Novel design of bicyclic β-turn dipeptides on solid-phase supports and synthesis of [3.3.0]-bicyclo[2,3]-leu-enkephalin analogues

Article abstract of DOI:10.1021/ol0488183

(Chemical Equation Presented) External bicyclic β-turn dipeptide mimetics provide an excellent design approach that can offer a rich chiral ensemble of structures with different backbone conformations. We report herein a novel design of a convergent combinatorial synthetic methodology, which is illustrated by the solid-phase synthesis of a series of [3.3.0]-bicyclo ^[2,3]-Leu-enkephalin analogues. The reactions were optimized and the epimeric configurations were determined by 2D NMR spectroscopy. Biological assays show that these analogues have more potent δ binding affinity and bioactivity for δ vs μ opioid receptor, which may be related to the different conformations preferred by these analogues in our modeling studies.

Full text of DOI:10.1021/ol0488183

ORGANIC
LETTERS
2
004
Vol. 6, No. 19
285-3288
Novel Design of Bicyclic â-Turn
Dipeptides on Solid-Phase Supports and
Synthesis of
3
[2,3]
[3.3.0]-Bicyclo -Leu-enkephalin
Analogues
†
†
Xuyuan Gu, Jinfa Ying, Richard S. Agnes, Edita Navratilova, Peg Davis,
†
†
†
Gannon Stahl, Frank Porreca, Henry I. Yamamura, and Victor J. Hruby*
Department of Chemistry, UniVersity of Arizona, 1306 East UniVersity BouleVard,
Tucson, Arizona 85721, and Department of Pharmacology, UniVersity of Arizona
Health Science Center, Tucson, Arizona 85724
hruby@u.arizona.edu
Received June 21, 2004
ABSTRACT
External bicyclic â-turn dipeptide mimetics provide an excellent design approach that can offer a rich chiral ensemble of structures with
different backbone conformations. We report herein a novel design of a convergent combinatorial synthetic methodology, which is illustrated
by the solid-phase synthesis of a series of [3.3.0]-bicyclo[2,3]-Leu-enkephalin analogues. The reactions were optimized and the epimeric
configurations were determined by 2D NMR spectroscopy. Biological assays show that these analogues have more potent δ binding affinity
and bioactivity for δ vs µ opioid receptor, which may be related to the different conformations preferred by these analogues in our modeling
studies.
Numerous efforts have been focused on building a variety
of â-turn peptidomimetics. Among them, bicyclic â-turn
and æ dihedral angles of the backbone but also the ø
1
and
1
ø
2
dihedral angles of side chain groups (Figure 1). Different
dipeptide (BTD) mimetics have been generally classified as
internal or external â-turn mimetics based on the covalent
kinds of external bicyclic scaffold synthetic methodologies
have been developed by several groups, but a systematic
2a
3
scaffold support located inside or outside the turn structure.
application of these methods to solid-phase synthesis has not
been reported. Due to the lack of efficient synthetic
methodologies for preparing structurally diverse bicyclic
Several types of internal â-turn-stabilizing structures have
been developed,1
c,d,2
a few of which have been used to
generate combinatorial libraries by using solid-phase
2
a,c,e
2a
(2) (a) Eguchi, M.; Lee, M. S.; Nakanishi, H.; Stasiak, M.; Lovell, S.;
Kahn, M. J. Am. Chem. Soc. 1999, 121, 12204. (b) Eguchi, M.; Shen, R.
Y. W.; Shea, J. P.; Lee, M. S.; Kahn, M. J. Med. Chem. 2002, 45, 1395.
synthesis.
External bicyclic â-turn dipeptides are es-
pecially interesting because they can control not only the ψ
(c) Acharya, A. N.; Nefzi, A.; Ostresh, J. M.; Houghten, R. A. J. Comb.
†
University of Arizona Health Science Center.
Chem. 2001, 3, 189. (d) Ostresh, J. M.; Schoner, C. C.; Hamashin, V. T.;
Nefzi, A.; Meyer, J.-P.; Houghten, R. A. J. Org. Chem. 1998, 63, 8622. (e)
Souers, A. J.; Virgilio, A. A.; Rosenquist, A.; Fenuik, W.; Ellman, J. A. J.
