Fluorescent analogues of the insect neuropeptide helicokinin I: Synthesis, photophysical characterization and biological activity

Article abstract of DOI:10.2174/092986610790963672

In insects numerous physiological processes are regulated by neuropeptides. Two fluorescent analogues of the amino acids tryptophan and tyrosine were synthesized and incorporated in the diuretic neuropeptide helicokinin I from the moth Heliothis zea. By fluorescence emission measurements it was shown that both fluorescent helicokinin I analogues react sensitive on the dielectricity of their microenvironment. A helicokinin I analogue containing the fluorescent tryptophan mimic β-[6'-(N,N-dimethyl)-amino-2'-naphthoyl]alanine (Ald) was shown to bind to dodecylphosphocholine (DPC) micelles by the Ald residue. A membrane binding model for helicokinin I is proposed based on data from related mammalian and insect-neuropeptides.

Full text of DOI:10.2174/092986610790963672

Protein & Peptide Letters, 2010, 17, 431-436
431
Fluorescent Analogues of the Insect Neuropeptide Helicokinin I: Synthesis,
Photophysical Characterization and Biological Activity
Heru Chena, Jürgen Scherkenbecka,*, Tino Zdobinskya and Horst Antonicekb
aFachgruppe Chemie, Bergische Universität Wuppertal, Gaußstraße 20, D-42119 Wuppertal, Germany; bBayer
CropScience AG, Alfred-Nobel-Straße 50, D-40789 Monheim, Germany
Abstract: In insects numerous physiological processes are regulated by neuropeptides. Two fluorescent analogues of the
amino acids tryptophan and tyrosine were synthesized and incorporated in the diuretic neuropeptide helicokinin I from the
moth Heliothis zea. By fluorescence emission measurements it was shown that both fluorescent helicokinin I analogues
react sensitive on the dielectricity of their microenvironment. A helicokinin I analogue containing the fluorescent trypto-
phan mimic ꢀ-[6’-(N,N-dimethyl)-amino-2’-naphthoyl]alanine (Ald) was shown to bind to dodecylphosphocholine (DPC)
micelles by the Ald residue. A membrane binding model for helicokinin I is proposed based on data from related mam-
malian and insect-neuropeptides.
Keywords: Insect neuropeptides, helicokinin, fluorescent amino acids, micelles, peptides.
INTRODUCTION
the fluorescence properties are influenced by the polarity of
the microenvironment [10,11]. Neuropeptides react very
sensitive on structural modifications at the N- and in particu-
lar at the C-terminus. Thus, the attachment of a linker cou-
pled fluorescent dye is expected to result in a complete loss
of biological activity. A more promising strategy appears to
be the replacement of a natural amino acid by a structurally
related fluorescent amino acid analogue [12]. Examples of
small extrinsic fluorophores are the 1-dimethylamino-
naphthalene-5-sulfonyl (dansyl) group, the anthraniloyl
group or the 6-dimethyl-amino-2-acyl-naphthalene (DAN)
group. DAN is an environmentally sensitive probe and has
recently been incorporated into the side chain of L-alanine,
resulting in the fluorescent amino acid aladan (Ald, 1)
[13,14]. Both the fluorescence quantum yield and the fluo-
rescence emission maximum of aladan (1) are exceptionally
sensitive to the polarity of its environment, ranging from
409 nm in heptane to 542 nm in water. Another fluorescent
amino acid (Fig. 1), carrying the ꢀ-(4’-hydroxy-2’-
benzoyl)alanine (Alb, 2) residue in the side-chain has re-
cently been described, unfortunately without any details on
its dielectric sensitivity [14]. Structure activity studies of
helicokinin I revealed Tyr1 the most flexible residue with
respect to structural modifications [15]. Thus Alb (2) is sup-
posed to be a valuable replacement for Tyr1 and Ald (1) for
the critical Trp5 residue in helicokinin I. Following the
‘Schwyzer model’ lipophilic peptides interact with the cell
membrane prior to receptor-binding [16-18]. Thus, dielectric
sensitive fluorescence probes such as the helicokinin ana-
logues 8 and 9 should be useful to identify the amino acids,
which anchor a peptide in the cell membrane.
