Syntheses of depsipeptide analogues of the insect neuropeptide proctolin.

Article abstract of DOI:10.1016/S0960-894X(02)00214-7

Four depsipeptide analogues of the insect neuropeptide proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) have been prepared, containing a single ester linkage between Arg(1) and Tyr(2), Tyr(2) and Leu(3), and between Pro(4) and Thr(5), respectively. A didepsipentapeptide containing an ester linkage between Tyr(2) and Leu(3) and between Pro(4) and Thr(5), has also been prepared. The depsipeptide 4 is the first example of a backbone-modified proctolin analogue which shows full myotropic activity.

Full text of DOI:10.1016/S0960-894X(02)00214-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1625–1628
Syntheses of Depsipeptide Analogues of the Insect Neuropeptide
Proctolin
Jurgen Scherkenbeck,a,* Andrew Plant,a Frank Stieber,b Peter Losela and Hubert Dykera
aBayer AG, Central Research, 51368 Leverkusen, Germany
bDepartment of Chemistry, University of Dortmund, Germany
Received 14 February 2002; revised 29 March 2002; accepted 3 April 2002
Abstract—Four depsipeptide analogues of the insect neuropeptide proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) have been prepared,
containing a single ester linkage between Arg1 and Tyr2, Tyr2 and Leu3, and between Pro4 and Thr5, respectively. A didepsipenta-
peptide containing an ester linkage between Tyr2 and Leu3 and between Pro4 and Thr5, has also been prepared. The depsipeptide 4
is the first example of a backbone-modified proctolin analogue which shows full myotropic activity. # 2002 Elsevier Science Ltd.
All rights reserved.
Insect neuropeptides control almost all physiological
processes in insects and thus are potential targets for a
novel generation of selective insecticides.1 Unfortu-
nately, insect neuropeptides suffer from inadequate in
vivo efficacy due to poor absorption, lack of transpor-
tation, or rapid metabolic degradation. The incorpora-
tion of peptide mimetics has been established as a
method of circumventing the resulting insufficient
bioavailability of the native peptides.2À7
information.15 In only a few cases have proctolin ana-
logues been prepared with modifications to the peptide
backbone. For example, the incorporation of a-methyl-
l-Tyr,16 N-methyl amino acids,17 the replacement of the
amide bond between Tyr and Leu with the isosteric –
CH2–O–moiety,18 and cycloproctolin,19 have been
reported.
We were interested in probing the hydrogen bonding
requirements in proctolin by replacing one or more of
the peptide bonds with peptide bond isosteres or
mimics. Furthermore, we speculated that introducing
less hydrophilic, protease resistant functionality, might
lead to proctolin analogues exhibiting insecticidal activ-
ity. We chose to synthesize the following four depsi-
peptides: H-Arg-c[CO–O]-Tyr-Leu-Pro-Thr-OH (2) H-
Arg-Tyr-c[CO–O]-Leu-Pro-Thr-OH (3) H-Arg-Tyr-Leu-
Pro-c[CO–O]-Thr-OH (4) H-Arg-Tyr-c[CO–O]-Leu-Pro-
c[CO–O]-Thr-OH (5)
Since the insect neuropeptide Proctolin (1) was first iso-
lated from extracts of the cockroach Periplanata ameri-
cana8 and later shown to be a pentapeptide, H-Arg-Tyr-
Leu-Pro-Thr-OH (1) (RYLPT),9,10 it has received con-
siderable attention from scientists spanning a range of
disciplines (Fig. 1).11 Proctolin has been identified in
species from six orders of insect, as well as other inver-
tebrates, where it has been shown to exert myotropic
effects on visceral and skeletal muscle.11 Proctolin has
variously been assigned roles as a neurotransmitter, a
neuromodulator, and a neurohormone.12
Proctolin has been the subject of structure–activity
studies by a number of research groups.13 The majority
of the published data are concerned with the exchange
of one or more of the five constituent amino acids with
other natural or non-natural a-amino acids,14 affording
a considerable amount of structure–activity relationship
*Corresponding author. Tel.: +49-214-307-1157; fax: +49-214-305-
0070; e-mail: juergen.scherkenbeck.js@bayer-ag.de
Figure 1. Proctolin (1).
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00214-7

