Stereoselective synthesis of individual isomers of Leu-enkephalin analogues containing substituted beta-turn bicyclic dipeptide mimetics.

Article abstract of DOI:10.1039/b302235h

Novel constrained beta-turn dipeptide mimetics, 8-phenyl thiaindolizidinone amino acids 3, have been synthesized stereoselectively and incorporated into Leu-enkephalin peptides as a replacement of dipeptide Gly3-Phe4 to afford four individual isomers of L

Full text of DOI:10.1039/b302235h

Stereoselective synthesis of individual isomers of Leu-enkephalin
analogues containing substituted b-turn bicyclic dipeptide mimetics†
Chiyi Xiong,a Junyi Zhang,a Peg Davis,b Wei Wang,ac Jinfa Ying,a Frank Porrecab and Victor J.
Hruby*a
a Department of Chemistry, University of Arizona, 1306 E. University Blvd., Tucson, AZ 85721, USA
b Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA
c
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA
92121, USA
Received (in Corvallis, OR, USA) 21st February 2003, Accepted 1st May 2003
First published as an Advance Article on the web 4th June 2003
Novel constrained b-turn dipeptide mimetics, 8-phenyl
thiaindolizidinone amino acids 3, have been synthesized
stereoselectively and incorporated into Leu-enkephalin pep-
tides as a replacement of dipeptide Gly3–Phe4 to afford four
individual isomers of Leu-enkephalin analogues 6.
tors.5,2b For example, Nagai and co-workers have incorporated
unsubstituted thiaindolizidinone amino acid as a Gly-Gly
replacement in Leu-enkephalin to produce an analogue, which
exhibited 1/500 of the activity of the parent peptide.2b Lubell et
al.employed the indolizidin-9-one amino acids as a constrained
Gly-Gly surrogate in a Leucine-enkephalin mimic, and their
compound showed affinities for opioid receptors that were three
orders of magnitude lower than that of Leu-enkephalin.5f
However due to the lack of a robust methodology, to the best of
our knowledge, there is no report of the incorporation of
8-substituted BTDs into a biologically active peptide to make
individual isomers which could truly mimic the peptide
functions. In this communication we wish to report a flexible
synthesis which allows for the preparation of diastereoisomers
of Leu-enkephalin analogues containing the substituted chiral
b-turn bicyclic dipeptide mimetics.
In recent years, a great deal of effort has focused on the design
of b-turn peptidomimetics, which are conformationally con-
strained to provide a better understanding of the stereostructural
basis of peptide and protein interactions.1 One of the pharma-
ceutically interesting biological targets for which the peptido-
mimetic approach has been applied recently is Leu-enkepha-
lin.2
Previous studies on Leu-enkephalin (H-Tyr-Gly-Gly-Phe-
Leu-OH) and related cyclic analogues have suggested that it
adopts, predominantly, a b-turn centered at Gly3-Phe4.2a,3
These studies also suggested that for opioid peptides, the turn
position residues not only play a role of conformation
restriction, but also are involved in direct interaction with
receptor(s).2b,3 Furthermore, our studies have indicated that the
conformational requirements for optimal interaction with the m
and d receptors differ in a subtle manner.4 With these criteria in
mind, we propose that conformationally restricted, substituted
bicyclic b-turn dipeptide (BTD) mimetics with different
stereochemical configurations (Figure 1) would be a desirable
peptidomimetic moiety. The compounds were designed to
constrain two backbone angles (y2 and o3), of the four torsion
angles that define a b-turn, and most importantly, to place the
critical phenyl side chain of the Phe4 residue of Leu-enkephalin
correctly in 3D space. Incorporations of unsubstituted BTD into
biologically active peptides have been used to investigate the
structure–activity relationship between peptides and recep-
The synthesis of 8-phenyl-substituted thiaindolizidinone
amino acids was accomplished using the convergent synthetic
strategy in Scheme 1. The b-phenylcysteine derivatives (2R,
3S)-2a and (2S, 3R)-2b, were prepared according to our new
protocol.6 The doubly protected glutamic acid g-aldehyde 1 was
prepared according to a procedure previously described.5a
Aldehyde 1 was converted to a mixture of thiazolidines by
condensation with the b-phenylcysteine derivative (2R, 3S)-2a,
or (2S, 3R)-2b in buffered aqueous ethanol. Sequential fluoride-
mediated deprotection and cyclization of the resulting amino
acids with the aid of carbodiimide coupling reagent resulted in
