Enhancing the Cell Permeability and Metabolic Stability of Peptidyl Drugs by Reversible Bicyclization

Article abstract of DOI:10.1002/anie.201610888

Therapeutic applications of peptides are currently limited by their proteolytic instability and impermeability to the cell membrane. A general, reversible bicyclization strategy is now reported to increase both the proteolytic stability and cell permeabil

Full text of DOI:10.1002/anie.201610888

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201610888
German Edition: DOI: 10.1002/ange.201610888
Drug Delivery
Enhancing the Cell Permeability and Metabolic Stability of Peptidyl
Drugs by Reversible Bicyclization
Ziqing Qian, Curran A. Rhodes, Lucas C. McCroskey, Jin Wen, George Appiah-Kubi,
David J. Wang, Denis C. Guttridge, and Dehua Pei*
Abstract: Therapeutic applications of peptides are currently
limited by their proteolytic instability and impermeability to the
cell membrane. A general, reversible bicyclization strategy is
now reported to increase both the proteolytic stability and cell
permeability of peptidyl drugs. A peptide drug is fused with
a short cell-penetrating motif and converted into a conforma-
tionally constrained bicyclic structure through the formation of
a pair of disulfide bonds. The resulting bicyclic peptide has
greatly enhanced proteolytic stability as well as cell-perme-
ability. Once inside the cell, the disulfide bonds are reduced to
produce a linear, biologically active peptide. This strategy was
applied to generate a cell-permeable bicyclic peptidyl inhibitor
against the NEMO-IKK interaction.
in a number of autoimmune diseases (for example, rheuma-
toid arthritis) and cancer (such as diffuse large B-cell
lymphoma), among others.[6] Canonical NF-kB signaling is
mediated by the interaction between the inhibitor of kB
(IkB)-kinase (IKK) complex and regulatory protein NF-kB
essential modifier (NEMO).[7] Binding to NEMO activates
IKK, which in turn phosphorylates IkB, promoting the
proteasomal degradation of IkB and release of active NF-
kB. Modulators targeting various steps of the NF-kB signaling
pathway have been reported, and some of them have
progressed into the clinic.[6,8] One attractive strategy for
ameliorating the NF-kB activity is to selectively disrupt the
IKK-NEMO interaction. Previous studies generated a weak
NEMO inhibitor (KD ꢀ 37 mm), Antp-NBD (Table 1, peptide
Compared to small-molecule drugs, peptides are highly
Table 1: Sequences of peptides in this work.[a]
selective and efficacious and, at the same time, relatively safe
and well-tolerated. A particularly exciting application of
peptides is the inhibition of protein–protein interactions
(PPIs), which remain challenging targets for small mole-
cules.[1] Consequently, there is an increased interest in
peptides in pharmaceutical research and development, and
about 140 peptide therapeutics are currently being evaluated
in clinical trials.[2] However, peptides are inherently suscep-
tible to proteolytic degradation. Additionally, peptides are
generally impermeable to the cell membrane, largely limiting
their applications to extracellular targets. Although N-meth-
ylation of the peptide backbone and formation of intra-
molecular hydrogen bonds have been shown to improve the
proteolytic stability and membrane permeability of certain
cyclic peptides,[3,4] alternative strategies to increase both the
metabolic stability and cell permeability of peptide drugs are
clearly needed.
Peptide ID
Sequence
1
2
3
4
5
6
[a] BMB=3,5-bis(mercaptomethyl)benzoyl; F=l-2-naphthylalanine;
MP=3-mercaptopropionyl. See the Supporting Information, Figure S1
for detailed structures.
NF-kB is a transcription factor that controls the expres-
sion of numerous gene products involved in immune, stress,
inflammatory responses, cell proliferation, and apoptosis.[5]
Aberrant activation of NF-kB signaling has been implicated
1), which consists of the 11-residue NEMO-binding domain
(NBD) of IKKb covalently linked to a cell-penetrating
peptide (CPP), Antp.[9] Interestingly, Antp-NBD blocks the
IKK activity stimulated by different pro-inflammatory stim-
uli, but does not affect the basal NF-kB activity, thus
providing a potentially safe and effective mechanism for
reducing aberrant NF-kB activity.[9] In several pre-clinical
studies, Antp-NBD demonstrated in vivo efficacy for treating
Duchenne muscular dystrophy and large B-cell lymphoma in
mouse and canine models.[10] However, to achieve clinical
utility, Antp-NBD would benefit significantly from improve-
ments in its NEMO-binding affinity, metabolic stability, and
cell permeability.
