A highly efficient type i β-turn mimetic simulating an asx-pro-turn-like structure

Article abstract of DOI:10.1021/ol201313q

Asx-Pro-turns have been identified with high frequency in protein structures nucleating type I β-turns. By bridging the amino acid side chain in position i with a nitrogen substituent in position i+2 by ring-closing olefin metathesis (RCM), peptide mimeti

Full text of DOI:10.1021/ol201313q

ORGANIC
LETTERS
2011
Vol. 13, No. 13
3502–3505
A Highly Efficient Type I β-Turn Mimetic
Simulating an Asx-Pro-Turn-Like
Structure
€
€
Andrea Pinsker, Jurgen Einsiedel, Steffen Harterich, Reiner Waibel,
and Peter Gmeiner*
Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University,
Schuhstrasse 19, 91052 Erlangen, Germany
peter.gmeiner@medchem.uni-erlangen.de
Received May 16, 2011
ABSTRACT
Asx-Pro-turns have been identified with high frequency in protein structures nucleating type I β-turns. By bridging the amino acid side chain in
position i with a nitrogen substituent in position iþ2 by ring-closing olefin metathesis (RCM), peptide mimetics of type 1 could be developed. NMR
based conformational investigations indicated a stable intramolecular H-bond constraining a U-turn conformation that was predicted to simulate a
type I β-turn.
Reverse-turn motifs are known as major recognition
elements for receptorꢀligand interactions.1 As an exam-
ple, more than 100 peptide-activated G-protein-coupled
receptors bind biological effectors with U-turn-type
structures.2 Drug research has been directed to design
conformationally restricted scaffolds nucleating reverse
turns that allow a stable prearrangement of crucial
pharmacophores.3,4 Thus, pharmacological properties in-
cluding binding affinity, target selectivity, and metabolic
stability can be improved. Statistical analysis documented
the preferential incorporation of amino acid residues in
turn structures5 when the tendency of asparagine, which is
known to form a functionally relevant Asx-turn,6 was
found to predispose a type I β-turn.7 Taking advantage
of its frequent occurrence in the positions iþ1 and iþ2 of
β-turns of type I/II and VI, respectively, powerful reverse-
turn mimetics were derived from the functionally important
amino acid proline.5 Asx-Pro-turns exhibit a H-bonding
network featuring both the i to iþ3 backbone H-bond of
the classical β-turn and a side chain to iþ2 interaction
forming the Asx-turn.5 In fact, Asx-Pro-turns have been
identified with high frequency in protein structures nucle-
ating type I β-turns.7 As a key recognition element for
initiation of biological recognition events at the surface of
proteins, the type I β-turn, which is by far the most
common, has attracted considerable interest for the design
of peptidomimetic moieties.8 Bridging the amino acid side
chains in positions iþ1 and iþ2 by ring-closing olefin
metathesis (RCM) has been demonstrated to result in type
I β-turn inducingscaffolds by J. A. Katzenellenbogen.4a As
a complementary strategy that allows the incorporation of
individual side chains into the positions iþ1 and iþ2, we
aimed to replace the Asx-turn forming side-chain element
in position i by a cis-olefin unit.
Employing the results of molecular dynamics simula-
tions, we recently designed a highly efficient proline-based
type VI β-turn mimetic.9 Our design of length and geome-
try of the olefin linker for homologous and isomeric ring
systems of type A was also based on molecular dynamics
studies. Thus, a family of structures of type A including 9-
to 12-membered ring systems were subjected to an energy
minimization followed by molecular dynamics simulations
€
(1) Muller, G.; Hessler, G.; Decornez, H. Y. Angew. Chem., Int. Ed.
2000, 39, 894–896.
(2) Tyndall, J. D. A.; Pfeiffer, B.; Abbenante, G.; Fairlie, D. P. Chem.
Rev. 2005, 105, 793–826.