Am. Chem. Soc. 1999, 121, 1817. (f) Virgilio, A. A.; Ellman, J. A. J. Am.
Chem. Soc. 1994. 116, 11580.
(3) For a bicyclic dipeptides review, see: Hanessian, S.; McNaughton-
Smith, G.; Lombart, H.-G.; Lubell, W. D. Tetrahedron 1997, 53, 12789.
(1) For recent reviews of â-turn mimetics, see: (a) Souers, A. J.; Ellman,
J. A. Tetrahedron 2001, 57, 7431. (b) Halab, L.; Gosselin, F.; Lubell, W.
D. Biopolymers (Pept. Sci.) 2000, 55, 101. For â-turn definitions and their
significance in biological activity, see: (c) Rizo, J.; Gierasch, L. M. Annu.
ReV. Biochem. 1992, 61, 387. (d) Rose, R. D.; Gierasch, L. M.; Smith, J.
A. AdV. Protein Chem. 1985, 37, 1.
1
0.1021/ol0488183 CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/24/2004

7
peptide. Enkephalin analogues with δ and µ opioid agonist
8
activities have been extensively studied. Previous work
indicated that although enkephalin itself has a random
2
3
structure in water, a â-turn conformation at the Gly -Gly
9
positions is important for its biological activity. A (3S,6S,9R)-
[
2,3]
10a
[
[
4.3.0]-azobicyclo -Leu-enkephalin and a (2R,5S,8S)-
3.4.0]-thiazobicyclo[ -Leu-enkephalin have been pre-
2,3]
10b
1
0
pared previously by conventional methods. However, one
single isomer inserted in the enkephalin structure gives a
very limited picture. Here we report a novel synthesis of
several [3.3.0]-bicyclo[ -Leu-enkephalin analogues with
different geometries by nonconventional solid-phase syn-
thesis. The synthesis of S -Fm-cysteine was accomplished
by a modified method. S -Fm protection was chosen so
that both the N -Fmoc and S -Fm protecting groups can be
2,3]
1
1
â
1
2
â
R
â
simultaneously deprotected by piperidine. Allylglycine was
R
protected by an N -Fmoc, and the terminal alkene was
1
3
oxidized to the aldehyde by OsO
4
/NaIO
4
.
R
N -Fmoc-Leu-Wang resin was used to begin our synthesis
of the [3.3.0]-bicyclo -Leu-enkephalin analogues (Scheme
). N -Fmoc deprotection and the subsequent coupling steps
[2,3]
R
1
Figure 1. Design of external BTD and its retrosynthetic analysis
4
on the solid phase.
Scheme 1. Total Synthesis of (2R,5R,7S)- and
2R,5S,7S)-[3.3.0]-Bicyclo[ -Leu-enkephalin Analogues on a
2,3]
(
Solid-Phase Supporta
dipeptides, most of this work has been limited to obtaining
only a few of the possible isomers of bicyclic dipeptides in
a particular scaffold.
Recently, we have been working on the asymmetric
5
synthesis of different kinds of BTD mimetics to obtain a
better understanding of the molecular basis of peptide
ligand-protein receptor interactions that lead to high potency
and receptor selectivity. The BTD design 2 (Figure 1)
maintains the original peptide backbone. This design is
general enough to be used for all peptides which may involve
â-turn structures. The stereochemistry of these BTDs gener-
ally has been predetermined by the nature of the novel amino
acid building blocks which are used for the synthesis. We
envisioned that bicyclic dipeptides could be synthesized on
a solid-phase support in conjunction with synthesis of larger
peptides in a manner similar to solid-phase peptide chemistry.