[*] Dr. Z. Qian, C. A. Rhodes, L. C. McCroskey, J. Wen, G. Appiah-Kubi,
Prof. Dr. D. Pei
Department of Chemistry and Biochemistry
The Ohio State University
484 West 12th Avenue, Columbus, OH 43210 (USA)
E-mail: pei.3@osu.edu
Dr. D. J. Wang, Prof. Dr. D. C. Guttridge
Department of Molecular Virology, Immunology, and Medical
Genetics, The Ohio State University
We previously reported cyclo(FFRRRRQ) (cFFR4,
where F is l-2-naphthylalanine) as a member of a novel
class of cyclic CPPs.[11] These CPPs bind directly to the
membrane phospholipids, enter cells by endocytosis, and
Columbus, OH 43210 (USA)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201610888.
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

Angewandte
Communications
Chemie
efficiently escape from the early endosome into the cytosol by
inducing budding of small, unstable vesicles.[11,12] With
a cytosolic delivery efficiency (defined as the ratio of cytosolic
over extracellular cargo concentration) of 20%, cFFR4 is an
order of magnitude more active than Tat, one of the most
widely used CPPs.[12] Most importantly, cFFR4 and other
cyclic CPPs are capable of efficiently delivering a variety of
cargo molecules including small molecules, peptides, and
proteins into the cytosol of mammalian cells.[11] For example,
short peptidyl cargos were directly incorporated into the
cFFR4 ring (endocyclic delivery) and the resulting cyclic
peptides were cell-permeable.[11–13] cFFR4 was also fused with
5.7 million different cyclic peptides to generate a library of
cell-permeable bicyclic peptides (bicyclic delivery).[14] How-
ever, many peptide ligands
Scheme 1. A reversible peptide bicyclization strategy. GSH=gluta-
thione.
must be in their extended con-
formations to be biologically
active and are not compatible
with the above cyclization
approaches. To this end, we
recently developed a reversible
cyclization strategy for intra-
cellular delivery of linear pep-
tidyl ligands, by fusing them
with FFR4 and cyclizing the
fusion peptides through a disul-
fide bond.[15] Unfortunately,
the previous approach is lim-
ited to relatively short pep-
Scheme 2. Solid-phase synthesis of disulfide-mediated bicyclic peptides.
tides, as cyclization of longer
peptides results in large rings,
whose conformational flexibility limits the gains in metabolic
stability and cell-permeability.[11] Cyclization via an internal
cysteine results in smaller rings and better cellular uptake, but
leaves a portion of the peptidyl cargo in the linear form, which
remains susceptible to proteolytic degradation. To overcome
this limitation, we report here a reversible bicyclization
strategy, which allows the entire CPP-cargo fusion to be
converted into a bicyclic structure by the formation of a pair
of disulfide bonds (Scheme 1). When outside the cell, the
peptide exists as a highly constrained bicycle, which possesses
enhanced cell permeability and proteolytic stability. Upon
entering the cytosol, the disulfide bonds are reduced by the
intracellular glutathione (GSH) to produce the linear, bio-
logically active peptide. The bicyclic system permits the
formation of a small CPP ring for optimal cellular uptake[11]
and a separate cargo ring to accommodate peptides of
different lengths.
To test the validity of the reversible bicyclization strategy,
we first designed two model peptides consisting of the CPP
motif (RRRRFF or FFRRRR) and a mock cargo motif
(SASAS) fused to its N- or C-terminus (Table 1, peptides 2
and 3; see the Supporting Information, Figure S1 for detailed
structures). Two cysteine residues were also incorporated into
the sequences for later cyclization, one at the junction
between the CPP and cargo motifs and one at the C-terminus.
The linear peptides were synthesized by standard Fmoc solid-
phase peptide synthesis (SPPS) chemistry on Rink amide
resin (Scheme 2). The acetamidomethyl (Acm) groups on the
two cysteine side chains were selectively removed by treat-
ment with Hg(OAc)2 and the exposed free thiols were then
reacted on-resin with 3,5-bis((pyridin-2-yldisulfanyl)methyl)-
benzoic acid, which was readily prepared from commercially
available starting materials (Supporting Information,
Scheme S1). Formation of two disulfide bonds between the
cysteine side chains and the 3,5-bis(mercaptomethyl)benzoic
acid (BMB) scaffold resulted in cyclization of the peptide.
