Expanding the accessible chemical space by solid phase synthesis of bicyclic homodetic peptides

Article abstract of DOI:10.1039/c1cc15704c

Norbornapeptides (bicyclo[2.2.1]heptapeptides) and related bicyclic homodetic peptides were prepared by solid-phase peptide synthesis using an orthogonal protection scheme. These conformationally rigid peptides cover an almost pristine area of peptide top

Full text of DOI:10.1039/c1cc15704c

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COMMUNICATION
Expanding the accessible chemical space by solid phase synthesis of
bicyclic homodetic peptidesw
Marco Bartoloni, Rameshwar U. Kadam, Julian Schwartz, Julien Furrer, Tamis Darbre and
Jean-Louis Reymond*
Received 15th September 2011, Accepted 11th October 2011
DOI: 10.1039/c1cc15704c
Norbornapeptides (bicyclo[2.2.1]heptapeptides) and related bicyclic
homodetic peptides were prepared by solid-phase peptide synthesis
using an orthogonal protection scheme. These conformationally rigid
peptides cover an almost pristine area of peptide topological space
and adopt globular shapes similar to those of short a-helical peptides.
The discovery of bioactive molecules interfering with protein–
ligand interactions is indispensable to the progress of modern
medicine.1 While the chemical space of drug-like small molecules
has been well delineated,2 there is a pressing need to explore bigger
molecules to tackle interaction sites that are too large for small
organic ligands.3 Peptides are particularly well suited for this task
since they are readily accessible by solid-phase peptide synthesis
(SPPS).4 Although linear peptides may act as drugs, they often
show poor bioavailability and rapid proteolysis. These limitations
can be overcome by rigidifying their structure through cyclisation
by peptide bond formation,5 or by side-chain bridges such as
disulfides,6 thioethers,7 alkenes,8 or triazoles.9 Cyclisation may also
increase biological activity by stabilising secondary structures and
by reducing the entropic penalty of binding to a protein target.
Fig. 1 Bicyclic peptides as topological analogs of bicyclic alkanes. Arrows
Compared to the structural diversity of organic molecules
assembled by connecting atoms,10 the topological diversity of
indicate the a-chain (N-terminus - C-terminus, CO–NH), red lines
indicate side chain connectivity. One-letter codes are used for amino acids
peptides assembled by connecting amino acids is rather poor
(upper case = L-, lower case = D-, B = (S)-2,3-diaminopropanoic acid).
and mostly confined to linear,4 monocyclic,5 and dendritic11
sequences. Polycyclic topologies occur in sulfur-bridged peptides
such as lantibiotics7b and cysteine knot proteins,6a and in natural
and synthetic peptides involving at least one side-chain cross
link.5b,12 Only a few cases of homodetic (in which amino acids are
connected by amide bonds only) peptide analogs of polycyclic
alkanes are known,13 with only three cases of ‘‘non-zero bridged’’
bicyclic peptides prepared by solution phase synthesis (Table S1,
ESIw).14 Herein we report the synthesis of such bicyclic
‘‘non-zero bridged’’ homodetic peptides by SPPS using an ortho-
gonal protection scheme and their structural determination by
NMR (Fig. 1). Analysis of molecular shapes shows that these
conformationally rigid structures resemble short segments of
folded a-helices.
Bicyclic homodetic peptides were prepared by SPPS on an
acid-labile Wang resin followed by two subsequent cycliza-
tions (Scheme 1). The linear hepta-, octa- and deca-peptides
contained an a- or g-allyl protected glutamate at the 2nd or
Department of Chemistry and Biochemistry, University of Berne,
Freiestrasse 3, CH-3012 Berne, Switzerland.
E-mail: jean-louis.reymond@ioc.unibe.ch; Fax: +41 31 631 80 57;
Tel: +41 31 631 43 25
w Electronic supplementary information (ESI) available: Details of
synthesis, NMR, and shape analysis. See DOI: 10.1039/c1cc15704c
Scheme 1 Synthesis of bicyclic peptide 4b. Conditions: (a) Fmoc SPPS;
(b) Pd(PPh3)4, PhSiH3, CH2Cl2; (c) piperidine/DMF (1 : 4); (d) HATU,
DIEA, NMP/DMSO (4 : 1); (e) TFA/TIS/H2O (94 : 5 : 1); (f) PyAOP,
DIEA, DCM/DMF (6 : 1).
c
12634 Chem. Commun., 2011, 47, 12634–12636
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Table 1 Synthesis of cyclic and bicyclic peptidesa
No. Length Sequenceb mg % [M + H]+ calc. obs.