clean formation of the bicyclic lactams 3a/3b–3c/3d as epimers
at the bridgehead. These diastereoisomers can be readily
separated. The ratio of 3a/3b, 3c/3d ranged from 1.8–1.3 :1. The
reaction behavior of the b-phenylcysteines is quite different
than the unsubstituted counterpart, in which only one epimer
was formed during the cyclization step.5a In addition, in the case
of b-phenyl-substituted cysteines, the addition of the carbodii-
mide coupling reagent was necessary for efficient cyclization,
Fig. 1
† Electronic supplementary information (ESI) available: experimental
Scheme 1 Reagents: a, HOAc, EtOH, H2O; b, PhCH2N(CH3)3F; c, DCC/
details. See http://www.rsc.org/suppdata/cc/b3/b302235h/
HOBt
1598
CHEM. COMMUN., 2003, 1598–1599
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Scheme 2 Reagents: a, BBr3, DCM; b, Leu-OMe·HCl, BOP, HOBt, DIPEA, DMF; c, NH2NH2, EtOH,CHCl3, H2O; d, (BOC)2O, THF, TEA; e, TFA, 0 °C,
20 min; f, Boc-Tyr-Gly-OH, HOBt, BOP, DIPEA, DMF; g, BBr3, DCM, 210 °C, 4 h, 9–15% overall yields.
while the unsubstituted bicyclic lactam can be formed through
catalysis of dilute HCl in the case of unsubstituted cysteine.5a
The stereochemistries at the bridgehead carbons (C-6) of the
8-substituted bicyclic thiazolidine lactams were assigned on the
basis of selective 1D transient NOE experiments performed on
3a–d (Figure 2). For example, in the experiment performed on
3a, relatively strong NOEs were observed between H6 and H8,
H9, H3 respectively, whereas weaker NOEs were observed
between H6 and H8, H9, H3 respectively in the case of 3b.
Similar results were seen with bicyclic lactams 3c and 3d. These
data support the assignment of a R-configuration at C-6 in 3a/3c
and a S-configuration at C-6 in 3b/3d.
Compounds 6a–d were evaluated in the isolated mouse vas
deferens (MVD, for d-receptor) and guinea pig ileum (GPI, for
m receptor) bioassays. At 1mM concentration, compounds 6a–c
showed 3.3, 8.9, 0 and 1% agonist activity for MVD
respectively, and 2.9, 2.6, 4 and 4.6% agonist activity for GPI
respectively. These correspond to a loss of potency of
approximately three orders of magnitude compared with Leu-
enkephalin, and suggest that the correct spatial distances and
orientations of the two aromatic pharmacophores (Tyr1 phenol
group and Phe4 phenyl group) in Leu-enkephalin are critical for
high potency.
In summary, this communication described the synthesis of
novel, conformationally constrained 8-phenyl substituted b-
turn dipeptide mimetics (3S, 6R, 8S, 9R)-3a, (3S, 6S, 8S, 9R)-3b,
(3S, 6R, 8R, 9S)-3c and (3S, 6S, 8R, 9S)-3d. Incorporation of
these substituted dipeptide mimetics into Leu-enkephalin was
accomplished by using modified solution phase peptide synthe-
sis methods. Extensive structure–activity relationship studies
are under the investigation.
Incorporation of the 8-phenyl BTD into Leu-enkephalin
using similar methodology as described before5a suffered some
problems. For example, after converting the phthalyl protecting
group to the Na-Boc protecting group (Scheme 2), we tried to
hydrolysize the ethyl ester group using standard base saponifi-
cation conditions, However this failed because of the steric
hindrance provided by the neighboring phenyl group, which is
on the same face as the carboxylate function, and when longer
reaction times were employed, racemization occurred. The
problem was solved by using boron tribromide to selectively
cleave the ethyl ester group,7 while keeping the phthalyl
protecting group and thiazolidine ring intact. Subsequently, the
leucine methyl ester was treated with Pht-8-phenyl-BTD-OH in
DMF using BOP and HOBt as the coupling reagent to yield the
tripeptide Pht-8-phenyl-BTD-Leu-OMe. Then the phthalyl
protecting group was removed by hydrazinolysis and the a-
amino group reprotected with di-tert-butyl dicarbonate to afford
tripeptide 4 (Scheme 2). Cleavage of the Na-Boc group of 4 and
coupling with Na-Boc-Tyr-Gly-OH gave the desired Leu-
enkephalin analogues 5 in a protected form which were
deprotected by treatment with BBr3 to give peptides 6 (Scheme
2). After purification by RP-HPLC, the Leu-enkephalin ana-
logues were isolated in 9–15% overall yield. There was no
The authors thank the USPHS grants DA 06284 and DA
13449 for support.
Notes and references
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1
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Fig. 2 NOEs observed for the bicyclic thiazolidine lactam intermediates 3a-
d.
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