(3) (a) Blomberg, D.; Hedenstrom, M.; Kreye, P.; Sethson, I.;
€
Brickmann, K.; Kihlberg, J. J. Org. Chem. 2004, 3500–3508. (b)
Bittermann, H.; Gmeiner, P. J. Org. Chem. 2006, 71, 97–102.
r
10.1021/ol201313q
Published on Web 06/09/2011
2011 American Chemical Society

(100 ps) to probe the stability of the observed β-turn-like
conformations. ThelengthofthepresumedH-bondduring
the resulting trajectory was plotted against the number of
conformations observed with a specific distance when a
˚
clear prevalence for a H-bond (distance < 2.25 A) was
mainly observed for compounds bearing a cis configura-
tion at the double bond. Especially, 9- and 10-membered
ring systems showed a very narrow distance distribution
indicating a stable H-bond. These molecules were selected
for further ab initio geometry optimization with the Har-
treeꢀFock algorithm at a 6-31G(d) level of theory. Dihe-
dral angles of the calculated structures fit well to the ideal
values for the βI-turn (Figure 1). Because the parent Asx-
turn is formed by a pseudo-10-membered ring and struc-
ture A clearly fulfilled the stringent criteria for assigning
a type I β-turn, we chose to approach the potential reverse
turn mimetics of type 1 incorporating a diazecine based
ring system. We herein present a highly practical synthetic
approach to 10-membered unsaturated lactams as βI-
turn mimetics, conformational investigations, and solid
phase supported application toward artificial neuropep-
tide analogues.
Figure 1. Design and molecular dynamics investigations.
Grubb’s ring-closing olefin metathesis has been proven
as an elegant technique for the synthesis of 10-membered
rings when cyclization through RCM was explored as an
alternative to the classic methods of lactam formation.10
We intended to take advantage of ring-closing olefin
metathesis to access the novel molecular scaffolds of
type 1.
Our initial investigations were directed to the synthe-
sis of the Asx-Pro-turn mimetic 6a starting from the
N-allylglycine derivative 2a, which could be easily prepared
by N-alkylation (Scheme 1). Coupling of Fmoc-protected
proline acid chloride11,12 with the secondary amine 2a led
to the synthetic intermediate 3a. Subsequent N-deprotec-
tion and coupling with Fmoc-protected C-allylglycine acid
chloride afforded the cyclization precursor 5a. Ring clo-
sure metathesis was performed using a second generation
Grubbs catalyst in refluxing dichloromethane to give
access to the cyclic olefin 6a in 41% yield. The control of
cis/trans-geometry in generating double bonds of medium
size, particularly 10-membered rings, is difficult and hard
to predict13 when the ratios of E/Z-isomers can be influ-
enced by reaction temperature10 or groups neighboring the
olefins asdescribedby Grubbs.13 Some publications report
the formation of only the trans-isomer,3a,14 while others
observe the generation of a cis-double bond exclusively.15
For our 10-membered system, careful NMR analysis un-
ambiguously proved the structure of 6a with a diagnostic
3J-coupling of 9.9 Hz for the cis-alkenyl partial structure
(for trans-configuration, a coupling constant >15 Hz
would be expected).16 To demonstrate the versatility of
the synthesis, the tyrosine analog 6b was prepared
(4) (a) Kahn, M.; Chen, B. Tetrahedron Lett. 1987, 28, 1623–1626. (b)
Kahn, M.; Wilke, S.; Chen, B.; Fujita, K. J. Am. Chem. Soc. 1988, 110,
1638–1639. (c) Ball, J. B.; Alewood, P. F. J. Mol. Recog. 1990, 2, 55–64.
(d) Hinds, M. G.; Welsh, J. H.; Brennand, D. M.; Fisher, J.; Glennie,
M. J.; Richards, N. G. J.; Turner, D. L.; Robinson, J. A. J. Med. Chem.
1991, 34, 1777–1789. (e) Hanessian, S.; McNaughton-Smith, G.; Lombart,
H.-G.; Lubell, W. D. Tetrahedron 1997, 38, 12789–12854. (f) Fink,
B. E.; Kym, P. R.; Katzenellenbogen, J. A. J. Am. Chem. Soc. 1998, 120,
4334–4344. (g) Souers, A. J.; Virgilio, A. A.; Rosenquist, A.; Fenuik, W.;
Ellman, J. A. J. Am. Chem. Soc. 1999, 121, 1817–1825. (h) Eguchi, M.;
Lee, M. S.; Nakanishi, H.; Stasiak, M.; Lovell, S.; Kahn, M. J. Am.
