Structural prerequisites for receptor binding of helicokinin I, a diuretic insect neuropeptide from helicoverpa zea

Article abstract of DOI:10.1002/ejoc.201301773

In insects essential physiological processes such as muscle activity or water balance are controlled by neuropeptides. However, owing to their metabolic instability and adverse physicochemical properties peptides are unsuited as crop protection agents. Helicokinin I, a diuretic neuropeptide of cotton pest Helicoverpa zea represents a most promising target for the design of neuropeptide mimetics. Several helicokinin analogues containing scaffolds with varying rigidity and different orientations of the N- and C-terminal peptide chains were synthesized and tested for receptor binding. Additional conformational analyses by NMR spectroscopy in a membrane-mimicking environment together with MD simulations provide a deeper insight into the structural requirements for receptor binding and explain the remarkable activity of a macrocyclic helicokinin I derivative. A delicate balance of rigidity and flexibility makes the difference between activity and inactivity. Detailed conformational studies of analogues of the diuretic insect-neuropeptide helicokinin I containing diverse cyclic scaffolds with different flexibility and orientation of the N- and C-termini provide a better understanding of the structural requirements for receptor binding. Copyright

Full text of DOI:10.1002/ejoc.201301773

FULL PAPER
DOI: 10.1002/ejoc.201301773
Structural Prerequisites for Receptor Binding of Helicokinin I, a Diuretic
Insect Neuropeptide from Helicoverpa zea
Chien Tran Van,[a] Tino Zdobinsky,[b] Guiscard Seebohm,[c] Dirk Nennstiel,[d]
Oliver Zerbe,*[e] and Jürgen Scherkenbeck*[a]
Keywords: Synthetic methods / Amino acids / Peptidomimetics / Insect neuropeptides / Helicokinin I
In insects essential physiological processes such as muscle
activity or water balance are controlled by neuropeptides.
However, owing to their metabolic instability and adverse
physicochemical properties peptides are unsuited as crop
protection agents. Helicokinin I, a diuretic neuropeptide of
cotton pest Helicoverpa zea represents a most promising tar-
get for the design of neuropeptide mimetics. Several helicok-
inin analogues containing scaffolds with varying rigidity and
different orientations of the N- and C-terminal peptide chains
were synthesized and tested for receptor binding. Additional
conformational analyses by NMR spectroscopy in a mem-
brane-mimicking environment together with MD simulations
provide a deeper insight into the structural requirements for
receptor binding and explain the remarkable activity of a
macrocyclic helicokinin I derivative.
have also been reported to inhibit weight gain by larvae of
the tobacco budworm (Heliothis virescens), a serious agri-
cultural pest.[5]
Introduction
Neuropeptides control specific physiological processes in
insects.[1] Thus, insect neuropeptides and their receptors
present promising targets for a new generation of insectici-
dal agents that offer levels of selectivity and environmental
compatibility that are absent from conventional insecti-
cides.[2] Despite of all progress in insect-neuropeptide re-
search a major obstacle remains to be solved. Peptides are
metabolically unstable and display physicochemical and
pharmacological properties that render them unsuitable for
any application as crop-protection agents. Nevertheless,
their short sequences and established structure-activity rela-
tionships make insect neuropeptides prime candidates for
the design of peptide mimetics with improved drug proper-
ties. The large family of myokinins appears most promising
as lead structures for peptidomimetic approaches owing to
their short chain lengths of 6–13 residues and their potent
diuretic activity with EC50 values for receptor activation in
the low nanomolar range.[3,4] Recently, the helicokinins
One strategy to find analogues of a biologically active
peptide comprises high-throughput screenings of large com-
pound collections. Following this approach ten thousand
compounds were tested for agonistic activity on the diuretic
helicokinin receptor from Heliothis virescens. Surprisingly,
not a single hit was found during this mass screening cam-
paign, calling into questions, once again, the value of this
strategy.[6] Obviously, the helicokinin receptor only tolerates
very limited structural variations of the natural peptide mo-
tif. Therefore, a rational strategy holds much more promise
to identify active peptidomimetic analogues of helicoki-
nin I.[7]
The ideal basis for the rational design of peptidomimetics
is of course the knowledge of the receptor-bound neuropep-
tide conformation. Unfortunately, this remains a challenge
because neuropeptides bind to membrane-bound G pro-
tein-coupled receptors, which are notoriously difficult to
crystallize.[8,9] In fact, there have been no reports describing
the conformation of a receptor-bound insect neuropeptide.
As a consequence only indirect information on biologically
active conformations of insect neuropeptides is available.
Several efforts to get a better insight into the conforma-
tional prerequisites for receptor binding of kinins have been
undertaken. For instance, macrocyclic analogues of the ach-
etakinin core (Phe-Phe-Pro-Trp-GlyNH2) were prepared in
order to freeze potentially relevant conformations into a
small, conformationally restricted structure that can easily
be analyzed by conventional NMR spectroscopy. Based on
those cyclic neuropeptide analogues together with peptido-
mimetic approaches and molecular dynamic (MD) simula-
[a] Institute of Organic Chemistry, University of Wuppertal,
Gaußstraße 20, 42119 Wuppertal, Germany
http://www.bioorganik.uni-wuppertal.de/
[b] Department of Organic Chemistry, Weizmann Institute of
Science,
234 Herzl Street, 76100 Rehovot, Israel
[c] Department of Myocellular Electrophysiology, University of
Münster,
Albert-Schweitzer Campus 1, 48149 Münster
[d] Bayer CropScience AG,
Alfred-Nobel-Straße 50, 40789 Monheim, Germany
[e] Institute of Organic Chemistry, University of Zürich,
Winterthurerstraße 190, 8057 Zürich, Switzerland
http://www.chem.uzh.ch/zerbe/index.html
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301773.
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Structural Prerequisites for Receptor Binding of Helicokinin I
tions Nachman and Coast proposed a VI β-turn involving unnecessary additional protecting-group manipulations. Di-
the C-terminal residues Phe-Phe-Pro-Trp as a general struc- rect azidation of the enolate, formed from compound 3 with
tural feature of the kinins.[10]
trisyl azide, as employed by Evans, resulted in yields lower
A completely different approach to obtain information than 20%.[15] Far more efficient for the preparation of azide
on the biologically active conformation of a peptide is 5 turned out to be a three-step procedure starting with a
based on the structure determination of a peptide in the highly
diastereoselective
enolate
oxidation
with
membrane-bound state. According to the membrane-com- MoO5·Py·DMPU (MoOPD) followed by mesylation and
partment theory, the target cell surface influences the recep- nucleophilic substitution with NaN3.[16]
tor selection of regulatory peptides by increasing peptide
concentration in the vicinity of the receptor and by in-
ducing preferred conformations and orientations.[11] This
means the membrane-bound structure at least resembles the
conformation that is recognized by the receptor in the first
stage of the binding event. By using spin-labeling tech-
niques and detailed NMR spectroscopic studies it was
shown that hexapeptide helicokinin I (Tyr-Phe-Ser-Pro-Trp-
GlyNH2, 1) forms in both dodecylphosphocholine (DPC)
and sodium dodecyl sulfate micelles a C-terminal type-I β-
turn including the amino acid residues Ser3 (i), Pro4 (i+1),
Trp5 (i+2) and Gly6 (i+3) with a trans Ser-Pro amide bond.
Remarkably, in aqueous solution helicokinin I does not
adopt any preferred conformation, proving that the forma-
tion of the β-turn is induced by the interaction of the pept-
ide with the membrane.[12]
Both strategies, synthesis of constrained kinin mimics
and conformational analyses of membrane-bound helicoki-
nin I revealed a C-terminal β-turn. However, the exact na-
ture and flexibility of the β-turn, required for receptor bind-
ing, remain to be elucidated. To get a deeper insight into
these questions a series of helicokinin I analogues contain-
ing scaffolds with varying rigidity and different orientations
of the N- and C-terminal peptide chains was synthesized,
studied in a membrane-mimicking environment and tested
for receptor binding.
Scheme 1. Synthesis of the trans 4-amino-pyroglutamic acid scaf-
fold and construction of helicokinin I analogues. Reagents and con-
ditions: (a) MeOH, SOCl2, room temp., 18 h, toluene, reflux, 2 h,
98%; (b) NaBH4, iPrOH, room temp., 20 h, 83%; (c) benzaldehyde,
p-toluenesulfonic acid, toluene, reflux, 6 h, 62%, (d) Lithium diiso-
propylamide, –78 °C, 50 min, MoOPD, –40 to –35 °C, 3 h, 73%;
(e) MsCl, triethylamine (TEA), room temp., 18 h, 84%; (f) Part 1.
NaN3, dimethyl sulfoxide, 60 °C, 1 h, 82%; Part 2. TFA, THF/
H2O, room temp., 18 h, 86%; (g) RuCl3·xH2O (2 mol-%), NaIO4,
CCl4/MeCN/H2O (2:2:3), room temp., 16 h, 87%; (h) H-Trp-Gly-
NH2, HATU, DIPEA, DMF, room temp., 20 h, 60%; (i) Pd/C, H2,
MeOH, room temp., 16 h, 95%; (j) Part 1. HATU, DIPEA, DMF,
room temp., 22 h, Boc-Tyr(tBu)-Phe-OH (68%) or Boc-Tyr(tBu)-
Phe-Ser(tBu)-OH (66%); Part 2. TFA/triisopropylsilylane (TIPS)/
H2O, 0 °C, 4 h, 85–92%.