A retrosynthetic analysis of this concept indicates that these
bicyclic dipeptides actually need only two nonstandard amino
acids (Figure 1). They are â-substituted ω-unsaturated amino
acids 4 and the â-substituted cysteines 5, respectively. Both
of these enantiomerically pure nonproteinous amino acids
can be synthesized using synthetic methodologies we recently
a
Key: (i) 25% piperidine in DMF; (ii) Fmoc-Phe-OH, HBTU,
HOBT, DIEA; (iii) identical to i; (iv) Fmoc-L-Cys-S(Fm)-OH,
DCM/DMF, HBTU, HOBT, DIEA; (v) 50% piperidine, 5% TIPS,
DMF; (vi) S-2-(9H-fluorenyl-9-methoxycarbonylamino)-4-oxo-
butyric acid, DMF, DIEA; (vii) HBTU, HOBT, DIEA; (viii)
6
have developed.
To demonstrate this novel concept, we chose Leu-
enkephalin, an endogenous opioid peptide as a target
t
identical to i; (ix) Fmoc-Tyr-(O- Bu)-OH, HBTU, HOBT, DIEA;
(
x) identical to i; (xi) 90%TFA, 5% H
2
O, 5% TIPS.
(4) Eight diastereomer peptides are possible for external bicyclic dipep-
tides with no side chain groups involved, while 32 possible isomers are
available if the side-chain groups are included.
(
5) (a) Zhang, J.; Xiong, C.; Wang, W.; Ying, J.; Hruby, V. J. Org. Lett.
13
were accomplished by conventional methods. After cysteine
2
002, 4, 4029. (b) Gu, X.; Tang, X.; Cowell, S.; Ying, J.; Hruby, V. J.
R
â
coupling, the N -Fmoc and S -Fm were deprotected using
50% piperidine in DMF and 5% TIPS to guarantee the
completeness of the reaction in 30 min. The Kaiser test14
for Cys residues does not show a deep blue color in the
Tetrahedron Lett. 2002, 43, 6669. (c) Wang, W.; Yang, J.; Ying, J.; Xiong,
C.; Zhang, J.; Cai, C.; Hruby, V. J. J. Org. Chem. 2002, 67, 6353. (d)
Wang, W.; Xiong, C.; Hruby, V. J. Tetrahedron Lett. 2001, 42, 3159. (e)
Qiu, W.; Gu, X.; Soloshonok, V. A.; Carducci, M. D.; Hruby, V. J.
Tetrahedron Lett. 2001, 42, 145.
3286
Org. Lett., Vol. 6, No. 19, 2004

presence of the unprotected thiol because the sulfur reacts
in an intramolecular reaction15 to give a thiazolidine. As a
matter of fact, a positive color indicated an incomplete
â
deprotection of the S -Fm on sulfur. The N,S-thiazolidine
formation with R- or S-2-(9H-fluorenyl-9-methoxycarbonyl-
amino)-4-oxobutyric acid followed and was optimized in
DMF with 2 equiv of DIEA within 2 h. A similar strategy
for thiazolidine synthesis on a solid-phase support has been
reported previously.16 Diastereomeric peptides with the
epimeric bridge head H were formed at this stage. The
lactamization of the secondary amine using HBTU and
HOBT in the presence of DIEA progressed quickly. Finally,
R
t
N -Fmoc-Tyr-(O- Bu)-OH was introduced by conventional
13
R
peptide synthesis. After N -Fmoc deprotection, the peptide
was cleaved from resin. The peptide solution was evaporated
and neutralized, and the diastereomeric analogues were
Figure 2. Stereochemistry of bicyclic dipeptides and the bridgehead
ROEs.
1
3
isolated and purified on a reversed-phase HPLC column.