Chem. Soc. 1999, 121, 12204–12205. (i) Haskell-Luevano, C.;
Rosenquist, A.; Souers, A.; Khong, K. C.; Ellman, J. A.; Cone, R. D.
J. Med. Chem. 1999, 4380–4387. (j) Souers, A. J.; Ellman, J. A. Tetra-
hedron 2001, 7431–7448. (k) Qiu, W.; Gu, X.; Soloshonok, V. A.;
Carducci, M. D.; Hruby, V. J. Tetrahedron Lett. 2001, 42, 145–148. (l)
Eguchi, M.; Lee, M. S.; Stasiak, M.; Kahn, M. Tetrahedron Lett. 2001,
1237–1239. (m) Wang, W.; Xiong, C.; Hruby, V. J. Tetrahedron Lett.
2001, 18, 3159–3161. (n) Freidinger, R. M. J. Med. Chem. 2003, 46 (26),
5553–5566. (o) Banfi, L.; Basso, A.; Guanti, G.; Riva, R. Tetrahedron
Lett. 2003, 44, 7655–7658. (p) Jeanotte, G.; Lubell, W. D. J. Am. Chem.
Soc. 2004, 126, 14334–14335. (q) Kaul, R.; Surprenant, S.; Lubell, W. D.
ꢀ
J. Org. Chem. 2005, 10, 3838–3844. (r) Stepien, A.; Loska, R.; Cmoch,
P.; Stalinsky, K. Synlett 2005, 1, 83–86. (s) Pawar, V. G.; De Borggraeve,
ꢀ
W. M.; Maes, V.; Tourwe, D. A.; Compernolle, F.; Hoornaert, G. J.
Tetrahedron Lett. 2005, 1707–1710. (t) Surprenant, S.; Lubell, W. D.
Org. Lett. 2006, 13, 2851–2854. (u) Albericio, F.; Arvidson, P. I.; Bisetty,
K.; Giralt, E.; Govender, T.; Jali, S.; Kongsaeree, P.; Kruger, H. G.;
Prabpai, S. Chem. Biol. Drug Des. 2008, 71, 125–130. (v) Ko, E.; Burgess,
K. Org. Lett. 2011, 5, 980–983.
(5) (a) Hutchinson, E. G.; Thornton, J. M. Protein Sci. 1994, 3, 2207–
2216. (b) Guruprasad, K.; Rajkumar, S J. Biosci. 2000, 25, 143–156.
(6) (a) Mcharfi, M.; Aubry, A.; Boussard, G; Marraud, M. Eur.
Biophys. J. 1986, 14, 43–51. (b) Imperiali, B.; Shannon, K. L. Biochem-
istry 1991, 30, 4374–4380.
(11) Beyermann, M.; Bienert, M.; Niedrich, H.; Carpino, L. A.;
Sadat-Aalaee, D. J. Org. Chem. 1990, 55, 721–728.
(12) Oku, A.; Yamaura, Y.; Harada, T. J. Org. Chem. 1986, 52, 3732–
3734.
(13) Mohapatra, D. K.; Ramesh, D. K.; Giardello, M. A.;
Chorghade, M. S.; Gurjar, M. K.; Grubbs, R. H. Tetrahedron Lett.
2007, 48, 2621–2625.
(7) Richardson, J. S. Adv. Protein Chem. 1981, 34, 167–339.
€
(8) (a) Bieraugel, H.; Jansen, P. T.; Schoemaker, H. E.; Hiemstra, H.;
van Maarseveen, J. H. Org. Lett. 2002, 16, 2673–2674. (b) David, O.;
Meester, W. J. N.; Bieraugel, H.; Schoemaker, H. E.; Hiemstra, H.; van
(14) Srihari, P.; Kumaraswamy, B.; Somaiah, R.; Yadav, J. S. Synth-
esis 2010, 1039–1045.
€
Maarseveen, J. H. Angew. Chem., Int. Ed. 2003, 42, 4373–4375.
(9) Hoffmann, T.; Lanig, H.; Waibel, R.; Gmeiner, P. Angew. Chem.,
Int. Ed. 2001, 40, 3361–3364.
(15) (a) Hoffmann, T.; Waibel, R.; Gmeiner, P. J. Org. Chem. 2003,
68, 62–69. (b) Szostak, M.; Aube, J. Org. Lett. 2009, 11, 3878–3881.