Neuropeptides comprise a versatile class of extracellular
messenger molecules that function as chemical commu-
nication signals between the cells of an organism. In insects,
essential physiological processes, such as the embryonal-
and post-embryonal development, the ion-homeostasis, the
osmoregulation, and muscle activity are regulated by neu-
ropeptides and peptide hormones [1,2]. Peptide messengers
exert their biological functions via specific signal-
transduction membrane receptors [3,4]. Thus, the receptors
of insect-neuropeptides represent promising targets for a
novel generation of selective and environmentally beneficial
insecticides. Agonists or antagonists, acting on these recep-
tors may interfere with the development, growth, behaviour
and homeostasis of insects [5,6]. The regulation of water
balance is a crucial aspect of homeostasis in terrestrial in-
sects involving excretion of excess water via the Malphigian
tubules and resorption in posterior regions of the hindgut.
The process of urine production has been shown to be under
endocrine control, primarily by corticotrophin-releasing-
factor (CRF) related peptides and kinins, also called leucoki-
nins or myokinins [7,8]. In particular the helicokinins I-III
from the moth H. zea have been shown to stimulate fluid
secretion in isolated Malphigian tubules in vitro and to in-
crease mortality in vivo following injection of helicokinin I
(Tyr-Phe-Ser-Pro-Trp-Gly-NH2) into the haemolymph of
Heliothis virescens larvae [9]. Their short sequences and
potent diuretic effects render the kinins prime candidates for
the peptidomimetic design of metabolically stable neuropep-
tide based insect control agents.
Fluorescent probes positioned in the pharmacophoric
domain of a peptide can provide important insights into the
molecular basis of membrane and receptor binding because
O
NH2
O
NH2
COOH
HO
COOH
Alb (2)
Ald (1)
N
*Address correspondence to this author at the Fachgruppe Chemie, Bergi-
sche Universität Wuppertal, Gaußstraße 20, D-42119 Wuppertal, Germany;
E-mail: scherkenbeck@uni-wuppertal.de
Figure 1. Fluorescent analogues of tryptophan and tyrosine.
0929-8665/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

432 Protein & Peptide Letters, 2010, Vol. 17, No. 4
Chen et al.
MATERIALS AND METHODS
General
tor from Lymnea stagnalis [21, 22]. The helicokinin receptor
(HKR) was functionally expressed in a CHO line. The acti-
vation of the HKR was analyzed in living cells by measuring
the induced calcium ion flux in the cytosol after activation of
the second messenger cascade via G-proteins [23, 24]. Cal-
cium ion dyes were obtained from Molecular Devices and
were used according to the supplier’s protocol. Cells were
plated in 384-well plates (4x103 cells per well) and incubated
over night (37oC, 5% CO2). Cells were removed from the
incubator, allowed to reach room temperature over the
course of 10 min, and then washed with 50 ꢀl of HBSS. Sub-
sequently, the medium was replaced with 50 ꢀl of calcium
ion dye solution in HBSS, and cells were loaded for 1 h in
the dark. Then, the plates were read on a Flexstation fluores-
cence plate reader (Molecular Devices). Excitation and emis-
sion wavelengths were set to 485 nm and 525 nm, respec-
tively. Measurements were made every 2.7 s intervals for
100 s. Basal fluorescence was determined for 15–30 s, fol-
lowed by addition of 25 ꢀl test compounds (to assess agonist
activity). Helicokinins I or II were used as standards and
were tested in four replicates once for evaluating EC50 val-
ues. All test compounds were measured twice in ten dilution
steps from 0.5 nM to 10 ꢀM. EC50 values with test com-
pounds and peptides were calculated after concentration-
dependent induction of calcium flux analyzed in a Flexsta-
tion (Molecular Devices). Finally, EC50-values were calcu-
lated as Fmax (maximal agonist signal) minus Fmin (base-
line) according to Softmax-Pro 5.0 software (Molecular De-
vices).