Results
Syntheses of Helicokinin I Analogues
Inspired by the high activities of 4-amino-pyroglutamate
achetakinin-analogues employed by Nachman we prepared
analogous derivatives of helicokinin I (1).[13] In addition, we
used 4-amino-pyroglutamate not only as a β-turn motif, re-
placing the central dipeptide Ser3 and Pro4 of the “Nach-
man” β-turn, but also as a substitute for the single residue
From structure-activity studies of helicokinin I it is well
proline. In both cases a sufficient length of at least five resi- known that proline can be replaced by (L)-alanine without
dues for the resulting helicokinin I analogues is guaranteed. any loss of receptor binding.[17] Thus we devised the (S)-
According to the results of Nachman, only the two trans- 3-isopropylpiperazin-2-one scaffold as a stretched proline
isomers of 4-amino-pyroglutamate were prepared in enan- analogue in which the isopropyl side-chain is intended to
tiomerically pure form, starting either from l- or d-glut- mimic parts of the lipophilic pyrrolidine ring of proline.[18]
amic acid (2) according to Scheme 1. Despite several litera- In contrast to proline, which favors the formation of a type-
ture reports on the synthesis of substituted pyroglutamates I β-turn, 2-oxo-piperazine analogue 15 is expected to induce
we found our modified route most efficient for the prepara- a more open, bowl-like conformation in which the amino
tion of 4-amino pyroglutamate.[14] In particular, we kept the acid residues are oriented in opposite directions (Scheme 2).
azido function in compound 5 intact throughout the syn- The synthesis of the piperazinone-Trp dipeptide was ac-
thesis and reduced it only at the tripeptide stage prior to complished in a straightforward sequence commencing with
chain elongation with either dipeptide Boc-Tyr(tBu)-Phe- Val-OtBu (8). The ethyl-bridge was introduced by alkyl-
OH or tripeptide Boc-Tyr(tBu)-Phe-Ser(tBu)-OH to avoid ation of dipeptide NosNH-Val-Trp-OMe (9) with 1,2-di-
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Scheme 2. Synthesis of the 2-oxo-piperazine scaffold and helicokinin I mimetic 15. Reagents and conditions: (a) 2-nitrophenylsulfonyl
chloride, TEA, CH2Cl2, room temp., 20 h, 98%; (b) TFA/CH2Cl2 (1:1), 0 °C, 4 h, 87%; (c) l-tryptophan methyl ester, TBTU, DIPEA,
CH2Cl2, room temp., 24 h, 91%; (d) 1,2-dibromoethane, K2CO3, DMF, 60 °C, 48 h, 74%; (e) 4% LiOH, THF, room temp., 3 h, 97%;
(f) glycinamide hydrochloride, TBTU, DMF, room temp., 24 h, 72%; (g) PhSH, K2CO3, room temp., 4 h, 96%; (h) Fmoc-Ser(tBu)-OH,
TBTU, DIPEA, DMF, 95%; (i) piperidine, CH2Cl2, 98%; (j) Boc-Tyr(tBu)-Phe-OH, TBTU, DMF, DIPEA, 87%; (k) TFA/TIPS/H2O
(95:2.5:2.5), 0 °C, 3 h, 90%.
bromoethane in 73% yield, followed by conventional O- placement, which could at least explain the formation of
(benzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium
tetra- coupling product 22. Hydrogenolysis of compound 22 with
fluoroborate (TBTU)-mediated amino acid couplings and Pd(OH)2/C yielded completely deprotected product 24 and
protecting group removals to afford helicokinin I analogue the mono-deprotected PMB-derivative in an overall yield of
15.
92%. (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo-
The synthesis of helicokinin I analogue 28 provides a for- [4,5-b]pyridinium 3-oxide hexafluorophosphate) (HATU)-
midable challenge because reports concerning related struc- mediated ring closure afforded monocycle 25 to which
tures suffer from either low yields and diastereoselectivities Fmoc-Ser(tBu)-OH was attached in a yield of 70% after
or inconvenient starting materials.[19,20] In our synthesis we three days without any epimerization. The extended reac-
used valine derivative 16 and glycidol 17 as convenient chi- tion time is probably caused by the steric hindrance of the
ral starting materials (Scheme 3).[21] Ring opening of pro- isopropyl and the bulky silyl-protecting group.[24] Fmoc
tected glycidol 17 turned out to be a critical reaction with cleavage and a second coupling with dipeptide Boc-
respect to the Lewis-acid needed to facilitate the epoxide Tyr(tBu)-Phe-OH gave peptide 26 in 77% yield, which after
opening. After extensive testing we found Mg(OTf)2 best cleavage of the methyl ester, was coupled with glycine amide
for the nucleophilic ring opening with PMB-Val-OBn. to afford peptide 27. Construction of the second ring was
Stronger Lewis-acids such as SnCl4 or BF3*Et2O caused ex- accomplished by mesylation of the primary hydroxy group
tensive decomposition of the starting materials and weaker in 27 and subsequent cyclization in almost quantitative
Lewis acids as Cu(OTf)2 were found to be unreactive or yield in less than 30 min.[25] Removal of all protecting
gave incomplete conversion. Under carefully controlled groups in one-step (TFA/TIS/H2O, 95:2.5:2.5) finally af-
conditions with 1.1 equiv. Mg(OTf)2 in MeCN a 12:88 mix- forded desired helicokinin I analogue 28 in quantitative
ture of coupling products 18 and 19 was obtained in 85% yield as the TFA salt. All biological and NMR spectro-
yield along with 13% unreacted epoxide 17.[22] Mesylation scopic studies were conducted with the free amine obtained
and subsequent nucleophilic substitution with Trp-OMe by column chromatography with ethyl acetate/ethanol in
unexpectedly afforded the coupling product as a mixture of the presence of 0.1% triethylamine.
two regioisomers 22 and 23 through aziridinium intermedi-
Our synthesis of the flexible-macrocyclic-bridged helico-
ate 21 in a 1:1.5 ratio and an overall yield of 69% over the kinin I analogues 34 and 36 started with the coupling of
two steps.[23] Detailed NMR spectroscopic studies on the alanine derivative 29 and Trp-OMe with (benzotriazol-
stereochemistry revealed unambiguously that both isomers 1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
22 and 23 were formed by ring-opening of the postulated (PyBOP) in 82% yield, followed by ester cleavage with
aziridinium intermediate and not by simple nucleophilic re- LiOH and a second coupling with N-allylglycine amide
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Structural Prerequisites for Receptor Binding of Helicokinin I
Scheme 3. Synthesis of a rigid bicyclic β-turn mimetic and assembly of helicokinin I analogue 28. Reagents and conditions: (a) Mg-
(OTf)2, MeCN, 75 °C, 36 h, 85%; (b) MsCl, TEA, CH2Cl2, 2 h, room temp., 75%; (c) Trp-OMe, MeCN, 60 °C, 3 d, 22 (26%), 23 (43%);
(d) H2, Pd(OH)2/C, EtOH, room temp., 1 h, 92%; (e) HATU, MeCN, room temp., 17 h, 32%; (f) Part 1. Fmoc-Ser(tBu)-OH, HATU,
DMF, DIPEA, 3 d, 40 °C, 70%; Part 2. Piperidine, DMF, 2 h, room temp., 87%; Part 3. Boc-Tyr (tBu)-Phe-OH, HATU, DIPEA, CH2Cl2,
18 h, room temp., 89%; (g) Part 1. LiOH, THF/MeOH/H2O (3:1:1), 13 h, room temp.; Part 2. Gly-NH2·HCl, HATU, DMF, DIPEA, 15
h, room temp., 89% (two steps); Part 3. Tetra-n-butylammonium fluoride (TBAF), THF, 19 h, 81%; (h) Part 1. MsCl, TEA, CH2Cl2,
30 min, 85%; Part 2. NaH, THF, 30 min, room temp., 97%; Part 3. TFA/TIPS/H2O (95:2.5:2.5), 30 min, 0 °C to room temp., 100%.