(16) (a) Smith, G. V.; Kriloff, H. J. Am. Chem. Soc. 1963, 85, 2016–
2017. (b) Pawar, M. D. J. Am. Chem. Soc. 1996, 118, 12821–12825.
ꢀ
€
€
(10) Furstner, A.; Muller, T. Synlett 1997, 8, 1010–1012.
Org. Lett., Vol. 13, No. 13, 2011
3503

Scheme 1. Synthesis of the Molecular Scaffold 6a,b and the Neurotensin Analogs 8 and 9
following ananalogous reaction protocol. Thus, westarted
from bis-tert-butyl protected tyrosine which was N-alky-
lated to give the secondary amine 2b. Subsequent ligation
with proline, N-deprotection, coupling with allylglycine,
and RCM gave rise to the conformationally restricted
peptide mimetic 6b via intermediates 3b, 4b, and 5b.
when the impurities of the final products 8 and 9 could be
separated by preparative HPLC.
Aspartate is the most prevalent amino acid in position
iþ2 of type I β-turns in naturally occurring proteins.5
Thus, we envisioned investigating a further model system
that incorporates an aspartate derivative adjacent to the
proline residue. Because we planned to investigate this
model peptide surrogate for its conformational properties
emphasizing its capability to form a stable type I β-turn, N-
acetylation and N-methyl carboxamide functionalization
should be realized. Moreover, we were intrigued by the
question if our RCM precursor sequence can be also
constructed by solid phase supported peptide synthesis.
Thus, we chose N-methyl aminomethylindole resin as a
commercially available backbone amide linker (BAL) that
was N-acylated with Fmoc-protected γ-benzyl aspartate
by microwave assisted coupling when we employed Py-
BOB/HOBt as an activating mixture (Scheme 2).17a After
N-allylation and coupling with triphosgene-activated
Fmoc-proline, Fmoc-allylglycine was ligated. After N-
acetylation, TFA promoted cleavage in the presence of
triisopropylsilane gave access to the fully functionalized
cyclization precursor 10. Employing microwave irradia-
tion at 120 °C, the target compound 11 could be synthe-
sized upon treatment of the bis-olefine 10 with a Grubbs II
catalyst.18 The stereochemistry of the proline-allylglycine
peptide bond was assigned trans by NOE experiments.
To investigate the ability of our molecular scaffold to
nucleate a stable type I β-turn, NMR spectroscopic studies
were performed comparing the model peptide surrogate 11
with the N-acetylated tripeptidyl carboxamides 12, 13, and
14 that were expected to show the differential prevalence of
We have recently shown that peptide backbone mod-
ifications of neurotensin analogues substantially control
their receptor affinity and subtype selectivity.17 To take
advantage of our novel peptide mimetic for the investiga-
tion of receptor ligand interactions and to demonstrate the
suitability of our molecular scaffold for solid phase sup-
ported peptide synthesis (SPPS), the conformationally
constrained neurotensin mimetics 8 and 9 were prepared
starting from Wang resin preloaded with the sequence Ile-
Leu. Deprotection of our scaffold 6b was done by tri-
methylsilyl trifluoromethansulfonatetogivethecarboxylic
acid 7. Attachment of the building block to the functiona-
lized Wang resin and subsequent ligation with Fmoc-Arg
and Fmoc-Lys followed by deprotection and cleavage led
to the neurotensin mimetics 8 and 9, respectively. Standard
solid phase protocols for microwave assisted SPPS were
employed using PyBOP/HOBt/DIPEA for the coupling
steps. Epimerization could be observed to a minor extent
€
(17) (a) Bittermann, H.; Einsiedel, J.; Hubner, H.; Gmeiner, P.
J. Med. Chem. 2004, 47, 5587–5590. (b) Bittermann, H.; Boeckler, F.;
Einsiedel, J.; Gmeiner, P. Chem.;Eur. J. 2006, 12, 6315–6322. (c)
Einsiedel, J.; Lanig, H.; Waibel, R.; Gmeiner, P. J. Org. Chem. 2007,
72, 9102–9113. (d) Einsiedel, J.; Huebner, H.; Hervet, M.; Haerterich, S.;
Koschatzky, S.; Gmeiner, P. Bioorg. Med. Chem. Lett. 2008, 18, 2013–
€
2018. (e) Harterich, S.; Koschatzky, S.; Einsiedel, J.; Gmeiner, P. Bioorg.