Table 2 Half-lives t1/2 [min] of H/D exchange in D2O (298 K, pH 7.4)
measured by fitting residual peak intensities to an exponential function
(f = fast exchange, t1/2 o 5 min)
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
epi-5a
epi-5b
6a
7
7
7
7
7
7
8
8
8
8
8
8
10
10
L1GBFPE1gA
L1GB2FPE1gA2
G1GBGPE1gV
G1GB2GPE1gV2
G1GBGPE1gF
G1GB2GPE1gF2
L1AkGPE1FG
L1Ak2GPE1FG2
G1LAKFPE1gG
45.2 26 701.36
14.0 37 683.35
9.0 12 583.28
3.3 45 565.27
701.36
683.35
583.28
565.27
631.28
613.28
800.43
782.42
800.43
782.42
800.43
782.42
1004.52
986.51
1a
L
f
L
f
L
f
G
f
G
f
G
52
A
f
L
f
L
f
aB
f
bB
f
bB
397
ek
f
aK
43
ak
165
F
f
F
f
G
f
eK
f
E
103
E
1164
E
713
F
f
F
39
A
f
A
f
F
f
E
275
E
f
1b
aB
128
ak
142
A
f
A
f
3.8 8 631.28
4b
G
f
G
22
G
f
1.1 39 613.27
48.1 31 800.43
24.6 62 782.42
24.0 12 800.43
5b
epi-5b
G
f
ek
f
G1LAK2FPE1gG2 11.2 55 782.42
G1LAkFPE1gG
43.5 21 800.43
5.1 14 782.42
G1LAk2FPE1gG2
A1FGkVFPE1AG 67.5 27 1004.52
6b
A1FGk2VFPE1AG2 22.8 38 986.51
a
b
See Scheme 1 and Fig. 1 for details. The sequence is indicated in the
order used for SPPS with standard one-letter amino acid codes (B =
(S)-2,3-diaminopropanoic acid). g = amidation at the g-carboxyl group of
E in the SPPS sequence. Upper case 1 and 2 indicate residues connected in
the 1st and 2nd cyclization, respectively, similarly to the notation for cycles
in small molecule SMILES.15 Branching residues are indicated in italics.
3rd position and a L- or D-lysine or L-2,3-diaminopropanoic
acid (Dap) at the 6th or 7th position to engage in cyclizations,
and proline at the 3rd or 4th position to facilitate ring formation.
Following SPPS, the first cyclization was performed on the solid
support by amide bond formation between the a- or g-carboxyl
group of glutamate and the N-terminus after cleavage of the allyl
ester with Pd(PPh3)4/PhSiH3 and Fmoc removal, respectively.
The cyclic peptides were then cleaved from the resin, purified by
RP-HPLC, and subjected to amide bond formation conditions
under high dilution to perform the second cyclization, which
provided the bicyclic peptides in good yields after preparative
RP-HPLC purification (Table 1).
The bicyclic peptides exhibited sharp peaks in the 1H NMR
spectra and NH resonances spread over at least 2 ppm with
several amide protons showing slow exchange rates in D2O
(Fig. 2, Table 2). These results are consistent with an organized
backbone and strong intramolecular H-bonds indicating the
presence of a single, stable conformation on the NMR time scale.
All proton resonances were assigned by standard 1D and 2D
NMR experiments (see ESIw).
Fig. 3 Fingerprint regions of the ROESY spectrum (0.15 s mixing
time) of 1b. Assignments of rOe peaks between protons of bridgehead
amino acids and those included in the bridging loop: 1 NHGlu to
aNHDap, 2 NHGlu to bNHDap, 3 NHAla to bNHDap, 4 bNHDap to
bCH3Ala, 5 bNHDap to g0CHGlu, 6 bNHDap to aCHAla
.
several rOe cross-peaks between protons of bridgehead amino
acids and those of the bridging loop (Fig. 3), by implementing
the rOe distance restraints and refinement using SANDER
module of AMBER 10.0.16 In all cases only one of the two
possible geometries for closing the 2nd ring resulted in a structure
compatible with the rOe distance restraints. The structures
displayed a rigid backbone with RMSD 0.25–0.65 A and
conformational flexibility in the side chains (Fig. 4).