Solvents, reagents, amino acids (Bachem) and TentaGel
S RAM resin (Fluka) were purchased from commercial
sources. Dodecylphosphoryl-choline was synthesized accor-
ding to literature [19]. The concentrations of SDS and DPC
were either 0.4 or 0.8 wt% in 20 nmol L-1 Hepes-HCl buffer,
pH 6.5 [20]. HPLC was performed on a Varian 500 IonTrap
LC-ESI-MS system (column: 5 ꢀm RP18). For preparative
HPLC separations a Gilson HPLC system with UV detection
(column: 2.3x25 cm, 10 ꢀm RP18) was used. 1H and
13C NMR spectra were recorded at 25°C on a Bruker
ARX400 (400 MHz) spectrometer using tetramethylsilane as
internal standard. Fluorescence emission spectra were re-
corded on a Varian Fluorescence Spectrometer (parameter:
excitation slit 5 nm; emission slit 5 nm; excitation filter in
auto state; emission filter open). Water was deionized and
distilled prior to use. Hepes sodium salt was purchased from
AppliChem (Germany) and used directly. All fluorescence
measurements were performed with 1ꢀ10-6 mol L-1 of
[Alb1]helicokinin (8) and [Ald5]helicokinin I (9). Abbrevia-
tions: Alb, ꢀ-(4’-hydroxy-2’-benzoyl)alanine; Ald, ꢀ-[6’-
(N,N-dimethyl)-amino-2’-naphthoyl]alanine; CRF, cortico-
trophin-releasing-factor; dansyl, 1-dimethyl-amino-naph-
thalene-5-sulfonyl; DAN, 6-dimethyl-amino-2-acyl-naph-
thalene; DCM, dichloromethane; DPC, dodecylphosphoryl-
choline; DIEA, diisopropylethylamine; DMF, dimethylfor-
mamide; DMSO, dimethylsulfoxide; Fmoc, fluorenyl-
methoxycarbonyl; PyBOP, benzotriazol-1-yl-oxytripyrroli-
dinophosphonium-hexafluorophosphate; THF, tetrahydro-
furane; TFA, trifluoro acetic acid; TIPS, triisopropylsilane.
RESULTS
The synthesis of Alb 2 (Scheme 1) followed a route simi-
lar to the literature procedure used for the preparation of Ald
1 [13,14]. The overall yields and ee values strongly de-
pended on the careful control of the reaction conditions.
With cinchonidinium bromide as a chiral phase transfer cata-
lyst e.e. values of 90% for Alb (2) and 92% for Ald (1) were
obtained after a reaction time of 48 h at -90°C [25].
General Procedure for the Synthesis of Helicokinin I De-
rivatives
TentaGel S RAM resin (0.565 g, 0.13 mmol) was filled
in a syringe equipped with a filter and a stopper. The resin
was dispensed twice with 5 ml of a 30% piperidine solution
in DMF, shaken for 30 min each and then washed with dry
DMF. 2 equivalents amino acid and coupling reagent (Py-
BOP / HOBt / DIPEA) were used for Fmoc-Gly-OH, Fmoc-
Trp-OH, and Fmoc-Pro-OH and 3 equivalents for Fmoc-
Ser(tBu)-OH, Fmoc-Phe-OH, and Fmoc-Tyr(tBu)-OH.
Quantitative removal of the Fmoc protecting group was
achieved with two treatments (15 min and 5 min) of the resin
with a solution of piperidine in DMF (30%). The peptides
were cleaved from the resin by adding a TFA / TIPS solution
and shaking for 2 h. After evaporation of the TFA / TIPS
reagent the residue was purified by reversed-phase prepara-
tive HPLC (flow rate: 3.6 mL min-1, gradient: 10% CH3CN
to 40% CH3CN within 40 min). The product containing frac-
tions were lyophilized. [Alb1]helicokinin I (8): LC-MS pu-
rity: 91% (220 nm); HR-ESI-MS: calcd for C40H46N8O9:
782.3461; found for [M+H]+: 783.3471. [Ald5]helicokinin I
(9): LC-MS: purity 92% (220 nm); HR-ESI-MS: calcd for
C44H52N8O9: 836.3930; found for [M+H]+: 837.3955.