(75% yield) to afford tripeptide 31 (Scheme 4).[26] The key- ford peptide 33. Unfortunately, the more convergent direct
step in our sequence is the ring-closing metathesis (RCM) attachment of the tripeptide Boc-Tyr(tBu)-Phe-Ser(tBu)-
reaction between the two allyl residues.[27] The best results OH to the macrocyclic scaffold failed even with coupling
(43% of trans-product 32 and 9% of corresponding cis-iso- reagents such as bromotripyrrolidinophosphonium hexa-
mer) were obtained in the cyclization of tripeptide 31 with fluorophosphate (PyBrop), 2-bromo-3-ethyl-4-methylthia-
Hoveyda–Grubb’s II catalyst, whereas the RCM reaction of zolium tetrafluoroborate (BEMT), HATU, 2-bromo-1-eth-
linear hexapeptide Boc-Tyr(tBu)-Phe-Ser(tBu)-N-allyl-Ala- ylpyridinium tetrafluoroborate (BEP), bis(trichloromethyl)-
Trp-N-allyl-GlyNH2 with Zhan catalyst afforded only 19% carbonate (BTC), BMIT, and 4-(4,6-bis[2,2,2-trifluoro-
cyclization product.[28,29] The N-terminal tripeptide chain ethoxy]-1,3,5-triazin-2-yl)-4-methylmorpholinium
tetra-
was built up stepwise by HATU-mediated couplings of fluoroborate (DFET) developed for especially difficult se-
Fmoc-Ser(tBu)-OH (67%) followed, after protecting group quences. Finally, the unsaturated cyclic hexapeptide 33 was
removal, by dipeptide Boc-Tyr(tBu)-Phe-OH (76%) to af- converted into desired helicokinin I derivative 34 by acidic
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Scheme 4. Synthesis of macrocyclic-bridged helicokinin I analogues 34 and 36. Reagents and conditions: (a) tryptophan methyl ester
hydrochloride, PyBop, DIPEA, MeCN, 20 h, 84%; (b) 4% LiOH, THF, room temp., 4 h, 79%; (c) BOP-Cl, DIPEA, MeCN, –10 °C to
room temp., 16 h, 75%; (d) Hoveyda–Grubb’s II catalyst (10 mol-%), CH2Cl2, reflux, 56 h, 43%; (e) HSCH2CH2OH, 1,8-diazabicycloun-
dec-7-ene, DMF, room temp., 1 h, 95%; (f) Fmoc-Ser(tBu)-OH, HATU, DIPEA, DMF, room temp., 67%; (g) piperidine, CH2Cl2, 4 h,
room temp., 99%; (h) Boc-Tyr(tBu)-Phe-OH, HATU, DIPEA, DMF, 24 h, 76%; (i) Pd/C, H2, MeOH/EtOAc, room temp., 24 h, 97%;
(j) TFA/TIPS/H2O (95:2.5:2.5), 0 °C, 4 h, 91% for 36 and 86% for 34.
protecting-group removal (95%) or to saturated analogue Table 1. EC50 values of helicokinin analogues.
36 by hydrogenation of 33 (97%) and final acidic deprotec-
tion (91%).
Receptor assay EC50 [μm]
YFSPWG-NH2
0.003
0.24
(helicokinin I)
FSPWG-NH2
(helicokinin I pentapeptide)
YFSPWNMeG-NH2
(NMe-Gly-helicokinin I)
6a
6b
7a
7b
15
28
34
36
Biological Data
Ͼ 100
The complete hexapeptide helicokinin I was used as ref-
erence and not the pentapetide core-sequence lacking the
N-terminal Tyr, which showed already significantly reduced
activity by a factor of approximately 80 (Table 1). We define
“inactive” as an EC50 value higher than 100 μm correspond-
ing to a loss of receptor binding by a factor of more than
30.000 relative to helicokinin I.
Ͼ 100
Ͼ 100
Ͼ 100
25.1
Ͼ 100
Ͼ 100
1.9
12.5
The two diastereoisomeric helicokinin analogues 6a and
6b, in which the amino-pyroglutamate residue serves as a β-
turn inducing dipeptide surrogate, were inactive. Com- sponding (2R,4S)-diastereomer 7a was inactive again, indi-
pound 7b containing the (2S,4R)-4-amino-5-oxopyrrolid- cating a certain influence of the stereochemistry on receptor
ine-2-carboxylic building block as a simple replacement for binding. For comparison, native helicokinin I activates the
Pro expressed only low activity (EC50 25.1 μm). The corre- receptor with an EC50 value of 3.0 nM (Table 1).
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Structural Prerequisites for Receptor Binding of Helicokinin I
Owing to the 1,4-connection piperazinone scaffold 10
For mimetics 7a and 7b again only sequential NOEs were
can be regarded as an even more expanded proline ana- observed, in agreement with a stretched or disordered con-
logue than 4-amino-pyroglutamic acid (APy), forcing the formation with the exception of NOEs between the δ-and
attached C- and N-terminal peptide-chains of hexapeptide NH protons of Trp and the Apy ring in 7b. Compound 7a
15 in opposite directions. Not unexpected, helicokinin I an- mainly displays sequential contacts between the NH of Trp
alogue 15 was found inactive, too (Table 1). The rigid, bicy- to the Apy ring and between the β-protons of Ser and the
clic scaffold in helicokinin derivative 28 prevents any con- NH of the Apy. Both mimetics 7a and 7b do not assume
formational adaptation to the receptor during the binding unique structures. However they clearly adopt preferentially
process, which again results in a loss of activity.
On the other hand, the more flexible macrocyclic helico- tures were detected for either helicokinin I mimetic.
kinin I analogue 34 retains considerable activity in the low In contrast, oxo-piperazine derivative 15 displays a
extended conformations. In particular, no turn-like struc-
micromolar range (EC50 1.9 μm) confirming that it is pos- number of additional medium-range NOEs between β- and
sible to freeze the biologically active conformation needed δ-protons of Phe and the isopropyl group of the oxo-piper-
for receptor binding in a cyclic structure which can be ana- azine. In addition, a high number of NOEs between the
lyzed conveniently with conventional NMR spectroscopic Trp-side chain and the isopropyl group are found. The re-
methods (Table 1). Interestingly, the highly flexible macro- strained MD calculations suggest the presence of two con-
cyclic helicokinin I analogue 36 shows a factor ten reduced formers for 15 in micelles, both of which show a turn-like
activity (EC50 12.5 μm), suggesting a very specific receptor- structure at the C-terminal end and a disordered N-ter-
bound conformation tolerating only minor deviations in minus (Figure 2). The two conformers mainly differ in the
flexibility and rigidity.
conformation of the Ser residue. The replacement of Pro
with the oxopiperazine residue results in changes of the di-
hedral angles in the critical turn region comprising residues
Ser to GlyNH2. In Pro the angle φ is fixed at –75°, whereas
it is –126° on average in the oxo-piperazine moiety of 15. φ
and ψ torsions of Trp are also significantly different from
native helicokinin (Table 2). In fact, the observed structure
Conformational Studies
A higher number of sequential NOEs were identified in
truncated Apy helicokinin analogues 6a and 6b relative to
7a and 7b, but no structure-relevant medium-range or long-
range contacts. Helicokinin analogue 6b displays a signifi-
cant number of NOEs between protons of Apy and the δ
and NH protons of Trp, as well as strong correlations be-
tween Trp and Gly. Helicokinin analogue 6a possesses a
high number of NOEs between protons of Apy and NH-,
α-, β- and δ-protons of Phe. Structure calculations revealed
that both peptides 6a and 6b form linear, flexible peptide
chains with little conformational preferences. Owing to the
trans arrangement of the carboxy- and amino-function in
the 4-Apy residue, formation of a I β-turn is impossible.
Moreover, average distances between the α-protons of Phe
and Trp of 8.0 Å for 6b and 9.2 Å for 6a are so high that
they exclude the presence of a VI β-turn (Figure 1).
Figure 2. Low-energy conformations of helicokinin I analogues
containing monocyclic (15), rigid bicyclic (28), and more flexible
macrocyclic scaffolds (34 and 36).
Table 2. Dihedral angles of the main conformer of helicokinin ana-
logue 15.
φ
ψ
Tyr
143.7 +/–100.4
149.8 +/–7.9
157.5 +/–1.4
–20.9
Phe
–106.8 +/–108.0
–97.1 +/–5.7
–126.0
–150.3, –136.9
–171.5 +/–35.9
Ser
Oxo-piperazine
Trp
Gly
–87.5, 92.0
Figure 1. Superpositions of the lowest-energy conformers of APy
containing helicokinin I analogues.
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does not fit to the I β-turn of helicokinin I nor to any other minal residues Tyr and Phe as seen in the NMR spectro-
standard β-turn. scopic studies. As expected, helicokinin I formed a I β-turn
Bicyclic diketopiperazine derivative 28 exists as a 1:6 involving the residues Ser-Pro-Trp-GlyNH2 as found by
cis/trans-amide mixture about the Ser-pseudoVal peptide NMR spectroscopic measurements in DPC micelles. The I
bond. Several sequential and a few medium-range contacts β-turn structure was found to be most stable in the mem-
were observed in the trans-form, particularly between the brane core (RMSF in simulation type A: 2.79 +/– 0.37 Å;
side-chains of Trp and Val, but also between the aromatic type B: 1.24 +/– 0.14 Å; type C: 0.87 +/– 0.06 Å; errors as
protons of Phe and the Val side-chain. The N-terminus is standard error of the mean). Both, the alkenyl- and alkyl-
more flexible, but the carbonyl group and side-chain of Ser bridges in compounds 34 and 36 (Figure 2) stabilize the
clearly occupy positions different from those in helicoki- loop-structure of the C-terminal residues. However, owing
nin I. Whereas the isopropyl-bearing ring has a similar to its larger flexibility cyclopeptide 36 (containing the alkyl
bulkiness and orientation of the Pro ring, the Cα–Cβ bond bridge) adopts a more stretched overall conformation in the
of Val, shows a different orientation relative to the Cα–Cβ membrane whereas the rather rigid trans-alkenyl bridge in
bond of Pro in helicokinin I. In addition, the Trp side-chain helicokinin analogue 34 induces a loop conformation that
in rigid helicokinin I analogue 28 is directed to an equato- is very similar to the helicokinin I conformation in a mem-
rial position relative to helicokinin I in which the Trp side- brane-mimicking environment.
chain adopts a pseudo-axial position. Nevertheless, the Phe
side-chain is unaffected and together with the Trp side-
chain and the C-terminal Gly amide, all residues that were
were shown to be critical in structure-activity studies oc-
Discussion
cupy similar positions in 28 and in helicokinin I (see Fig-
ures 2 and 3, b).