Bicyclic peptides define a very large structural family. For
instance, there are 25 possible bicyclic peptide scaffolds up to
10 amino acids forming 3.3 ꢀ 1012 possible products using 20
different amino acids at the variable non-bridgehead positions
(for 15 amino acids: 97 scaffolds, 3.1 ꢀ 1019 products, Fig. S1,
ESIw). Analysis of molecular shapes17 by means of determina-
tion of the principal axes of inertia shows that bicyclic peptides
populate a distinct area of peptide shape space (Fig. 5). Thus,
while linear peptides in extended or b-sheet conformation
are rod-like (PC1 = 86.7 ꢁ 5.5% or 84.1 ꢁ 5.0% variance
of atomic coordinates) and monocyclic peptides are disk-like
(PC1 = 54.1 ꢁ 4.9%, PC2 = 37.7 ꢁ 4.5%), ‘‘non-zero bridged’’
bicyclic peptides have a significant third molecular dimension
indicative of a globular shape (PC1 = 55.6 ꢁ 7.5%, PC2 =
28.6 ꢁ 5.5%, PC3 = 15.8 ꢁ 4.5%), and ‘‘zero-bridged’’
bicyclic peptides are elliptical (PC1 = 63.4 ꢁ 7.9%, PC2 =
26.6 ꢁ 6.0%). The shapes of bicyclic peptides resemble
those of short a-helical segments (PC1 = 55.6 ꢁ 7.3%,
PC2 = 28.3 ꢁ 5.4%, PC3 = 16.1 ꢁ 3.9%). Similarly to the
examples shown here (Table 1) and those from the literature
The structure of peptides 1b, 4b, 5b, epi-5b and 6b was
determined from the 2D ROESY NMR data, which revealed
Fig. 2 Selected regions from 1H NMR spectra of 1a/1b, recorded in
90% H2O/D2O (blue) and D2O (green) at 298 K, 10 min after dissolution.
Red arrows indicate slow exchanging NH. Only the glutamic acid NH
signal shows a slow H/D exchange in the monocyclic species, but several
amide protons of the bicyclic one still appear in D2O.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 12634–12636 12635

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This work was supported financially by the University of
Berne, the European Union FP7-ITN-238434 and the Swiss
National Science Foundation.
Notes and references
1 K. H. Bleicher, H. J. Bohm, K. Muller and A. I. Alanine, Nat. Rev.
¨
Drug Discovery, 2003, 2, 369–378.
¨
2 (a) N. Singh, R. Guha, M. A. Giulianotti, C. Pinilla,
R. A. Houghten and J. L. Medina-Franco, J. Chem. Inf. Model.,
2009, 49, 1010–1024; (b) J. L. Reymond, R. Van Deursen,
L. C. Blum and L. Ruddigkeit, Med. Chem. Commun., 2010, 1,
30–38.
3 (a) J. A. Robinson, ChemBioChem, 2009, 10, 971–973;
(b) M. Rubinstein and M. Y. Niv, Biopolymers, 2009, 91, 505–513.
4 (a) C. L. Stevenson, Curr. Pharm. Biotechnol., 2009, 10, 122–137;
(b) N. H. D. Jakube, Peptides: Chemistry and Biology, Wiley-VCH,
2002, pp. 403–424.
5 (a) C. J. White and A. K. Yudin, Nat. Chem., 2011, 3, 509–524;
(b) S. Jiang, Z. Li, K. Ding and P. P. Roller, Curr. Org. Chem.,
2008, 12, 1502–1542; (c) C. Blackburn and S. A. Kates, Methods
Enzymol., 1997, 289, 175–198.
6 (a) L. Cascales and D. J. Craik, Org. Biomol. Chem., 2010, 8,
5035–5047; (b) V. Goncalves, B. Gautier, C. Garbay, M. Vidal and
N. Inguimbert, J. Pept. Sci., 2008, 14, 767–772; (c) S. C.
Shankaramma, Z. Athanassiou, O. Zerbe, K. Moehle, C. Mouton,
F. Bernardini, J. W. Vrijbloed, D. Obrecht and J. A. Robinson,
ChemBioChem, 2002, 3, 1126–1133.
7 (a) Y. Sako, Y. Goto, H. Murakami and H. Suga, ACS Chem.
Biol., 2008, 3, 241–249; (b) C. Chatterjee, M. Paul, L. Xie and
W. A. van der Donk, Chem. Rev., 2005, 105, 633–684; (c) A. A.
Aimetti, R. K. Shoemaker, C. C. Lin and K. S. Anseth, Chem.
Commun., 2010, 46, 4061–4063.
8 C. E. Schafmeister, J. Po and G. L. Verdine, J. Am. Chem. Soc.,
2000, 122, 5891–5892.