Next, the N-terminal Fmoc group was removed by 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) and the peptide was
bicyclized by forming a lactam between the carboxyl group of
BMB and the N-terminal amine (Scheme 2). BMB is ideally
suited as the scaffold, because its structural symmetry ensures
that a single bicyclic product is formed following the disulfide
exchange reactions. Additionally, the rigidity of the scaffold
prevents the formation of any intramolecular disulfide bond,
simplifying both the synthesis of the scaffold and its reaction
with the cysteine-containing peptides.
To monitor their cellular uptake, peptides 2 and 3 were
labeled with fluorescein isothiocyanate (FITC) on the side
chain of a C-terminal lysine. Flow cytometry analysis of HeLa
cells treated with 5 mm peptides cFFR4, 2 and 3 for 2 h
showed mean fluorescence intensity (MFI) values of 3020,
5180, and 4100, respectively (Figure 1a). Thus, bicyclic
peptides 2 and 3 entered HeLa cells with 72% and 36%
higher efficiencies, respectively, than cFFR4.
We next applied the reversible bicyclization strategy to
generate a cell-permeable, biologically active peptidyl inhib-
2
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!

Angewandte
Communications
Chemie
(d2), binding of GST-
NEMO to a biotinylated
IKKb fragment (amino
acids 701–745)[17] results in
a resonance energy trans-
fer. Addition of a NEMO
inhibitor
blocks
the
NEMO-IKKb interaction
and reduces the HTRF
signal. In the presence of
5 mm tris(carboxylethyl)-
phosphine (TCEP), which
is expected to completely
reduce the disulfide bonds
in peptides 4 and 5, peptide
4
inhibited the NEMO-
IKKb interaction in a con-
centration-dependent
manner, with a half-maxi-
mal inhibitory concentra-
tion (IC50) value of 3.5 Æ
0.2 mm (Figure 1b). Under
the same conditions, Antp-
NBD showed an IC50 value
of about 50 mm, in agree-
ment with the previously
reported binding affinity.[16]
As expected, up to 100 mm
Figure 1. a) MFI of HeLa cells after 2 hour treatment with 5 mm FITC-labeled peptide cFFR4 or 1–5, as
determined by flow cytometry analysis. Blank=no peptide. b) Inhibition of the NEMO-IKKb interaction by
peptides 1, 4, and 5 as monitored by the HTRF assay. c) Dose-dependent inhibition of TNFa-induced
activation of NF-kB signaling in HEK293 cells by peptides 1, 4, and 5. d) Comparison of the serum stability of
peptides 1, 4, and 6. Data reported are the meanÆSD of three independent experiments.
peptide
5
caused only
minor inhibition of the
itor against the NEMO-IKK interaction. Despite its in vivo
efficacy, the linear Antp-NBD peptide has poor pharmaco-
kinetics, which is due to rapid proteolytic degradation in
serum (t1/2 ꢀ 15 min).[10] We envisioned that conversion of
Antp-NBD into a conformationally constrained bicyclic
structure would substantially increase its proteolytic stability.
The CPP motif RRRRFF was fused to the N-terminus of
NBD, TALDWSWLQT, and the N- and C-terminal threonine
residues were replaced with two cysteines (Table 1, peptide 4;
see the Supporting Information, Figure S2 for the detailed
structure). The peptide fusion was bicyclized around the BMB
scaffold by two disulfide bonds as described above, to give
bicyclic peptide 4 as the predominant product (Supporting
Information, Figure S3A). As a control, we also prepared
peptide 5 (see the Supporting Information, Figure S2 for
detailed structure), which is structurally similar to peptide 4
but contains two Ala residues in place of the two Trp residues.