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J. Scherkenbeck et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1625–1628
We opted for a strategy employing orthogonal protect-
ing groups, protecting amino and guanidino function-
ality with the Boc group, and hydroxyl and carboxyl
groups were protected as their benzyl ethers and benzyl
esters, respectively (vide infra). H-Arg-c[CO–O]-Tyr-
Leu-Pro-Thr-OH (2) which contains an ester linkage
between Arg and Tyr was prepared as depicted in
Scheme 1.
cleavage of the benzyl ester by catalytic hydrogenolysis,
followed by removal of the three Boc groups on Arg by
treatment with trifluoroacetic acid, to afford the first
depsipeptide analogue of proctolin 2 (Scheme 1).
The synthesis of H-Arg-Tyr-c[CO–O]-Leu-Pro-Thr-OH
(3) commenced from H-Leu-OH (10), which was con-
verted into the corresponding (S)-a-hydroxycarboxylic
ester 11 via the tandem desamination-hydroxylation
procedure described above. DCC-mediated coupling
with Boc-Tyr(Bzl)-OH (6) gave the depsipeptide in very
good yield. Catalytic hydrogenolytic removal of the
benzyl protecting groups afforded the acid 12. For the
key fragment coupling reaction of 12 with the dipeptide
H-Pro-Thr-(Bzl)-OBzl (13) we chose BOP-Cl, with which
we have previously enjoyed success in the synthesis
of (cyclic)depsipeptides containing N-alkyl amino
acids.25,26
With a slight modification to a procedure from the lit-
erature used for analogous a-amino acids,20 H-Tyr(Bzl)-
OH (6) was desaminated and hydroxylated to the
corresponding a-hydroxycarboxylic acid with overall
retention of the desired configuration. Alkylation of the
crude product, via its caesium carboxylate,21 afforded
the benzyl ester 7 in 40% yield over two steps. The next
step was to couple a-hydroxycarboxylic ester 7 with
Boc-Arg(Boc)2-OH. Attempted ester formation using
the BOP reagent22 met with failure, as did the use of
water soluble carbodiimide/hydroxybenzotriazole.23
Fortunately, the DCC/DMAP protocol, according to
Gilion and Klausner,24 afforded the desired depsipep-
tide in 61% yield. Hydrogenolytic removal of the benzyl
protecting groups gave the acid 8 in high yield. This acid
was coupled to the tripeptide H-Leu-Pro-Thr-OBzl (9),
mediated by EDCI/HOBt.
No attempt to optimise the moderate yield of 42% for
this fragment coupling was made. The protecting
groups were removed in two high yielding steps, firstly
Scheme 1. Synthesis of H-Arg-c[CO–O]-Tyr-Leu-Pro-Thr-OH (2).
Scheme 2. Synthesis of H-Arg-Tyr-c[CO–O]-Leu-Pro-Thr-OH (3).

J. Scherkenbeck et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1625–1628
1627
The N-terminus of tetradepsipeptide 14 was liberated by
treatment with trifluoroacetic acid, setting up the final
coupling reaction with Boc-Arg(Boc)2-OH, to give the
protected pentadepsipeptide. Removal of the protecting
groups as described above afforded the depsipeptide
analogue 3 of proctolin with an ester linkage between
Tyr and Leu (Scheme 2).
H-Arg-Tyr-Leu-Pro-c[CO–O]-Thr-OH (4) was pre-
pared in a similar fashion to 3. Key steps were the forma-
tion of the ester linkage by DCC-mediated coupling of 16
with Boc-Pro-OH to afford, after deprotection of the N-
protecting group with trifluoroacetic acid, the didepsi-
peptide 17, and subsequent BOP-Cl-mediated coupling
of 17 with the dipeptide Boc-Tyr-Leu-OH 18 affording
the tetradepsipeptide 19. N-Deprotection and attach-
ment of the N-terminal amino acid Boc-Arg(Boc)2-OH
using EDCI/HOBt (56%), and subsequent removal of
all protecting groups led to the desired pentadepsipep-
tide 4 (Scheme 3).
The pentadidepsipeptide H-Arg-Tyr-c[CO–O]-Leu-Pro-
c[CO–O]-Thr-OH (5) was constructed from building
blocks already employed in the syntheses of 3 and 4.
Thus the BOP-Cl-mediated coupling of H-Pro-c[CO–
O]-Thr(Bzl)-OBzl (17) with Boc-Tyr-c[CO–O]-Leu-OH
(12) afforded the tetradidepsipeptide 20 in modest
(unoptimised) yield. The final four steps were identical
to those in the abovementioned syntheses of 3 and 4,
giving rise to the final depsipeptide analogue of procto-
lin (1), compound 5, modified at two positions of the
native peptide’s backbone (Scheme 4).
Scheme 3. Synthesis of H-Arg-Tyr-Leu-Pro-c[CO–O]-Thr-OH (4).
Myotropic effects of the new proctolin analogues were
assessed in vitro, using an isometric muscle-contraction
bioassay employing isolated hindguts from the locust
Locusta migratoria, based on procedures described by
Osborne and co-workers (Table 1).27
Introduction of ester functionality between Arg and Tyr
(2) resulted in a maximal contraction response of
approximately two-thirds that recorded for proctolin.
This is an interesting, as well as perplexing result when
one considers that [NMe-Tyr2]-proctolin has previously
been reported to lack myotropic activity.16 These con-
flicting results regarding the importance of hydrogen
bonding at this position require further investigation. In
contrast compound 3 was found to have 30% of the
agonistic activity of proctolin, a value not far removed
from that found for [NMe-Leu3]-proctolin (52%).17
Compound 4 showed a similar maximal contraction
response to proctolin, indicating that hydrogen bonding
Table 1. Preliminary results obtained using a test compound con-
centration of 1 mmol/L; an arbitrary value of 100% was assigned to
proctolin
Compd
Isometric contraction (%)
Proctolin
100
66
30
106
27
2
3
4
5
Scheme 4. H-Arg-Tyr-c[CO–O]-Leu-Pro-c[CO–O]-Thr-OH (5).