This 11-step synthesis of bicyclo[2,3]-Leu-enkephalin thus
was completed, and two bridgehead diastereomeric peptides
were generated in about 12 h time. The combined yield of
The synthesized [3.3.0]-bicyclo[2,3]-Leu-enkephalin ana-
logues were examined for their binding affinities to opioid
3
3
receptors in competition with [ H]-deltorphin II (δ) and [ H]-
DAMGO (µ) (Table 1). All the analogues show binding
affinities with IC50 values in the micromolar range. In
comparing the δ and µ binding affinities, we observed they
13
the final products was 48% with clean HPLC spectra. This
optimized procedure was used to synthesize two other
diastereomers 13a and 13b from D-allylglycine in a combined
yield of 43%. The diastereomeric ratio of 12a (2R,5R,7S) to
1
2b (2R,5S,7S) was 47:53 while the ratio of 13a (2R,5R,7R)
13
to 13b (2R,5S,7R) was 40:60. The bridgehead configuration
3
Table 1. Binding Affinity in Competition with [ H]-DAMGO
were assigned on the basis of ROE connectivity patterns
3
and [ H]-Deltorphin II in Mouse Brain Membranes and Potency
(Figure 2). We were fortunate that the bridgehead hydrogens
in MVD and GPI/LMMP Bioassays
(4.8-5.0 ppm) are well separated from other protons in the
2
D O solution NMR spectra, allowing the relationship of these
peptide
Del II (δ) (%) DAMGO MVD GPI/LMMP
isomers to be assigned unambiguously.
analogues
IC50 (µM)
(µ) (%)
(δ) (%)
(µ) (%)
1
1
2a (2R,5R,7S)
2b (2R,5S,7S)
22.5a
47 ( 9
0.6a
0a
14.4b
10.3b
3.8b
6.1b
(
6) For a convenient asymmetric synthesis of â-aromatic and â-aliphatic
substituted cysteines, see: (a) Xiong, C.; Wang, W.; Cai, C.; Hruby, V. J.
J. Org. Chem. 2002, 67, 1399. (b) Xiong, C.; Wang, W.; Hruby, V. J. J.
Org. Chem. 2002, 67, 3514. For â-substituted γ,ꢀ-unsaturated amino acid,
see: (c) Gu, X.; Scott, C.; Ying, J.; Tang, X.; Hruby, V. J. Tetrahedron
Lett. 2003, 44, 5863.
13a (2R,5R,7R)
3b (2R,5S,7R)
2.4 ( 0.9
20.6a
15.5b
5.9b
33.1a
2.6a
11.5b
4.6b
1
a Percent decrease of maximum binding at 10 µM peptide. b Percent
(7) Hughes, J.; Smith, T. W.; Kosterlitz, H. W.; Fothergill, L. A.; Morgan,
decrease of maximum effect at 1 µM peptide.
B. A.; Morris, H. R. Nature 1975, 258, 577.
(
8) Hruby, V. J.; Gehrig, C. A. Med. Res. ReV. 1989, 9, 343.
(9) (a) Kreye, P.; Kihlberg, J. Tetrahedron Lett. 1999, 40, 6113. (b)
Malicka, J.; Groth, M.; Czaplewski, C.; Kasprzykowska, R.; Liwo, A.;
Lankiewicz, L.; Wiczk, W. Lett. Pept. Sci. 1998, 5, 445. (c) Hruby, V. J.;
Kao, L.-F.; Pettitt, B. M.; Karplus, M. J. Am. Chem. Soc. 1988, 110, 3351.
all are more potent δ opioid ligands. The binding affinity of
1
2
3a (2.4 µM) with a D-amino acid in position-2 is about
0-fold better than that of 12b (47 µM) with an L-amino
(10) (a) Gosselin, F.; Tourw e´ , D.; Ceusters, M.; Meert, T.; Heylen, L.;
Jurzak, M.; Lubell, W. D. J. Pept. Res. 2001, 57, 337. (b) Nagai, U.; Sato,
K. Prepr.: Struct. Funct.; Proc. Am. Pept. Symp., 9th 1985, 465.
acid at this position. The effect of D-amino acids in Leu-
enkephalin analogues DPDPE has been previously dis-
(11) It should be noted that the [3.4.0]- and [3.5.0]-bicyclic dipeptides
cannot be simply synthesized using the methodology we have developed
here due to the formation of 5- and 6-membered hemiaminals (see ref 5b
and 6c). The methodology for [3.4.0]- and [3.5.0]-bicyclic dipeptides can
be modified from the above strategy, and such studies currently are under
investigation in our laboratory.
17
cussed. The functional assays (MVD and GPI/LMMP) also
show more potent δ activity than µ activity (Table 1).