It was previously shown that replacement of the Trp residues
with alanine largely abolished NEMO binding.[9]
Peptides 4 and 5 were labeled with FITC at the side chain
of a lysine added to their C-termini and their cellular entry
was assessed by flow cytometry. Both peptides entered HeLa
cells efficiently, exhibiting MFI values that were 3- and 2-fold
higher than that of cFFR4, respectively (Figure 1a). The
NEMO-binding affinity of peptides 4 and 5 was determined
using a homogenous time-resolved fluorescence (HTRF)
assay.[16] Briefly, in the presence of an anti-glutathione-S-
transferase (GST) antibody labeled with a fluorescence donor
(Tb) and streptavidin labeled with a fluorescence acceptor
interaction. Since substitution of the two cysteine residues
for threonine did not significantly change the NEMO binding
affinity (Supporting Information, Figure S4), the enhanced
NEMO binding of peptide 4 relative to Antp-NBD is likely
caused by additional interactions between the phenylalanine
of the CPP motif (RRRRFF) and the NEMO protein surface.
IKKb contains a phenylalanine at the same position (Phe-
734). The crystal structure of the NEMO-IKKb complex
shows that the side chain of Phe-734 inserts into a hydrophobic
pocket on the NEMO surface.[16] Thus, the phenylalanine in
peptide 4 likely plays dual roles of cellular entry and NEMO
binding.
The ability of the bicyclic peptides to modulate the
NEMO-IKK interaction inside the cell was assessed by
monitoring the TNFa-induced activation of NF-kB.
HEK293 cells transfected with a luciferase reporter gene
under the control of NF-kB were first treated with varying
concentrations of a peptide for 2 h and then TNFa.[9,10e] In the
absence of any inhibitory peptide, treatment with 5 ngmLÀ1
TNFa increased the luciferase activity from a basal level of
177 arbitrary units (AU) to 715 AU (data not shown). Peptide
4 reduced the TNFa-induced luciferase activity in a dose-
dependent manner, with an IC50 value of about 20 mm
(Figure 1c). In contrast, the control peptide 5 had no
significant effect on NF-kB signaling at 20 mm and resulted
in about 10% inhibition at the highest concentration tested
(40 mm). Consistent with the earlier report,[9] Antp-NBD
(peptide 1) also caused concentration-dependent inhibition,
but showed an IC50 value of 140 mm. The higher potency of
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

Angewandte
Communications
Chemie
b) K. Fosgerau, T. Hoffmann, Drug Discovery Today 2015, 20,
122.
[3] T. Rezai, J. E. Bock, M. V. Zhou, C. Kalyanaraman, R. S. Lokey,
M. P. Jacobson, J. Am. Chem. Soc. 2006, 128, 14073.
[4] For selected examples on peptide N-methylation, see: a) J.
Chatterjee, C. Gilon, A. Hoffman, H. Kessler, Acc. Chem. Res.
2008, 41, 1331; b) T. R. White, C. M. Renzelman, A. C. Rand, T.
Rezai, C. M. McEwen, V. M. Gelev, R. A. Turner, R. G.
Linington, S. S. F. Leung, A. S. Kalgutkar, J. N. Bauman, Y. Z.
Zhang, S. Liras, D. A. Price, A. M. Mathiowetz, M. P. Jacobson,
R. S. Lokey, Nat. Chem. Biol. 2011, 7, 810.
bicyclic peptide 4 relative to Antp-NBD in the cellular assay
is likely the results of both improved cellular entry efficiency
(Figure 1a) and greater NEMO-binding affinity (Figure 1b).
In vitro treatment of bicyclic peptide 4 with 5 mm glutathione
for 2 h completely reduced the disulfide bonds (Supporting
Information, Figure S3b), suggesting that peptides 2–5 should
undergo complete reduction upon cytosolic entry.
Finally, the proteolytic stability of peptide 4 and Antp-
NBD was tested by incubating the peptides in human serum
for varying lengths of time and the remaining amounts of
intact peptides were quantitated by analytical HPLC. For
comparison, we synthesized a control peptide (Table 1,
peptide 6), which has the same sequence as peptide 4 but
only its CPP motif was cyclized. Peptide 6 also reduced the
[5] A. Oeckinghaus, S. Ghosh, Cold Spring Harbor Perspect. Biol.
2009, 1, a000034.
[6] a) V. Baud, M. Karin, Nat. Rev. Drug Discovery 2009, 8, 33; b) S.-
C. Sun, J.-H. Chang, J. Jin, Trends Immunol. 2013, 34, 282;
c) F. D. Herrington, R. J. Carmody, C. S. Goodyear, J. Biomol.
Screening 2016, 21, 223; d) G. Cildir, K. C. Low, V. Tergaonkar,
Trends Mol. Med. 2016, 22, 414.