Despite their enormous biological potential not a single
insect neuropeptide has been developed as an insecticidal
The spectra of macrocyclic analogues 34 and 36 dis- agent. The main reasons stem from unfavorable metabolic
played a reduced number of signals arising from exchange- stability and physicochemical properties, a feature common
mediated line broadening irrespective of temperature and to most peptides. Recently, intense efforts have been made
field and, despite several attempts, we were unable to deter- to establish structure-activity relationships and to identify
mine their structures. Such intermediate exchange between the biologically active conformation of several insect neuro-
different conformers is often observed for medium-sized peptides as a prerequisite for the design of non- or less-
rings. These effects resulted in low intensities for many sig- peptidic analogues with improved drug-like properties.[17]
nals, in particular for the inherently weak amide signals, in Owing to their small size the insect myokinins have been
both TOCSY and NOESY spectra. Therefore, we con- the favorites for most peptidomimetic strategies. The C-ter-
structed de novo models with the build module of YAS- minal pentapeptide constitutes the minimal sequence of the
ARA Structure (version 13.5.10) of helicokinin I as refer- kinins and follows the general formula FX1X2WGamide,
ence and cyclic helicokinin I analogues 34 and 36, after pa- where X1 is Ser, His, Asn, Tyr and X2 is Ser, Pro or Ala.
rameterizing the new building blocks by means of semi-em- However, for full activation of the helicokinin receptor the
pirical quantum mechanics in AM1/NOVA and energy min- hexapeptide sequence of helicokinin I (Tyr-Phe-Ser-Pro-
imizing the conformers in AMBER by using the Trp-GlyNH2, 1) is required.
AMBER03 force field. Several MD simulations were per-
Macrocyclic analogues of the achetakinin core (Phe-Phe-
formed placing the models (A) in water (explicit), (B) into Pro-Trp-GlyNH2) were prepared by the Nachman group to
the core, or (C) into the region of the membrane head- freeze potentially relevant conformations into a small, con-
groups (phosphate region).
formationally restricted structure that can easily be ana-
Analyses of the energies suggest that simulations of all lyzed by conventional NMR spectroscopy.[10] In fact, cy-
compounds reach stable energies within 50 psec. The ener- clo(Phe-Phe-Pro-Trp-Gly-Ala) was found to be highly
gies of helicokinin I and analogs 34 and 36 are similar in active with an EC50 value of 3 nM relative to 0.4 nM for
both membrane regions B and C, but are significantly the linear hexapeptide in a diuretic assay with isolated Mal-
higher in an aqueous environment suggesting that all three pighian tubules from crickets. On the contrary, cyclic helico-
compounds tend to dissolve in different membrane regions kinin I (cyclo[Tyr-Phe-Ser-Pro-Trp-Gly]) analogues of dif-
evenly rather than in water. Only in one of three helicoki- ferent sizes and flexibility tested in our helicokinin receptor
nin I simulations it was slowly (within 0.5 ns) expelled from assay were found to be at least four orders of magnitude
the membrane core and remained stable among phosphates less active than native helicokinin I.[12]
of the lipid head groups (5 ns simulations). Analogues 34
Further analogues of the C-terminal achetakinin I (Phe-
and 36 remained stable in the membrane core (1 ns simula- Tyr-Pro-Trp-GlyNH2) pentapeptide were synthesized by re-
tions). Rout-Mean-Square-Fluctuation analyses (RMSF) placing the cis-amide bond-forming dipeptide Tyr-Pro by
suggest that helicokinin I residues fluctuate stronger in the known type-VI β-turn mimics APy and tetrazole di-
water (2.2–3.8 Å) and slightly stronger among phosphates peptide Phe-ψ[CN]4Ala.[13,30] The resulting achetakinin an-
of the lipid head-groups (0.8–2.2 Å) than in the membrane alogues trans-Arg-Phe-(2R,4S)-APy-Trp-GlyNH2 and Phe-
core (0.7–1.4 Å). In all cases the helicokinin I C-terminal Phe-ψ[CN]4Ala-Trp-GlyNH2 showed a reduced but still re-
core (Ser-Pro-Trp-GlyNH2) is more stable than the N-ter- markable activity in a cricket Malpighian tubule assay with
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Eur. J. Org. Chem. 2014, 2714–2725

Structural Prerequisites for Receptor Binding of Helicokinin I
EC50 values of 7 nM and 340 nM relative to 0.055 nM for scopic studies, which proved trans-APy helicokinin I ana-
the C-terminal pentapeptide of achetakinin I.[10,13] The po- logues 7a and 7b to be flexible comparable with peptides 6a
tencies (EC50) of the two achetakinin analogues tested in and 6b (Figure 1), it can be concluded that a conformation
a leucokinin receptor assay from the arthropod Boophilus unsuitable for receptor binding is the main cause for the
microplus (southern cattle tick) were found to be 1.56 μm loss of biological activity and not simply insufficient length
and 11.08 μm relative to 0.57 μm for the core sequence Phe- of the peptide chain.
Phe-Ser-Trp-GlyNH2.[31] Remarkably, cis-APy-kinin ana-
The 1,3-arrangement of the attached amino acid side-
logue Ac-Arg-Phe-(2S,4S)-APy-Trp-GlyNH2 was found to chains in APy presents another principal difference to the
be less active (EC50 142 nM) than the trans-isomer in the 1,2-connectivity in Pro. To obtain a more detailed under-
Malpighian tubule assay. Although the studies by Nachman standing of the relative spatial orientations of the C-ter-
and Coast favor a C-terminal VI β-turn encompassing the minal Trp-GlyNH2 dipeptide and the N-terminal Tyr-Phe-
residues Phe-Phe-Pro-Trp as a general structural feature of Ser tripeptide for receptor binding we devised the bridged
the myokinins, our NMR spectroscopic measurements of helicokinin I analogue 15 containing a 3-isopropylpipera-
helicokinin I in a membrane-mimicking environment dem- zin-2-one as a substitute for Pro. The 2-oxo-piperazine 15
onstrate a definite preference for a I β-turn including amino provides 1,4-connectivity and thus places the incoming and
acids Ser-Pro-Trp-GlyNH2. The formation of a type-I β- outgoing peptide-chains in opposite directions. The objec-
turn is not unexpected, because this type of β-turn has fre- tive of the isopropyl group is to place lipophilicity in the
quently Pro and Gly residues at positions i+1 and i+3, as region of the pyrrolidine ring of Pro. Our NMR spectro-
in helicokinin I.
scopic data reveal that helicokinin I analogue 15 displays a
The homochiral (2S,4S) and (2R,4R) cis-stereoisomers of bowl-like, open structure absent of a β-turn accompanied
4-amino-5-oxopyrrolidine-2-carboxylic acid are described by complete loss of receptor binding demonstrating that the
as excellent VI β-turn mimetics, whereas the (2R,4S) and spatial orientation of the N- and C-terminal peptide-chains
(2S,4R) trans-stereoisomers (introduced in peptides 6 and indeed plays a crucial role in receptor binding (Figure 2).
7) have been used as conformationally restricted dipeptide
The octahydro-2H-pyrazino[1,2-a]pyrazin-3,6-dione moi-
analogues.[32–34] Inspired by the impressive activity of trans- ety in helicokinin I analogue 28 represents a new bicyclic
(2R,4S)-APy-kinin we introduced the two trans-APy stereo- scaffold designed to fit optimally onto the micelle-bound
isomers into helicokinin I in the same positions (Ser-Pro) as conformation of helicokinin I. The bicyclic scaffold is able
employed by Nachman. To our surprise, both helicokinin I to place the Val and Trp side-chains at positions corre-
analogues 6a (2R,4S) and 6b (2S,4R) lacked activity up to sponding to those of the central amino acids of a type-I β-
an EC50 value of 100 μm. A general prerequisite for any β- turn (Figure 3, b). In fact, related structures have been
turn mimic is its ability to direct the N- and C-terminal shown to induce either type-I β-turns or type-II β-turns de-
peptide chains in same directions. However, the trans-APy pending on the chirality at carbon 9a.[19,20]
orients the attached peptide chains into opposite directions
We attribute the inactivity of 28 to two reasons: First,
and hence it prevents formation of a C-terminal β-turn. In- owing to its bicyclic nature, the pyrazino-pyrazin-dione
deed, our NMR spectroscopic studies indicated flexible, scaffold is considerably more rigid than the β-turn in
stretched conformations for both diastereoisomers 6a and helicokinin I and thus does not allow any conformational
6b devoid of any β-turn (Figure 1).
adaptation during receptor binding. For bradykinin
It is well established that truncated helicokinin I se- (RPPGFSPFR), a mammalian peptide hormone, which is
quences lacking any other amino acid except the N-terminal structurally related to the helicokinin C-terminal region, it
tyrosine lose their activity completely.[17] To address the was previously shown that the C-terminal β-turn differs
question of the minimum chain length APy was introduced somewhat in the receptor- and membrane-bound state.[35]
as a substitute only for Pro. The resulting peptides 7a and Obviously, a certain conformational flexibility is needed for
7b were similar in size to helicokinin I. The (2S,4R)-isomer receptor binding. Second, the backbone conformation of
7b expressed weak activity (EC50 25 μm) and diastereomer Ser in rigid bicyclic analogue 28 differs significantly from
7a was again completely inactive. Based on NMR spectro- helicokinin I. It is well known from the weakly
Figure 3. Superpositions of helicokinin I (green) with (a) macrocycles 34 (purple) and 36 (blue) and (b) bicyclic helicokinin I analogue 28
(yellow).