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J. Scherkenbeck et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1625–1628
interaction involving the amide moiety between Pro4
and Thr5 is not essential for myotropic activity. Based
on NMR studies Osborne et al. had previously postu-
lated an intramolecular hydrogen bond between Thr4
(donor) and Leu3 (acceptor), giving rise to an inverse
g-Turn.17 Based on our findings, it would appear that
this conformation is not necessarily the one which
interacts with the putative proctolin receptor, leading to
an agonistic response. Didepsipeptide 5 showed a simi-
lar response to compound 3. This result can be
explained by considering the additive effects of com-
pounds 3 and 4, and also confirms the conclusions made
about the importance of hydrogen bonding interactions
at these positions.
7. Giannis, A.; Kolter, T. Angew. Chem. 1993, 105, 1303.
8. Brown, B. E. Science 1967, 155, 595.
9. Brown, B. E.; Starratt, A. N. J. Insect. Physiol. 1975, 21,
1879.
10. Brown, B. E.; Starratt, A. N. Life Sciences 1975, 17, 1253.
11. For a recent review see Konopinska, D.; Rosinski, G. J.
Peptide Sci. 1999, 5, 533.
12. Osborne, R. H. Pharmacol. Ther. 1996, 69, 117.
13. Starrat, A. N.; Brown, B. E. Can. J. Chem. 1977, 55, 4238.
14. The Konopinska group has published extensively in this
area; the reader is referred to ref 5 and literature cited therein.
15. A number of different organisms and tissues thereof have
been used to evaluate biological activity, which complicates
assessing the published data regarding SAR.
16. Gray, A. S.; Osborne, R. H.; Jewess, P. J. J. Insect Phy-
siol. 1994, 40, 595.
17. Osborne, R. H.; Odell, B.; Blagbrough, I. S. Bioorg. Med.
Chem. Lett. 1995, 5, 2085.
In summary, we have synthesised four new analogues of
the insect neuropeptide proctolin, containing modifi-
cations to the peptide backbone. These novel depsipep-
tides have been prepared using a building block
approach, and employing an orthogonal protecting
group strategy.28 Depsipeptide 4 is the first example of a
backbone-modified proctolin analogue which retains
full myotropic activity.
18. Cameron, S.; Khambay, B. P. S. In Insect: Chemical,
Physiological and Environmental Aspects (Proc. 1st Interna-
tional Conference on insects 1994, Poland, Konopinska, D.
Ed., Wydawnictwa Uniwersytetu Wroclawskiego: Wroclaw,
1995; p. 209.
19. Hinton, J. M.; Osborne, R. H.; Odell, B.; Hammond, S. J.;
Blagbrough, I. S. Bioorg. Med. Chem. Lett. 1995, 5, 3007.
20. Degerbeck, F.; Fransson, B.; Grehn, L.; Ragnarsson, U.
J. Chem. Soc., Perkin Trans. 1 1993, 1 1 .
21. Wang, S.; Gisin, B. F.; Winter, D. P.; Makofske, R.;
Kulesha, I. D. J. Org. Chem. 1977, 42, 1286.
22. Kim, M. H.; Patel, D. V. Tetrahedron Lett. 1994, 35, 5603.
23. Dhaon, M. K.; Olsen, R. K.; Ramasamy, K. J. Org.
Chem. 1982, 47, 1962.
24. Gilon, C.; Klausner, Y. Tetrahedron Lett. 1979, 20, 3811.
25. Scherkenbeck, J.; Plant, A.; Harder, A.; Mencke, N. Tet-
rahedron 1995, 51, 8459.
References and Notes
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