TNFa-induced luciferase activity in
a dose-dependent
manner, with an IC50 value of about 40 mm (Supporting
Information, Figure S5). In agreement with the previous
reports,[10] Antp-NBD was rapidly degraded by human serum,
with a half-life of about 15 min (Figure 1d). In contrast,
bicyclic peptide 4 showed a half-life of about 10 h, and 23% of
the peptide remained intact after 20 h of incubation at 378C.
The monocyclic control peptide 6 was also rapidly degraded
(with a half-life of about 30 min), which is most likely due to
proteolysis of the linear NBD sequence. We had previously
shown that linear peptidyl cargos attached to the Gln side
chain of cFFR4 were rapidly degraded in human serum.[15]
In conclusion, a simple method has been developed to
efficiently deliver peptidyl ligands into mammalian cells, by
fusing the peptide with a short CPP motif and reversibly
cyclizing the fusion peptide through disulfide bonds. The
resulting bicyclic peptide has greatly enhanced cellular
uptake as well as proteolytic stability. This strategy should
be applicable to delivering any linear peptides.
[7] a) S. Yamaoka, G. Courtois, C. Bessia, S. T. Whiteside, R. Weil, F.
Agou, H. E. Kirk, R. J. Kay, A. Israel, Cell 1998, 26, 1231;
b) D. M. Rothwarf, E. Zandi, G. Natoli, M. Karin, Nature 1998,
395, 297.
[8] a) S. C. Gupta, C. Sundaram, S. Reuter, B. B. Aggarwal,
Biochim. Biophys. Acta Gene Regul. Mech. 2010, 1799, 775;
b) T. M. Herndon, A. Deisseroth, E. Kaminskas, R. C. Kane,
K. M. Koti, M. D. Rothmann, B. Habtemariam, J. Bullock, J. D.
Bray, J. Hawes, T. R. Palmby, J. Jee, W. Adams, H. Mahayni, J.
Brown, A. Dorantes, R. Sridhara, A. T. Farrell, R. Pazdur, Clin.
Cancer Res. 2013, 19, 4559.
[9] M. J. May, F. DꢀAcquisto, L. A. Madge, J. Glockner, J. S. Pober,
S. Ghosh, Science 2000, 289, 1550.
[10] a) E. Jimi, K. Aoki, H. Saito, F. DꢀAcquisto, M. J. May, I.
Nakamura, T. Sudo, T. Kojima, F. Okamoto, H. Fukushima, K.
Okabe, K. Ohya, S. Ghosh, Nat. Med. 2004, 10, 617; b) S. Dai, T.
Hirayma, S. Abbas, Y. Abu-Amer, J. Biol. Chem. 2004, 279,
37219; c) W. Shibata, S. Maeda, Y. Hikiba, A. Yanai, T. Ohmae,
K. Sakamoto, H. Nakagawa, K. Ogura, M. Omata, J. Immunol.
2007, 179, 2681; d) S. H. Dave, J. S. Tilstra, K. Matsuoka, F. Li, T.
Karrasch, J. K. Uno, A. R. Sepulveda, C. Jobin, A. S. Baldwin,
P. D. Robbins, S. E. Plevy, J. Immunol. 2007, 179, 7852; e) A.
Gaurnier-Hausser, R. Patel, A. S. Baldwin, M. J. May, N. J.
Mason, Clin. Cancer Res. 2011, 17, 4661; f) J. M. Peterson, W.
Kline, B. D. Canan, D. J. Ricca, B. Kaspar, D. A. Delfin, K.
DiRienzo, P. R. Clemens, P. D. Robbins, A. S. Baldwin, P. Food,
P. Kaumaya, M. Freitas, J. N. Kornegay, J. R. Mendell, J. A.
Rafael-Fortney, D. C. Guttridge, P. M. Janssen, Mol. Med. 2011,
17, 508; g) D. A. Delfꢁn, Y. Xu, J. M. Peterson, D. C. Guttridge,
J. A. Rafael-Fortney, P. M. Janssen, J. Transl. Med. 2011, 9, 68;
h) D. P. Reay, M. Yang, J. F. Watchko, M. Daood, T. L. OꢀDay,
K. K. Rehman, D. C. Guttridge, P. D. Robbins, P. R. Clemens,
Neurobiol. Dis. 2011, 43, 598; i) J. N. Kornegay, J. M. Peterson,
D. J. Bogan, W. Kline, J. R. Bogan, J. L. Dow, Z. Fan, J. Wang, M.
Ahn, H. Zhu, M. Styner, D. C. Guttridge, Skeletal Muscle 2014,
4, 18; j) G. Habineza Ndikuyeze, A. Gaunier-Hausser, R. Patel,
A. S. Baldwin, M. J. May, P. Food, E. Krick, K. J. Propert, N. J.
Masson, PLoS One 2014, 9, e95404.