Eur. J. Org. Chem. 2014, 2714–2725
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2721

O. Zerbe, J. Scherkenbeck et al.
FULL PAPER
active d-Ser analogue of helicokinin I that a correct orienta-
Our results demonstrate that receptor binding of helicok-
tion in this region is important for receptor binding inin I analogues is highly sensitive even on minor structural
(Figure 3, b).[17]
modifications in the C-terminal β-turn.
We showed previously, that end-to-end cyclized ana-
logues of helicokinin I were inactive, which we attributed to
an unsuitable orientation of the critical Phe side-chain in Conclusions
these macrocycles.[12] An alternative to the end-to-end cycli-
Detailed NMR spectroscopic studies and MD calcula-
zation constitutes a backbone cyclization in which an alkyl
or alkenyl spacer is linked to the amide nitrogens. Following
this concept, Verdine applied the ring-closing olefin metath-
esis to stabilize α-helices in peptides and proteins.[36] Back-
bone-cyclizations have also been successfully used to freeze
certain conformations of the pheromone biosynthesis acti-
vating neuropeptide (PBAN) of Heliothis peltigera. How-
ever, in PBAN (Tyr-Phe-Ser-Pro-Arg-LeuNH2) the uncriti-
cal N-terminus was cyclized by linking Tyr and Gly as a
substitute for the native Pro.[37] We used the backbone-cycli-
zation strategy to stabilize the conformation of the inner β-
turn residues Ala (instead of Pro) and Trp by attaching a
bridge to the i+1 and i+3 nitrogens by using the metathesis
reaction, which works well even with difficult amino acids.
In addition, this kind of inner bridge allows the critical Phe
and GlyNH2 residues to retain their conformational free-
dom required for receptor binding. Both helicokinin I ana-
logues 34 and 36 differ considerably in their rigidity. The
rather rigid trans-alkenyl bridge in helicokinin analogue 34
induces a loop conformation which is very similar to the
helicokinin I conformation in micelles, and the saturated
analogue 36 displays a higher degree of conformational
freedom (Figure 3, a).
The cyclic helicokinin I analogue 34 possesses a consider-
able activity in the low micromolar range (EC50 1.99 μm),
which is remarkable because a simple N-methylation of the
amide bond between Gly and Trp causes complete loss of
activity.[17] Obviously, the trans-alkene bridge constrains the
Trp residue in a position that facilitates receptor binding
and preserves the required flexibility of the critical Phe and
GlyNH2 residues. It should be noted that macrocycle 34
represents the first micromolar helicokinin I analogue with
significant structural modifications in the critical C-ter-
minal region.
Notably, the structurally similar alkyl-bridged helicoki-
nin I analogue 36 is less active by a factor of approximately
6. The larger flexibility of the alkyl-spacer in cyclopeptide
36 induces a more disordered conformation. As a conse-
quence, in the lowest-energy conformation the N-terminal
tripeptide-chain of macrocycle 36 adopts a considerably dif-
ferent position relative to helicokinin I or macrocycle 34
(Figure 3, a). Differences can also be seen within the macro-
cycle. For instance, the Trp carbonyl group in helicokinin I
occupies an exo-position, which differs to macrocycles 34
and 36 in which the corresponding carbonyl groups are
found in endo-positions. In addition the double bond in the
bridge of macrocycle 34 adopts a position almost identical
to the Ser carbonyl group in helicokinin I, suggesting that
the double bond is able to mimic the carbonyl group and
thus contributes to the remarkable activity of compound 34
in contrast to saturated macrocycle 36 (Figure 3, a).
tions of several helicokinin I analogues containing scaffolds
of different flexibility and orientation of the N- and C-ter-
minal peptide chains provide a better understanding of the
structural requirements for receptor binding. Scaffolds in-
capable of adopting a β-turn structure at the C-terminus,
as for trans-APy (6 and 7), or inducing inappropriate orien-
tations of the N- and C-terminal peptides, as for piperazine-
2-one 15, immediately result in a complete loss of biological
activity. Additionally, even scaffolds that place the attached
peptide chains in correct orientations need appropriate
plasticity in certain positions. Bicyclic analogue 28 fits well
to the micelle-bound-conformation of helicokinin I, but its
rigidity apparently prevents sufficient adaptation during the
receptor-binding process. It becomes clear now that helico-
kinin I has a very narrow structure-activity window, tolerat-
ing only minor modifications. Nevertheless, as exemplified
with macrocycle 34, helicokinin I analogues with appropri-
ate scaffolds providing specific conformational freedom and
correct orientation of the N- and C-terminal peptides retain
significant biological activity. Macrocycle 34 is the first hel-
icokinin I analogue with modifications in the critical C-ter-
minal region that shows low-micromolar receptor binding.
Thus, peptide 34 provides an excellent starting-point for im-
proved, conformationally fine-tuned helicokinin I deriva-
tives and may pave the future way for non-peptidic insect-
neuropeptide analogues displaying levels of selectivity un-
known for conventional insecticides.
Experimental Section
General: All chemical reagents were purchased from commercial
suppliers and used without purification. Dichloromethane, acetoni-
trile, N,N-dimethylformamide (DMF) and N,N-diisopropylethyl-
amine (DIPEA) were heated at reflux for 1 h with calcium hydride
and distilled. Tetrahydrofuran (THF) was heated at reflux for sev-
eral hours with LiAlH4 and then distilled. The instrumentation
1
used was as follows: H NMR: Bruker Avance 400, Bruker Avance
III 600; 13C NMR: Bruker Avance 400, Bruker Avance III 600;
Infrared (IR) spectra were obtained with
a FTIR: Nicolet
PROTÉGÉ 460 E.S.P. MS: Bruker microTOF; MS (ESI): Varian
IT 500-MS Iontrap; LC: Preparative low-pressure chromatography
was performed with silica gel 60 μm (230–400 mesh, Macherey–Na-
gel) The abbreviations are used for the proton spectra multiplicities
are: s singlet, br. broad, br. s broad singlet, d doublet, dd double
doublet, dt double triplet, t triplet, q quartet, m multiplet. TLC
analyses were performed on silica gel 60 F254 (Merck). Detection
was conducted with UV light (254 nm). Flash column chromatog-
raphy was performed with silica gel (0.063–0.200 mm).
Synthesis of 2-Oxo-piperazine Helicokinin I analogue 15
tert-Butyl (S)-3-Methyl-2-(2-nitrophenylsulfonamido)butanoate: To
a
solution of l-valine tert-butyl ester hydrochloride (2.20 g,
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2714–2725

Structural Prerequisites for Receptor Binding of Helicokinin I
10.49 mmol) and triethylamine (2.9 mL, 20.98 mmol) in CH2Cl2
(15 mL) was added dropwise a solution of 2-nitrophenylsulfonyl
chloride (2.79 g, 12.59 mmol) in CH2Cl2 (20 mL) at 0 °C. The reac-
tion mixture was stirred at room temperature for 20 h and then
diluted with CH2Cl2 (100 mL). The organic phase was washed with
H2O (2ϫ 10 mL) and brine and dried with Na2SO4. The solvent
was removed, and the residue was purified by flash chromatog-
raphy (CyH/EtOAc, 20:1). The product (3.7 g, 98%) was obtained
as a brown solid. Rf = 0.4 (CyH/EtOAc, 15:1). 1H NMR
(600 MHz, CDCl3): δ = 8.07 (m, 1 H), 7.91 (m, 1 H), 7.70 (m, 2
moethane (0.4 mL, 2.4 mmol) in DMF (5 mL) was stirred at 60 °C
for 48 h. After that time, EtOAc (50 mL) was added. The solution
was washed with H2O and brine and dried with Na2SO4. The sol-
vent was evaporated in vacuo. Flash chromatography (EtOAc/CyH,
3:2) provided a solid product (83 mg, 74%). Rf = 0.11 (EtOAc/
CyH, 3:2). 1H NMR (600 MHz, CDCl3): δ = 8.2 (s, 1 H, NH), 7.96
(dd, J = 7.69 Hz, J = 1.55 Hz, 1 H), 7.68 (m, 1 H), 6.67 (m, 1 H),
7.61 (dd, J = 7.5 Hz, J = 1.7 Hz, 1 H), 7.52 (d, J = 7.9 Hz, 1 H),
7.33 (d, J = 8.2 Hz, 1 H), 7.17 (dt, J = 7.5 Hz, J = 1 Hz, 1 H), 7.09
(dt, J = 7.6 Hz, J = 0.8 Hz, 1 H), 7.02 (d, J = 2.2 Hz, 1 H), 5.18
H), 6.05 (d, J = 9.79 Hz, NH), 3.88 (dd, J = 9.82 Hz, J = 5.1 Hz, (dd, J = 11 Hz, J = 5 Hz, 1 H), 4.08 (dd, J = 7.8 Hz, J = 1.2 Hz,
1 H), 2.13 (m, 1 H), 1.4 (s, 9 H), 1.03 (d, J = 6.82 Hz, 3 H), 0.94
(d, J = 6.82 Hz, 3 H) ppm. 13C NMR (150 MHz, CDCl3): δ =
169.7, 147.8, 134.4, 133.4, 132.8, 130.5, 124.5, 82.4, 62.7, 31.6, 31.3,
1 H), 3.77 (dt, J = 3.8 Hz, J = 1 Hz, 1 H), 3.64 (s, 3 H, OCH3),
3.44 (dd, J = 4.9 Hz, J = 0.75 Hz, 1 H), 3.33 (m, 1 H), 3.20 (dd, J
= 15.5 Hz, J = 11.2 Hz, 1 H), 3.14 (dd, J = 7 Hz, J = 4.7 Hz, 2 H),
27.6, 4.0, 17.3 ppm. IR (KBr): ν
= 3340.5 (NH), 1712.4 (C=O) 1.83 (t, J = 7 Hz, 1 H), 0.72 (dd, J = 6.8 Hz, 6 H) ppm. 13C NMR
˜
max
cm–1. MS (ESI): m/z (%) = 257.0 (100), 376.1 (71) [M + NH4]+.
