material 8, resulting in the amine intermediate, was observed in 3
min (Supporting Information). Despite the fact that the acyl transfer
step occurs at a sterically hindered center, i.e., Ile-Thr, the rate of
the reaction was still rapid, and complete acyl transfer, to give the
unmodified NPY analogue 9, was accomplished within 30 min.
As measured by circular dichroism (CD), the resulting switch
peptide adopts a random-coil conformation; however, upon activa-
(10) Low, W.; Kang, J.; DiGruccio, M.; Kirby, D.; Perrin, M.; Fischer,
W. H. J. Am. Soc. Mass. Spectrom. 2004, 15, 1156–1160.
(11) Riedrich, M.; Harkal, S.; Arndt, H. D. Angew. Chem., Int. Ed. 2007,
46, 2701–2703.
(12) Mutter, M.; Chandravarkar, A.; Boyat, C.; Lopez, J.; Dos Santos,
S.; Mandal, B.; Mimna, R.; Murat, K.; Patiny, L.; Saucede, L.; Tuchscherer,
G. Angew. Chem., Int. Ed. 2004, 43, 4172–4178.
Org. Lett., Vol. 10, No. 22, 2008
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tion with TCEP a random-coil to R-helix transition was observed
(Figure 3A). The structural change of NPY monitored by CD at
220 nm yields t1/2 of ∼6 min at 37 °C in presence of 1 mM TCEP
(Figure 3B), which is in a good agreement with the time of
conversion as measured using HPLC.
We have shown a new transformation based on the
Staudinger reaction and demonstrated its application in the
design of a novel switch element to control the folding of
the NPY peptide from random coil to R-helix conformation.
The azido functionality in the depsipeptide unit is activated
rapidly in water using TCEP via the Staudinger reaction.13
Our findings expand the repertoire of uses of the Staudinger
reaction in chemical biology and the number of available
triggers for use in switch peptides. Current efforts in our
laboratories are focused on applying the azide switch, with
other known switches, in the design and characterization of
switch proteins14 and self-assembling systems.
Acknowledgment. This work was supported by the
Israel Science Foundation and the Marc Rich Foundation
(A.B., Ben-Gurion University) and by grants from the
Swiss National Science Foundation (H.L., 301000, EPFL).
We would like to thank Prof. Manfred Mutter for his
inspirational support, encouragement and critical review
of the manuscript.
Supporting Information Available: Synthetic procedures
for peptides (3-5, 8), procedures for triggering, pH rate
profiles. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL802268E
(13) Along these lines, it was reported that TCEP can induce a mild
and chemoselective peptide-bond cleavage of peptides and proteins at azido
homoalanine by initiating the reaction via reduction of the azide to triazene:
Back, J. W.; David, O.; Kramer, G.; Masson, G.; Kasper, P. T.; de Koning,
L. J.; de Jong, L.; van Maarseveen, J. H.; de Koster, C. G. Angew. Chem.,
Int. Ed. 2005, 44, 7946.
Figure 3. Triggering and characterization of the switch peptide of the
C-terminal NPY. (A) CD spectra recorded before (dashed line) and after
(straight line) 30 min of incubation with 10 equiv of TCEP. (B) The
kinetics of the structural change monitored at 220 nm yields t1/2 of ∼6
min.
(14) Vila-Perello´, M.; Hori, Y.; Ribo´, M.; Muir, T. W. Angew. Chem.,
Int. Ed. 2008, 47, 7764–7767.
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Org. Lett., Vol. 10, No. 22, 2008