Acknowledgements
Financial support from the National Institutes of Health
(GM110208) is gratefully acknowledged. We thank Dr. M.
Pellegrini for the IKKb(701–745) plasmid and W. Lian and
J. S. Stewart for technical assistance.
Conflict of interest
The authors declare no conflict of interest.
Keywords: bicyclization · cell-penetrating peptides ·
cyclic peptides · nemo inhibitors · protein–protein interactions
[11] a) Z. Qian, T. Liu, Y.-Y. Liu, R. Briesewitz, A. M. Barrios, S. M.
Jhiang, D. Pei, ACS Chem. Biol. 2013, 8, 423; b) Z. Qian, J. R.
LaRochelle, B. Jiang, W. Lian, R. L. Hard, N. G. Selner, R.
Luechapanichkul, A. M. Barrios, D. Pei, Biochemistry 2014, 53,
4034.
[12] Z. Qian, A. Martyna, R. L. Hard, J. Wang, G. Appiah-Kubi, C.
Coss, M. A. Phelps, J. S. Rossman, D. Pei, Biochemistry 2016, 55,
2601.
[1] For excellent reviews on protein–protein interactions, see: a) J.
Wells, C. McClendon, Nature 2007, 450, 1001; b) L. Nevola, E.
Giralt, Chem. Commun. 2015, 51, 3302 – 3315; c) M. Pelay-
Gimeno, A. Glas, O. Koch, T. N. Grossmann, Angew. Chem. Int.
Ed. 2015, 54, 8896; Angew. Chem. 2015, 127, 9022.
[13] P. Upadhyaya, Z. Qian, N. G. Selner, S. R. Clippinger, Z. Wu, R.
Briesewitz, D. Pei, Angew. Chem. Int. Ed. 2015, 54, 7602; Angew.
Chem. 2015, 127, 7712.
[2] For reviews on peptide therapeutics, see: a) D. J. Craik, D. P.
Fairlie, S. Liras, D. Price, Chem. Biol. Drug Des. 2013, 81, 136;
4
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!

Angewandte
Communications
Chemie
[14] T. B. Trinh, P. Upadhyaya, Z. Qian, D. Pei, ACS Comb. Sci. 2016,
18, 75.
H. Nagata, H. Kase, M. Shimonishi, M. Ido, Anal. Biochem.
2010, 405, 19.
[17] B. Gao, C. O. Audu, J. C. Cochran, D. F. Mierke, M. Pellegrini,
Biochemistry 2014, 53, 6776.
[15] Z. Qian, X. Xu, J. F. Amacher, D. R. Madden, E. Cormet-
Boyaka, D. Pei, Angew. Chem. Int. Ed. 2015, 54, 5874; Angew.
Chem. 2015, 127, 5972.
[16] a) M. Rushe, L. Silvian, S. Bixler, L. L. Chen, A. Cheung, S.
Bowes, H. Cuervo, S. Berkowitz, T. Zheng, K. Guckian, M.
Pellegrini, A. Lugovskoy, Structure 2008, 16, 798; b) Y. Gotoh,
Manuscript received: November 7, 2016
Revised: December 1, 2016
Final Article published: && &&, &&&&
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Drug Delivery
In the loop: Peptide bicyclization by a pair
of disulfide bonds increases its proteo-
lytic stability and cell permeability. This
method also allows for regeneration of
the functional linear peptide once inside
the cytosol of the cell. CPP=cell-pene-
trating peptide.
Z. Qian, C. A. Rhodes, L. C. McCroskey,
J. Wen, G. Appiah-Kubi, D. J. Wang,
D. C. Guttridge, D. Pei*
&&&& — &&&&
Enhancing the Cell Permeability and
Metabolic Stability of Peptidyl Drugs by
Reversible Bicyclization
6
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!