HRMS (ESI): calcd. for C15H22N2O6SNa [M + Na]+ 381.1091;
found 381.1092.
(150 MHz, CDCl3): δ = 170.5, 167.1, 148.1, 136.2, 133.8, 132.9,
131.9, 130.8, 126.9, 124.2, 122.8, 122.2, 118.1, 114.6, 111.4, 110.5,
64.5, 57.5, 52.1 (OCH3), 43.2, 40.6, 31.4, 24.1, 4.5, 4.3 ppm. IR
(KBr): νmax = 3411.3 (NH), 1739.6, 1652.5 (C=O) cm–1. MS (ESI):
˜
(S)-3-Methyl-2-(2-nitrophenylsulfonamido)butanoic Acid: A solution
of tert-butyl (S)-3-methyl-2-(2-nitrophenylsulfonamido)butanoate
(100 mg, 0.28 mmol) in TFA/DCM (1:1, 1.6 mL) was stirred at 0 °C
for 3 h. The solvent was evaporated under reduced pressure, and
the crude product was purified by silica gel column chromatog-
raphy (EtOAc/1% AcOH) to afford a pure product (74 mg, 87%).
m/z (%) = 529.2 [M + H]+, 546.2 [M + NH4]+. HRMS (ESI): calcd.
for C25H28N4O7SNa [M + Na]+ 551.1576; found 551.1571.
(S)-3-(1H-Indol-3-yl)-2-{(S)-3-isopropyl-4-[(2-nitrophenyl)sulfonyl]-
2-oxopiperazin-1-yl}propanoic Acid: A solution of methyl (S)-3-
(1H-indol-3-yl)-2-{(S)-3-isopropyl-4-[(2-nitrophenyl)sulfonyl]-2-ox-
opiperazin-1-yl}propaneoate (1.5 g, 2.84 mmol) in THF (30 mL)
was treated with a 4% LiOH solution (28.38 mmol). After stirring
at room temperature for 3 h, the reaction mixture was acidified
with 1 n HCl to pH ≈ 4 and extracted with EtOAc (3ϫ 100 mL).
The combined organic phases were washed with H2O (2ϫ 10 mL)
and brine solution and dried with Na2SO4. Removal of the solvent
under reduced pressure gave a pure solid (1.42 g, 97%). Rf = 0.22
1
Rf = 0.21 (EtOAc/AcOH, 10:1). H NMR (600 MHz, CDCl3): δ =
8.10 (m, 1 H), 7.94 (m, 1 H), 7.75 (m, 2 H), 6.04 (d, J = 9.67 Hz,
1 H, NH), 4.08 (dd, J = 9.46 Hz, J = 4.78 Hz, 1 H), 2.25 (m, 1 H),
1.06 (d, J = 6.83 Hz, 3 H), 0.96 (d, J = 6.83 Hz, 3 H) ppm. 13C
NMR (150 MHz, CDCl3): δ = 175.6, 147.6, 134, 133.8, 132.9,
130.3, 125.5, 61.7, 31.3, 18.9, 17.2 ppm. IR (KBr): ν
= 3350.2
˜
max
(NH), 1713.9 (C=O) cm–1. MS (ESI): m/z (%) = 320.1 (100) [M +
1
NH4]+, 303.1 (24) [M
+
H]+. HRMS (ESI): calcd. for
(DCM/MeOH, 5:1). H NMR (600 MHz, [D6]DMSO): δ = 10.77
C11H14N2O6SNa [M + Na]+ 325.0470, found 325.0465.
(s, 1 H, NH), 7.96 (dd, J = 7.96 Hz, J = 1.12 Hz, 1 H), 7.91 (dd, J
= 7.95 Hz, J = 1.16 Hz, 1 H), 7.86 (dt, J = 7.6 Hz, J = 1.23 Hz, 1
H), 7.77 (dt, J = 7.85 Hz, J = 1.3 Hz, 1 H), 7.48 (d, J = 7.9 Hz, 1
H), 7.3 (d, J = 8.1 Hz, 1 H), 7.07 (d, J = 2.1 Hz, 1 H), 7.04 (dt, J
= 7.96 Hz, J = 0.87 Hz, 1 H), 6.95 (dt, J = 7.8 Hz, J = 0.74 Hz, 1
H), 5.20 (dd, J = 11.3 Hz, J = 4.7 Hz, 1 H), 3.84 (d, J = 7.65 Hz,
1 H), 3.68 (m, 1 H), 3.35 (m, 1 H), 3.34 (m, 1 H), 3.25 (dd, J =
15.3 Hz, J = 4.5 Hz, 1 H), 3.1 (m, 1 H), 3.08 (m, 1 H), 1.70 (t, J =
7 Hz, 1 H), 0.6 (d, J = 6.8 Hz, 3 H), 0.51 (d, J = 6.7 Hz, 3 H) ppm.
13C NMR (150 MHz, [D6]DMSO): δ = 171.4, 165.0, 147.0, 136.0,
134.9, 132.4, 130.9, 130, 126.7, 124.2, 123.3, 120.8, 118.2, 117.9,
111.2, 109.4, 63.6, 56.0, 40.8, 40.7, 30.8, 23.6, 4.18, 18.9 ppm. IR
Methyl (S)-3-(1H-Indol-3-yl)-2-[(S)-3-methyl-2-(2-nitrophenylsul-
fonamido)butanamido]propanoate (9): To a solution of (S)-3-methyl-
2-(2-nitrophenylsulfonamido)butanoic acid (70 mg, 0.23 mmol)
and l-tryptophan methyl ester (60.65 mg, 0.28 mmol) in DCM
(5 mL) was added TBTU (89.2 mg, 0.28 mmol) followed by ad-
dition of DIPEA (0.11 mL, 0.69 mmol). After stirring at room tem-
perature for 24 h, the solvent was evaporated in vacuo to dryness.
The residue was dissolved in EtOAc (150 mL), washed with H2O
(2ϫ 10 mL) and brine solution and dried with MgSO4. Removal
of the solvent and column chromatography (CyH/EtOAc, 5:2) af-
forded product 9 (106 mg, 91%) as a yellow solid. Rf = 0.15 (CyH/
(KBr): ν
= 3418.9 (NH), 1716.9, 1646.4 (CO) cm–1. MS (ESI):
˜
max
1
m/z (%) = 515.2 (100) [M + H]+, 532.2 (10) [M + NH4]+. HRMS
(ESI): calcd. for C24H26N4O7SNa [M + Na]+ 537.1412; found
537.1414.