The two fluorescent peptides Alb-Phe-Ser-Pro-Trp-Gly-
NH2 8 ([Alb1]helicokinin I) and Tyr-Phe-Ser-Pro-Ald-Gly-
NH2 9 ([Ald5]helicokinin I) were prepared by solid phase
synthesis based on a Fmoc/tButyl protecting group strategy.
Twofold excesses of Fmoc amino acids and coupling rea-
gents were employed for the amino acids Fmoc-Gly-OH,
Fmoc-Trp-OH, and Fmoc-Pro-OH. Threefold excesses were
used for the amino acids Fmoc-Ser(tBu)-OH, Fmoc-Phe-OH,
and Fmoc-Tyr(tBu)-OH. Coupling times with PyBOP /
DIEA were usually 2 h, depending on the monitoring results.
Cleavage of the protecting groups and removal from the
resin was accomplished in one step with the TFA / TIPS
reagent (2 h, rt). The crude peptides were purified by prepa-
rative reverse-phase HPLC and characterized by LC-MS-ESI
and HR-MS.
The maximum emission wavelength and fluorescence
intensity of the peptides [Alb1]helicokinin I (8) and
[Ald5]helicokinin I (9) were measured in different solvents
(Table 1). As expected, the fluorescence intensity was the
lowest and the maximum emission wavelength (ꢁem) was the
largest for both peptides 8 and 9 in water and Hepes-buffer
(Table 1). With decreasing polarity of the solvent the fluo-
Receptor Assay
The helicokinin receptor of Heliothis virescens was
cloned from a Malpighian tubule cDNA library and showed
about 50% homology (amino acids) to the leucokinin recep-

Fluorescent Analogues of the Insect Neuropeptide Helicokinin I
Protein & Peptide Letters, 2010, Vol. 17, No. 4 433
O
O
b
I
a
+
I2
86%
53%
MeO
MeO
3
4
Ph
O
O
N
NH2
CO2H
Ph
CO2tBu
c
70%
MeO
HO
6
5
O
NHFmoc
CO2H
d
HO
49%
HO
7
t
Scheme 1. Synthesis of Alb (1). (a) LiN(SiMe3)2 in dried THF, -78°C, then addition of I2. (b) Ph2CNCH2CO2 Bu, cinchonidinium bromide,
CsOHꢀH2O, DCM, -90°C, 48 h. (c) 6 mol L-1 HI/HOAc, reflux, 6 h. (d) Fmoc-OSu, NaHCO3, dioxane/H2O (1:1), rt, 24 h.
Table 1. Dielectric constant (ꢁ), Solvents, Fluorescence Intensity (a.u.) and Fluorescence Emission Maximum (ꢂem) of
[Alb1]helicokinin I (8) and [Ald5]helicokinin I (9). [Alb1]helicokinin I (8): ꢂex = 280 nm for all solvents except for toluene (240
nm). Concentration of the peptide: 1ꢃ10-6 mol L-1. [Ald5]helicokinin I (9): ꢂex = 350 nm for all solvents. Concentration of the pep-
tide: 1ꢃ10-6 mol L-1.
[Alb1]helicokinin I (8)
[Ald5]helicokinin I (9)
Solvents
ꢁ
ꢂ
em (nm)
a.u.
ꢂ
em (nm)
a.u.
toluene
ethyl acetate
1-octanol
2.38
6.02
17.5
32.7
36.7
46.7
80.2
ꢄ80.2
295
344
299
346
339
349
358
358
17
64
37
28
65
93
18
8
457
463
482
506
472
475
532
532
743
945
755
515
840
705
88
methanol
DMF
DMSO
H2O
20 mM Hepes-HCl (pH 6.5)
36
rescence intensity increased (except toluene) and the maxi-
mum emission wavelength was blue-shifted. When the sol-
vent was changed from DMF to DMSO a red-shift of 10 nm
was observed for [Alb1]helicokinin (8) compared to only
3 nm for [Ald5]helicokinin I (9). [Alb1]helicokinin (8)
showed a higher fluorescence intensity in DMSO whereas
almost no differences were found for [Ald5]helicokinin I (9)
in DMSO and DMF. The aprotic toluene and ethyl acetate
caused a significant blue-shift of the maximum emission
wavelength for both peptides 8 and 9, respectively.
micelles and 0.8 wt% DPC micelles (Fig. 2). Only a weak
fluorescence was found for [Alb1]helicokinin 8 with a broad
maximum emission wavelength identical to that in Hepes
buffer.