Med. Chem. 2008, 16, 9359–9368. (f) Lomlim, L.; Einsiedel, J.;
Heinemann, F. W.; Meyer, K.; Gmeiner, P. J. Org. Chem. 2008, 73,
3608–3611. (g) Einsiedel, J.; Held, C.; Hervet, M.; Plomer, M.; Tschammer,
N.; Huebner, H.; Gmeiner, P. J. Med. Chem. 2011, 54, 2915–2923.
(18) Chapman, R. N.; Arora, P. S. Org. Lett. 2006, 8, 5825–5828.
Org. Lett., Vol. 13, No. 13, 2011
3504

Table 1. Amide Proton Chemical Shift (δ) of NH(iþ3) As a
Function of the Percentage of DMSO-d6 in CDCl3 for the
Peptide Mimetic 11 and the Reference Peptides 12, 13, 14
Scheme 2. Solid Phase Supported Synthesis of the Peptide
Mimetic 11
% DMSO
11
12
13
14
0
5
6.7
6.8
6.9
7.0
7.1
6.9
6.9
7.0
7.1
7.1
6.9
7.0
7.1
7.1
7.2
6.3
6.8
7.1
7.3
7.4
10
20
30
Δδ
0.4
0.2
0.3
1.1
H-bond in the presence of even 30% DMSO. Temperature
1
dependent chemical shift changes of H NMR signals
as a measure for the stability of secondary structures
corroborated the assumption that the peptide mimetic 11
might form a stable intramolecular H-bond when a
comparatively low Δδ/ΔT value of ꢀ4.7 ppb/K was
observed.3b
In summary, novel reverse-turn mimetics could be de-
veloped by bridging the amino acid side chain and a
nitrogen substituent in positions i and iþ2, respectively,
by ring-closing olefin metathesis. NMR based conforma-
tional investigations indicated a stable intramolecular
H-bond constraining a U-turn conformation that was
predicted to simulate a type I β-turn.
type I β-turns. These peptides were obtained by microwave
assisted Fmoc-solid phase peptide synthesis (see Support-
ing Information). The experiments were done in 2 mM
solution to exclude intermolecular interactions.19 Interest-
ingly, NMR-derived δ(NH) values demonstrated the ex-
istence of an intramolecular hydrogen bond when the
NH(iþ3) signals in the conformationally restricted peptide
mimetic 11 and the reference agents 12 and 13 resonated
significantly more downfield (6.8ꢀ6.9 ppm) than the Ala-
Pro-Ala derivative (6.2 ppm).15 To further investigate the
stability of the intramolecular hydrogen bond, increasing
amounts of DMSO-d6, which acts as a competitive hydro-
gen bond acceptor, were added to a solution of 11 and
12ꢀ14 in CDCl3 (Table 1). Interestingly, the peptide
mimetic 11 and the Asx-Pro-turn inducing sequences 12
and 13 did not reveal a substantial alteration of the NH
shift (Δδ 0.2ꢀ0.4), thus indicating a stable intramolecular
Acknowledgment. The Volkswagen Foundation is ac-
knowledged for finacial support (Project I/81 615). We
ꢀ
ꢀ
thank Prof. Ivana Ivanovic-Burmazovic and Leanne Nye
(Department of Chemistry and Pharmacy, Chair of
Bioinorganic Chemistry, Friedrich Alexander University
€
Erlangen-Nurnberg) for high resolution ESI-TOF mass
spectra.
Supporting Information Available. Additional informa-
tion regarding the synthesis and characterization of pep-
tide analogs by HPLC, MS, and NMR. This material
is available free of charge via the Internet at http://
pubs.acs.org.
(19) (a) Gellman, S. H.; Dado, G. P.; Liang, G.-B.; Adams, B. R.
J. Am. Chem. Soc. 1991, 113, 1164–1173. (b) Dado, G. P.; Gellman, S. H.
J. Am. Chem. Soc. 1993, 115, 4228–4245.
Org. Lett., Vol. 13, No. 13, 2011
3505