EtOAc, 3:2). H NMR (600 MHz, CDCl3): δ = 8.16 (s, 1 H, NH),
7.91 (dd, J = 7.89 Hz, 1.34 Hz, 1 H), 7.75 (dd, J = 8.0 Hz, 1.15 Hz,
1 H), 7.56 (dt, J = 7.7 Hz, J = 1.28 Hz, 1 H), 7.48 (d, J = 7.91 Hz,
1 H), 7.42 (dt, J = 7.79 Hz, J = 1.27 Hz, 1 H), 7.38 (d, J = 8.4 Hz,
1 H), 7.23 (dt, J = 7.24 Hz, J = 1 Hz, 1 H), 7.15 (dt, J = 7.92 Hz,
J = 0.95 Hz, 1 H), 7.03 (d, J = 2.30 Hz, 1 H), 4.67 (m, 1 H), 3.77
(dd, J = 8.45 Hz, J = 5.17 Hz, 1 H), 3.65 (s, 3 H, OCH3), 3.15 (d,
J = 5.87 Hz, 2 H), 2.10 (m, 1 H), 0.85 (d, J = 6.81 Hz, 3 H), 0.77
(d, J = 6.81 Hz) ppm. 13C NMR (150 MHz, CDCl3): δ = 171.7,
169.6, 147, 136.2, 133.8, 133.6, 132.7, 130.2, 127.3, 125.4, 122.9,
122.4, 14.7, 118.4, 111.4, 109.6, 63.0, 52.8 (OCH3), 52.4, 31.25,
(S)-N-(2-Amino-2-oxoethyl)-3-(1H-indol-3-yl)-2-{(S)-3-isopropyl-4-
[(2-nitrophenyl)sulfonyl]-2-oxopiperazin-1-yl}propanamide (10): To a
solution
of
(S)-3-(1H-indol-3-yl)-2-{(S)-3-isopropyl-4-[(2-ni-
trophenyl)sulfonyl]-2-oxopiperazin-1-yl}propanoic acid (730 mg,
1.42 mmol), H-Gly-NH2·HCl (235 mg, 2.13 mmol), TBTU
(592 mg, 1.84 mmol) and HOBt·H2O (282 mg, 1.84 mmol) in DMF
(10 mL) was added DIPEA (0.7 mL, 4.26 mmol) dropwise. The re-
action mixture was stirred at room temperature for 18 h, then di-
luted with EtOAc (250 mL), washed with H2O (3ϫ 15 mL) and
brine (2ϫ 15 mL). Flash chromatography (EtOAc/EtOH, 10:1)
yielded product 10 (580 mg, 72%) as a solid. Rf = 0.32 (DCM/
MeOH, 20:1). 1H NMR (600 MHz, CDCl3): δ = 8.25 (s, 1 H, NH),
27.6, 4.0, 17.1 ppm. IR (KBr): ν
= 3358 (NH), 1735.6, 1669.9
˜
max
(C=O) cm–1. MS (ESI): m/z (%) = 503.2 (100) [M + H]+, 525.2
[M + Na]+. HRMS (ESI): calcd. for C23H26N4O7SNa [M + Na]+
525.1414; found 525.1414.
Methyl (S)-3-(1H-Indol-3-yl)-2-{(S)-3-isopropyl-4-[(2-nitrophenyl) 8.03 (d, J = 8.1 Hz, 1 H), 7.7 (m, 1 H), 7.67 (overlap, 1 H), 7.6 (m,
sulfonyl]-2-oxopiperazin-1-yl}propanoate:
(110 mg, 0.22 mmol), K2CO3 (136 mg, 0.98 mmol) and 1,2-dibro-
A
suspension of
9
1 H), 7.52 (d, J = 7.7 Hz, 1 H), 7.33 (d, J = 8.1 Hz, 1 H), 7.18 (t,
J = 7.1 Hz, 1 H), 7.10 (t, J = 7.1 Hz, 1 H), 7.01 (d, J = 2.2 Hz, 1
Eur. J. Org. Chem. 2014, 2714–2725
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2723

O. Zerbe, J. Scherkenbeck et al.
FULL PAPER
H), 5.33 (q, J = 7.3 Hz, J = 8.9 Hz, 1 H), 4.0 (dd, J = 1.1 Hz, J =
0.8 Hz, 1 H), 3.78–3.73 (m, 2 H), 3.82 (m, 1 H), 3.36 (m, 1 H), 3.32
(m, 2 H), 3.27 (m, 2 H), 1.97–1.92 (m, 1 H), 0.85 (d, J = 6.7 Hz, 3
mide (13): A solution of 12 (170 mg, 226.4 µmol) in DCM (10 mL)
was treated with piperidine (335 µl, 340 µmol) at room temperature
for 5 h. The reaction mixture was concentrated and the residue
H), 0.71 (d, J = 6.8 Hz, 3 H) ppm. 13C NMR (150 MHz, CDCl3): purified by silica gel column chromatography (EtOAc/EtOH, 5:1)
δ = 174.0, 172.4, 169.8, 149.5, 138.2, 135.8, 133.5, 133.4, 132.2,
128.4, 125.9, 124.5, 122.6, 14.9, 14.2, 112.4, 110.6, 65.8, 58.6, 43.3,
to give product 13 (116 mg, 98%). Rf = 0.18 (DCM/MeOH, 3:1).
1H NMR (600 MHz, CD3OD): δ = 7.6 (d, J = 7.9 Hz, 1 H), 7.32
(d, J = 8.1 Hz, 1 H), 7.12 (s, 1 H), 7.08 (dt, J = 0.8 Hz, 7.8 Hz, 1
H), 7.0 (dt, J = 1.1 Hz, 7.1 Hz, 1 H), 5.3 (t, J = 7.7 Hz, 1 H), 4.55
(d, J = 8.8 Hz, 1 H), 3.9 (m, 1 H), 3.84 (overlap, 1 H), 3.84 (overlap,
2 H), 3.51 (m, 1 H), 3.49 (br., 2 H), 3.37 (d, J = 8.4 Hz, 2 H), 3.32
(m, 2 H), 1.76 (m, 1 H), 1.12 (s, 9 H), 0.70 (d, J = 6.9 Hz, 3 H),
0.59 (d, J = 6.7 Hz, 3 H) ppm. 13C NMR (150 MHz, CD3OD): δ
= 174.2, 174.1, 172.8, 170.7, 138.1, 128.6, 124.0, 122.0, 120.0, 114.3,
112.5, 110.8, 74.7, 65.7, 62.3, 59.4, 52.6, 44.3, 43.2, 42.7, 33.1, 27.7,
43.2, 41.7, 32.6, 25.2, 20.02 ppm. IR (KBr): ν
= 3392.0 (NH),
˜
max
1733.5 (CO) cm–1. MS (ESI): m/z (%) = 571.2 (100) [M + H]+,
588.2 (26.6) [M + NH4]+, 593.2 (28) [M + Na]+. HRMS (ESI):
calcd. for C26H30N6O7SNa [M + Na]+ 593.1794; found 593.1789.
(S)-N-(2-Amino-2-oxoethyl)-3-(1H-indol-3-yl)-2-[(S)-3-isopropyl-2-ox-
opiperazin-1-yl]propanamide (11): A suspension of compound 10
(20 mg, 0.04 mmol) and K2CO3 (14.5 mg, 0.11 mmol) in aceto-
nnitrile (3 mL) was treated with thiophenol (8 mg, 0.07 mmol) at
room temperature for 4 h. The reaction mixture was then filtered
through a Celite pad and washed with EtOAc. The filtrate was
concentrated, and the residue was purified by silica gel column
chromatography (DCM/MeOH, 5:1) to afford product 11 (13 mg,
96%) as a solid. Rf = 0.14 (DCM/MeOH, 6:1). 1H NMR
(400 MHz, CD3OD): δ = 7.52 (d, J = 7.8 Hz, 1 H), 7.32 (d, J =
8.0 Hz, 1 H), 7.11 (s, 1 H), 7.08 (dt, J = 1.1 Hz, J = 8.0 Hz, 1 H),
7.01 (dt, J = 1 Hz, J = 8 Hz, 1 H), 5.18 (dd, J = 5.9 Hz, J = 10 Hz,
1 H), 3.83 (dd, J = 17 Hz, J = 4 Hz, 2 H), 3.41 (d, J = 4.3 Hz, 1
H), 3.14 (d, J = 10.8 Hz, 1 H), 3.38 (br., 2 H), 3.29 (overlap, 1 H),
2.94 (dd, J = 2.1 Hz, J = 13 Hz, 1 H), 2.65 (dd, J = 3.7 Hz, J =
11.4 Hz, 1 H), 2.23–2.27 (m, 1 H), 0.87 (d, J = 7.1 Hz, 3 H), 0.54
(d, J = 6.9 Hz, 3 H) ppm. 13C NMR (100 MHz, CD3OD): δ =
174.4, 173.3, 173.1, 138.2, 128.6, 124.2, 122.5, 14.8, 14.2, 112.4,
111.1, 65.3, 60.1, 47.04, 43.3, 42.6, 31.2, 24.6, 4.7, 16.7 ppm. IR
25.3, 4.95 ppm. IR (KBr): νmax = 3416.2 (NH), 1657.1, 1641.1 (CO)
˜
cm–1. MS (ESI): m/z (%) = 529.3 (100) [M + H]+, 551.3 (16.3) [M
+ Na]+. HRMS (ESI): calcd. for C27H41N6O5 [M + H]+ 529.3138;
found 529.3133.