All three helicokinins (Table 2) activated the helicokinin
receptor in the low nanomolar range (EC50: 2-5 nM). The
fluorescent analogue [Alb1]helicokinin 8 was found to be
only slightly less active (EC50: 32 nM) than helicokinin I.
The replacement of the critical tryptophan residue by aladan
(9) resulted in an almost complete loss of receptor activity.
Measurements of the steady-state fluorescence of
[Ald5]helicokinin 9 in 20 mM Hepes-HCl buffer (pH 6.5),
0.4 wt% DPC in 20 mM Hepes-HCl (pH 6.5), and 0.8 wt%
DPC in 20 mM Hepes-HCl (pH 6.5) revealed a blue-shift of
the fluorescence emission maximum wavelength from 533
nm in buffer solution to around 490 nm in 0.4 wt% DPC
DISCUSSION
In addition to NMR studies peptide analogues containing
dielectric sensitive fluorescent probes can provide important
information on peptide-lipid interactions and may be useful
tools to identify peptide-receptors in living cells. Since in-

434 Protein & Peptide Letters, 2010, Vol. 17, No. 4
Chen et al.
sect-neuropeptides are very sensitive to structural modifica-
tions, the conventional coupling of a fluorescent dye to the
N- or C-terminus is expected to produce inactive peptide
analogues. A different and less common strategy is based on
fluorescent amino acid mimics. For helicokinin I it has been
shown that Tyr1 and to some extent also Trp5 can be modi-
fied without immediate loss of activity [15]. In sharp contrast
Phe2 and Gly6 tolerate almost no structural modifications
[15]. Thus, the fluorescent insect neuropeptide analogues
[Alb1]helicokinin I (8) and [Ald5]helicokinin I (9) have been
synthesized and their fluorescence properties were studied in
solvents of different polarities as well as in phospholipid-
micelles as membrane mimics.
[Alb1]helicokinin I (8) fluorescence reflects the contributions
of both the tryptophan- and the Alb-residue, which is a con-
siderably stronger chromophore than tyrosine. Compared to
helicokinin I the fluorescence sensitivity of [Alb1]helicokinin
I (8) is higher and the fluorescence shifts in solvents of dif-
ferent polarities are more pronounced.
A significant red-shift of 10 nm for [Alb1]helicokinin I
(8) compared to only 3 nm for [Ald5]helicokinin I (9) is ob-
served when the solvent was changed from DMF to DMSO.
For tyrosine and some of its derivatives it has been shown
that a very strong interaction of DMSO with the phenolic
hydroxy group causes substantial changes in the fluorescence
emission wavelength as well as in the fluorescence intensity.
The red-shift of the maximum emission wavelength in
[Alb1]helicokinin I (8) reflects the higher hydrogen-bond
acceptor strength of DMSO compared to DMF. On the other
side the dimethylamino group in the fluorophore 1 serves as
a proton acceptor and thus no difference is expected in the
interaction with the proton acceptors DMSO and DMF for
[Ald5]helicokinin I (9) [26].
Steady-state fluorescence measurements of [Ald5]heli-
cokinin I (9) in DPC micelles revealed a significant blue-
shift of 43 nm compared to a Hepes-HCl buffer solution.
With regard to pure solvents this blue-shift corresponds to 1-
octanol, which is another well-known membrane model.
These results demonstrate that the fluorophoric side chain of
[Ald5]helicokinin 9 sticks in the lipophilic core of the DPC
micelles close to the phosphate head group.