13
Modeling studies have shown that the backbones con-
formations of [3.3.0]-bicyclo[ -Leu-enkephalin are con-
trolled by different bicyclic chiralities. Although [3.3.0]-
BTDs have been proposed as â-turn mimetics,18 their
conformations may be modulated not only by the configura-
tions of the BTDs but also by their specific peptide sequence.
It is not surprising that no typical â-turn structure was
2,3]
(12) (a) Albericio, F.; Nicolas, E.; Rizo, J.; Ruiz-Grayo, M.; Pedroso,
E.; Giralt, E. Synthesis 1990, 119. (b) Campbell, A. S.; Fraser-Reid, B.
Bioorg. Med. Chem. 1994, 2, 1209.
(
13) For experimental details, see the Supporting Information.
(14) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, R. I. Anal.
Biochem. 1970, 34, 595.
15) An intermediate generated in Kaiser test prevents the formation of
Ruhemann’s purple. Also see ref 14.
(
(
17) (a) Mosberg, H. I.; Hurst, R.; Hruby, V. J.; Gee, K.; Yamamura, H.
J.; Galligan, J. J.; Burks, T. F. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 5871.
b) Mosberg, H. J.; Hurst, R.; Hruby, V. J.; Galligan, J. J.; Burks, T. F.;
Gee, K.; Yamamura, H. I. Life Sci. 1983, 32, 2565.
18) Sabasingh, N. L.; Bontems, R. J.; McIntee, E.; Mishra, R. K.;
Johnson, R. L. J. Med. Chem. 1993, 36, 2356.
(
(
(16) Patek, M.; Drake, B.; Lebl, M. Tetrahedron Lett. 1995, 36, 2227.
Org. Lett., Vol. 6, No. 19, 2004
3287

observed for the bicyclo[2,3]-analogues. However, a type I
â-turn conformation in 13a occurs at positions 3-4 (φ
46.0°, ψ ) -43.9°, φ ) -94.9°, ψ ) +7.3°), consistent
with the X-ray structure of Leu-enkephalin previously
reported.19 In addition, the distance (8.78 Å) between the
two aromatic ring centroids fits well with expected distances
for δ receptor ligands.20 In light of the type I turn structure
and the pharmacophore distance requirements, no other
analogues show a better fit, which may provide the structural
basis for the higher δ binding affinity and δ bioactivity of
diastereomeric libraries of these novel compounds. It should
provide a unique tool for studying structure-biological
activity relationships in combination with NMR and model-
ing techniques. The method is general enough for parallel
synthesis of all chiral side-chain containing bicyclic dipep-
tides using the appropriate amino acid precursors. The
syntheses of [3.3.0]-bicyclic dipeptides with side-chain
groups and [3.4.0]- and [3.5.0]-bicyclic dipeptides on solid-
phase supports are currently under investigation.
3
)
-
3
4
4
1
3a relative to the other analogues.
In summary, we have developed a novel methodology for
Acknowledgment. This work was supported by US
Public Health Service grants (DK 17420) and the National
Institute of Drug Abuse (DA 06284, DA 13449). The Bruker
DRX 500 MHz spectrometer was obtained by a grant from
the NSF (9729350).
the synthetic assembly of external bicyclic dipeptide contain-
ing peptides from chiral precursors on solid-phase supports.
The synthesis of four isomers of [3.3.0]-bicyclo[2,3]-Leu-
enkephalin represents the first example in this area. This fast
and efficient synthetic strategy makes it possible to obtain
Supporting Information Available: Experimental pro-
cedures, purification, and characterization of all compounds;
(
19) Aubry, A.; Birlirakis, N.; Sakarellos-Daitsiotis, M.; Sakarellos, C.;
Marraud, M. Biopolymers 1989, 28, 27.
20) Liao, S.; Alfaro-Lopez, J.; Shenderovich, M. D.; Hosohata, K.; Lin,
2D NMR and computational study. This material is available
(
free of charge via the Internet at http://pubs.acs.org.
J.; Li, X.; Stropova, D.; Davis, P.; Jernigan, K. A.; Porreca, F.; Yamamura,
H. I.; Hruby, V. J. J. Med. Chem. 1998, 41, 4767.
OL0488183
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