tert-Butyl {(S)-1-{[(S)-1-({(S)-1-[(S)-4-{(S)-1-[(2-Amino-2-oxoethyl)
amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl}-2-isopropyl-3-oxopiper-
azin-1-yl]-3-(tert-butoxy)-1-oxopropan-2-yl}amino)-1-oxo-3-phen-
ylpropan-2-yl]amino}-3-[4-(tert-butoxy)phenyl]-1-oxopropan-2-
yl}carbamate (14): To a solution of amine 13 (70 mg, 132.41 µmol)
and Boc-Tyr(tBu)-Phe-OH (81 mg, 165.52 µmol) in DMF/ACN
(6:4, 8 mL) were added HATU (68 mg, 179 µmol) and DIPEA
(66 µl, 397 µmol). After stirring at room temperature for 24 h, the
solvent was removed under reduced pressure. Flash chromatog-
raphy (EtOAc/EtOH, 20 1) afforded product 14 (115 mg, 87%) as
1
a yellow solid. Rf = 0.19 (EtOAc). H NMR (400 MHz, CD3OD):
(KBr): ν
= 3405.7 (NH), 1668.2, 1623.5 (CO) cm–1. MS (ESI):
˜
max
δ = 7.97 (s, 1 H, NH), 7.62 (d, J = 7.9 Hz, 1 H), 7.32 (d, J = 8.0 Hz,
1 H), 7.2 (m, 2 H), 7.2 (m, 2 H Phe), 7.1 (m, 2 H), 7.1 (s, 1 H),
7.08 (overlap, 1 H), 7.08 (m, 1 H Phe) 7.03 (t, J = 7.1 Hz, 1 H),
6.9 (d, J = 8.3 Hz, 2 H), 5.36 (d, J = 8.25 Hz, 1 H), 4.76 (overlap,
1 H), 4.6 (t, J = 6.9 Hz, 1 H), 4.47 (d, J = 8.4 Hz, 1 H), 4.21 (d, J
= 5 Hz, 1 H), 3.84–3.87 (m, 2 H), 3.82–3.86 (m, 1 H), 3.46–3.47
(m, 1 H), 3.48–3.52 (m, 2 H), 3.45–3.43 (m, 2 H), 3.36–3.38 (m, 2
H), 3.03 (m, 1 H), 2.85 (m, 1 H), 2.95 (m, 1 H), 2.66 (m, 1 H),
1.49–1.53 (m, 1 H), 1.34 (s, 9 H), 1.30 (s, 9 H), 1.11 (s, 9 H), 0.61
(d, J = 6.8 Hz, 3 H), 0.53 (d, J = 6.6 Hz, 3 H) ppm. 13C NMR
(100 MHz, CD3OD): δ = 174.1, 174.0, 172.8, 172.6, 171.8, 170.7,
157.6, 155.3, 138.1, 138, 133.7, 130.8, 130.4, 129.5, 128.6, 127.8,
125.2, 124.5, 122.6, 120, 14.3, 112.5, 110.8, 80.7, 79.5, 75.0 [C(CH3)
3)], 63.1, 62.6, 58.8, 57.4, 55.4, 43.7, 42.9, 43.2, 38.8, 38.5, 33.0,
29.2, 28.7, 27.7 [C(CH3)3], 25.3, 20.2, 4.9 (2 CH3) ppm. IR (KBr):
m/z (%) = 386.2 (100) [M + H]+, 408.2 (45) [M + Na]+. HRMS
(ESI): calcd. for C20H27N5O3Na [M + Na]+ 408.2012; found
408.2006.
(9H-Fluoren-9-yl)methyl {(S)-1-[(S)-4-{(S)-1-[(2-amino-2-oxoethyl)
amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl}-2-isopropyl-3-oxopiper-
azin-1-yl]-3-(tert-butoxy)-1-oxopropan-2-yl}carbamate (12): To
a
stirred solution of Fmoc-l-Ser(tBu)-OH (180 mg, 0.469 mmol) and
amine 11 (145 mg, 0.375 mmol) in DMF (12 mL) were added
HATU (46 mg, 0.516 mmol) and DIPEA (0.23 mL, 1.4 mmol). The
reaction mixture was stirred at room temperature overnight, then
extracted with EtOAc (300 mL), and washed with H2O and brine.
The solvent was removed, and the residue was purified by silica gel
column chromatography (EtOAc/EtOH, 20:1) to yield product 12
(335 mg, 95%) as a solid. Rf = 0.22 (EtOAc/EtOH, 15:1). 1H NMR
(400 MHz, CDCl3): δ = 8.67 (s, 1 H, NH), 8.0 (s, 1 H, NH), 7.74
(d, J = 7.32 Hz, 2 H), 7.55 (d, J = 7.3 Hz, 2 H), 7.55 (d, J = 7.3 Hz,
1 H), 7.38 (t, J = 7.32 Hz, 2 H), 7.3 (m, 1 H), 7.28 (overlap, 2 H),
7.1 (t, J = 7.6 Hz, 1 H), 7.09 (t, J = 7.5 Hz, 1 H), 7.0 (s, 1 H), 6.1
(s, 1 H, NH), 5.9 (s, 1 H, NH), 5.6 (d, J = 8.1 Hz, 1 H, NH), 5.3
(m, 1 H), 4.78 (d, J = 6.9 Hz, 1 H), 4.71 (d, J = 5.4 Hz, 1 H), 4.35
(d, J = 4 Hz, 2 H), 4.2 (t, J = 6.8 Hz, 1 H), 3.96 (m, 1 H), 3.84 (m,
1 H), 3.73 (m, 1 H), 3.54 (m, 1 H), 3.41 (m, 1 H), 3.41 (overlap, 2
H), 3.38 (m, 1 H), 3.37 (m, 1 H), 3.22 (m, 1 H), 1.92 (br., 1 H), 1.1
(s, 9 H), 0.82 (d, J = 6.3 Hz, 3 H), 0.77 (d, J = 6.3 Hz, 3 H) ppm.
13C NMR (100 MHz, CDCl3): δ = 171.5, 171.1, 170.6, 170.6, 169.0,
155.0, 143.6, 141.3, 136.2, 127.7, 127.0, 125, 122.9, 122.6, 114.9,
114.7, 110.0, 74.0, 67.1, 63.0, 60.7, 57.4, 51.1, 47.1, 43.0, 42.7, 41.6,
ν
max = 3834.2 (NH), 1655.2 (CO) cm–1. MS (ESI): m/z (%) = 995.6
˜
(39.4) [M + H]+. HRMS (ESI): calcd. for C54H75N8O10 [M + H]+
995.5606; found 995.5601.
(S)-2-Amino-N-[(S)-1-({(S)-1-[(S)-4-{(S)-1-[(2-amino-2-oxoethyl)
amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl}-2-isopropyl-3-oxopiper-
azin-1-yl]-3-hydroxy-1-oxopropan-2-yl}amino)-1-oxo-3-phenylpro-
pan-2-yl]-3-(4-hydroxyphenyl)propanamide (15): Hexapeptide 14
(50 mg, 50.24 µmol) was treated with TFA/TIPS/H2O (95:2.5:2.5,
2 mL) at 0 °C for 2 h. After removal of the solvent, the residue
was dissolved in ACN/H2O (1:1, 2 mL) and lyophilized to provide
product 15 (35.5 mg, 90%) as a white powder. 1H NMR (400 MHz,
[D6]DMSO): δ = 10.8 (d, J = 2 Hz, 1 H, NH), 9.2 (br. s, 1 H, NH),
8.4 (d, J = 8 Hz, 1 H, NH), 8.2 (t, J = 5.7 Hz, 1 H, NH), 8.1 (br.
s, 1 H, NH), 7.27 (s, 1 H, NH), 7.64 (d, J = 7.8 Hz, 1 H),7.31 (d,
J = 8.0 Hz), 7.4–7.13 (m, 6 H), 7.05 (t, J = 8.0 Hz), 6.9 (overlap, 1
H), 6.96 (d, J = 8.5 Hz, 2 H), 6.66 (d, J = 8.5 Hz, 2 H), 5.4 (dd, J
= 4.9 Hz, 11 Hz, 1 H), 4.7 (q, J = 6.7 Hz, 1 H), 4.6 (q, J = 8 Hz,
32.2, 27.3, 24.1, 4.6 ppm. IR (KBr): ν
= 3428.0 (NH), 1656.9
˜
max
(CO) cm–1. MS (ESI): m/z (%) = 751.4 (100) [M + H]+, 769.4 (4)
[M + NH4]+, 773.4 (10.6) [M + Na]+. HRMS (ESI): calcd. for
C42H50N6O7Na [M + Na]+ 773.3639; found 773.3633.
(S)-N-(2-Amino-2-oxoethyl)-2-{(S)-4-[(S)-2-amino-3-(tert-butoxy)pro- 1 H), 4.3 (d, J = 8.5 Hz, 1 H), 3.8, 3.4 (m, 2 H, NCH2CH2N), 3.66
panoyl]-3-isopropyl-2-oxopiperazin-1-yl}-3-(1H-indol-3-yl)propana- (d, J = 5.8 Hz, 1 H), 3.58 (m, 1 H), 3.50 (m, 1 H), 3.5, 3.38 (m, 2
2724
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2714–2725

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H, NCH2CH2N), 3.41 (m, 1 H), 3.3, 3.12 (m, 2 H), 2.92 (m, 1 H),
2.77 (m, 1 H), 2.73 (m, 1 H), 2.45 (m, 1 H), 1.44 (m, 1 H), 0.53,
0.40 (2 CH3) ppm. 13C NMR (100 MHz, [D6]DMSO): δ = 155.9,
130.1, 127.0, 114.9, 137.1, 129.2, 127.8, 126.2, 136.1, 126.8, 123.4,
120.7, 118.5, 118.1, 61.0, 60.3, 55.6, 55.4, 52.8, 50.9, 41.8, 41.2
(NCH2CH2N), 40.8 (NCH2CH2N), 38.5, 37.7, 30.9, 24.2, 4.3, 4.2
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= 3423.41 (NH), 1651.7 (CO) cm–1.
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˜
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Acknowledgments
The authors thank the Vietnam International Education Develop-
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Received: November 27, 2013
Published Online: February 26, 2014
Eur. J. Org. Chem. 2014, 2714–2725
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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