Fig. 4 NMR structures of bicyclic peptides, showing 10 lowest energy
9 (a) J. M. Beierle, W. S. Horne, J. H. van Maarseveen, B. Waser,
J. C. Reubi and M. R. Ghadiri, Angew. Chem., Int. Ed., 2009, 48,
4725–4729; (b) S. Punna, J. Kuzelka, Q. Wang and M. G. Finn,
Angew. Chem., Int. Ed., 2005, 44, 2215–2220.
NMR model structures from simulated annealing refinements.
10 (a) L. C. Blum and J. L. Reymond, J. Am. Chem. Soc., 2009, 131,
8732–8733; (b) M. J. Wester, S. N. Pollock, E. A. Coutsias,
T. K. Allu, S. Muresan and T. I. Oprea, J. Chem. Inf. Model.,
2008, 48, 1311–1324.
11 T. Darbre and J. L. Reymond, Acc. Chem. Res., 2006, 39,
925–934.
12 (a) J. W. Taylor, Biopolymers, 2002, 66, 49–75; (b) T. Karskela,
P. Virta and H. Lonnberg, Curr. Org. Synth., 2006, 3, 283–311;
¨
(c) Y. Sako, J. Morimoto, H. Murakami and H. Suga, J. Am.
Chem. Soc., 2008, 130, 7232–7234; (d) C. Heinis, T. Rutherford,
S. Freund and G. Winter, Nat. Chem. Biol., 2009, 5, 502–507.
13 (a) M. Teixido, M. Altamura, L. Quartara, A. Giolitti, C. A. Maggi,
E. Giralt and F. Albericio, J. Comb. Chem., 2003, 5, 760–768;
(b) R. Hirschmann, W. Q. Yao, B. Arison, L. Maechler, P. A.
Sprengeler and A. B. Smith, Tetrahedron Lett., 1996, 37,
5637–5640.
14 (a) J. C. Tolle, M. A. Staples and E. R. Blout, J. Am. Chem. Soc.,
1982, 104, 6883–6884; (b) C. Bracken, J. Gulyas, J. W. Taylor and
J. Baum, J. Am. Chem. Soc., 1994, 116, 6431–6432; (c) R. Oliva,
L. Falcigno, G. D’Auria, M. Saviano, L. Paolillo, G. Ansanelli and
G. Zanotti, Biopolymers, 2000, 53, 581–595.
15 D. Weininger, J. Chem. Inf. Comput. Sci., 1988, 28, 31–36.
16 D. A. Case, T. E. Cheatham, 3rd, T. Darden, H. Gohlke, R. Luo,
K. M. Merz, Jr., A. Onufriev, C. Simmerling, B. Wang and
R. J. Woods, J. Comput. Chem., 2005, 26, 1668–1688.
17 (a) W. H. Sauer and M. K. Schwarz, J. Chem. Inf. Comput. Sci.,
2003, 43, 987–1003; (b) A. Nicholls, G. B. McGaughey, R. P.
Sheridan, A. C. Good, G. Warren, M. Mathieu, S. W. Muchmore,
S. P. Brown, J. A. Grant, J. A. Haigh, N. Nevins, A. N. Jain and
B. Kelley, J. Med. Chem., 2010, 53, 3862–3886.
18 (a) R. M. Catalioto, M. Criscuoli, P. Cucchi, A. Giachetti,
D. Gianotti, S. Giuliani, A. Lecci, A. Lippi, R. Patacchini,
L. Quartara, A. R. Renzetti, M. Tramontana, F. Arcamone and
C. A. Maggi, Br. J. Pharmacol., 1998, 123, 81–91; (b) L. Quartara
and M. Altamura, Curr. Drug Targets, 2006, 7, 975–992.
Fig. 5 Molecular dimensions of homodetic peptides of 5–10 amino
acids (MW = 1010 ꢁ 187 Da). The fraction of total variance of atomic
coordinates for all non-H atoms in the 3D structure is shown along the
principal components PC2 and PC3. Reference segments of a-helices
and b-sheets were extracted from the crystal structures of proteins in
the Protein Data Bank. See ESIw for details on the peptide virtual
library generation and content (Table S7 and S8, ESIw).
including a Phase II clinical candidate18 (Table S1, ESIw), the
majority of bicyclic peptides should be conformationally rigid
and well suited to engage in molecular interactions with protein
targets. Future experiments will address adapting the protecting
group strategy to allow the use of amino acids with protected side
chains at the variable positions, and exploring the biological
potential of bicyclic homodetic peptides.
c
12636 Chem. Commun., 2011, 47, 12634–12636
This journal is The Royal Society of Chemistry 2011