A weak fluorescence detected for [Alb1]helicokinin I (8)
in DPC is identical with the maximum emission wavelength
of [Alb1]helicokinin I (8) in water or Hepes buffer. For a
membrane embedded Alb residue a significantly higher fluo-
rescence intensity and a blue-shift to around 300 nm similar
to octanol would be expected. The low fluorescence of
[Alb1]helicokinin I (8) in DPC micelles may be caused by a
partial quenching of the Alb fluorescence by phosphate
groups in close proximity to the phenolic hydroxy group,
according to a mechanism well known for tyrosine [27].
These findings suggest the fluorophoric side-chain of
[Alb1]helicokinin 8 to be exposed to the aqueous phase close
to the zwitterionic headgroups of the DPC micelles.
Figure 2. Fluorescence emission spectra of [Ald5]helicokinin (9) in
DPC micelles (ꢀex = 350 nm. Concentration of the peptide: 1ꢁ10-6
mol L-1).
Table 2. Receptor Assay Data of Helicokinins I-III and Fluo-
rescent Analogues 8 and 9
Sequence
EC50
(nM)
Helicokinin I
Helicokinin II
Helicokinin III
Tyr-Phe-Ser-Pro-Trp-Gly-NH2
2
5
5
Val-Arg-Phe-Ser-Pro-Trp-Gly-NH2
The presence of a C-terminal ꢂ-turn in the insect kinins
and its significance for receptor-binding has been discussed
since a couple of years. For the leucokinin active core Asp1-
Pro2-Gly3-Phe4-Ser5-Ser6-Trp7-Gly8-NH2 a ꢂ-turn encom-
passing the C-terminal amino acids Phe4 to Trp7 was ob-
served by molecular dynamics simulations. The essential
Phe4 and Trp7 side-chains were shown to be located in close
proximity on the same side of the turn [28]. Further conclu-
sions can be drawn from a comparison of helicokinin I with
the mammalian peptide hormone bradykinin (RPPG
FSPFRamide), which binds to an evolutionarily related
GPCR. Bradykinin and helicokinin I share the amino acids
Phe, Ser, Pro in the C-terminal positions 5, 4, 3 and both
carry lipophilic residues (Trp and Phe) in position 2 (counted
from the C-terminus for better comparison). The backbone-
structure of bradykinin has recently been elucidated in the
receptor-bound state and in empty DDM micelles by solid-
state NMR spectroscopy [29]. As for membrane-bound heli-
cokinin I the torsion angles of receptor-bound bradykinin are
Lys-Val-Lys-Phe-Ser-Ala-Trp-Gly-
NH2
Alb-helicokinin (8)
Ald-helicokinin (9)
Alb-Phe-Ser-Pro-Trp-Gly-NH2
Tyr-Phe-Ser-Pro-Ald-Gly-NH2
32
>10000
A profound blue-shift of the maximum emission wave-
length and increase in fluorescence intensity (except toluene)
is observed for both helicokinin analogues with increasing
lipophilicity of the solvent. Noteworthy is the high dielectri-
cal sensitivity of [Ald5]helicokinin I (9) with blue-shifts of
more than 100 nm between a water and a toluene environ-
ment. In case of [Ald5]helicokinin I (9) the contribution of
the tyrosine fluorescence emission is expected to be minimal
at the wavelengths observed. Additionally the tyrosine emis-
sion maximum is relatively insensitive to solvent polarity
changes. In contrast to [Ald5]helicokinin
I (9) the

Fluorescent Analogues of the Insect Neuropeptide Helicokinin I
Protein & Peptide Letters, 2010, Vol. 17, No. 4 435
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Figure 3. Model for the membrane-bound conformation of helicok-
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In conclusion, a qualitative membrane-binding model of
the insect neuropeptide helicokinin I has been suggested by
fluorescence measurements of neuropeptide analogues con-
taining dielectrically sensitive amino acid mimetics. Future
work will focus on biologically highly active fluorescent
tryptophan analogues as probes for the identification of heli-
cokinin receptors in Malpighian tubule cells.
ACKNOWLEDGEMENTS
This work was supported by a grant from the Heinrich-
Hertz foundation and the Deutsche Forschungsgemeinschaft
(SCHE 301/2-2)
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Received: July 21, 2009
Revised: October 15, 2009
Accepted: October 15, 2009