Oostatic peptides containing D-amino acids: Synthesis, oostatic activity, degradation, accumulation in ovaries and NMR study

Article abstract of DOI:10.1007/s00726-011-0882-6

Analogs of the H-Tyr-Asp-Pro-Ala-Pro-OH pentapeptide with D-amino acid residues either in differing or in all of the positions of the sequences were prepared and their oostatic potency was compared with that of the parent pentapeptide. The D-amino acid residue containing analogs exhibited an equal or even higher oostatic effect in the flesh fly Neobellieria bullata than the parent peptide. Contrary to the rapid incorporation of radioactivity from the labeled H-Tyr-Asp-[³H]Pro-Ala-Pro-OH pentapeptide into the ovaries of N. bullata in vitro, the radioactivity incorporation from the labeled pentapeptides with either D-aspartic acid or D-alanine was significantly delayed. As compared to the parent pentapeptide, also the degradation of both the D-amino acid-containing analogs mentioned above proceeded at a significantly lower rate. The decreased intake of radioactivity, the lower degradation and finally also the high oostatic effect may be ascribed to the decreased enzymatic degradation of the peptide bonds neighboring the D-amino acid residues in the corresponding peptides. The introduction of the non-coded D-amino acids thus enhances the oostatic effect in N. bullata owing to the prolonged half-life of the corresponding pentapeptides, which can thus affect more ovarian cells. Springer-Verlag 2011.

Full text of DOI:10.1007/s00726-011-0882-6

Amino Acids (2012) 42:1715–1725
DOI 10.1007/s00726-011-0882-6
ORIGINAL ARTICLE
Oostatic peptides containing D-amino acids: synthesis, oostatic
activity, degradation, accumulation in ovaries and NMR study
•
Jan Hlavacek Richard Tykva Josef Holık
•
•
´ˇ
Blanka Bennettova Milos Budesınsky
´
•
•
´
Vera Vlasakova Bohuslav Cerny Jirina Slaninova
ˇ
ˇˇ´
´
ˇ
•
•
ˇ
´
´
´
ˇ
´
Received: 21 December 2010 / Accepted: 4 March 2011 / Published online: 18 March 2011
Ó Springer-Verlag 2011
Abstract
Analogs of the H-Tyr-Asp-Pro-Ala-Pro-OH
peptides. The introduction of the non-coded D-amino acids
thus enhances the oostatic effect in N. bullata owing to the
prolonged half-life of the corresponding pentapeptides,
which can thus affect more ovarian cells.
pentapeptide with D-amino acid residues either in differing
or in all of the positions of the sequences were prepared
and their oostatic potency was compared with that of the
parent pentapeptide. The D-amino acid residue containing
analogs exhibited an equal or even higher oostatic effect in
the flesh fly Neobellieria bullata than the parent peptide.
Contrary to the rapid incorporation of radioactivity from
the labeled H-Tyr-Asp-[3H]Pro-Ala-Pro-OH pentapeptide
into the ovaries of N. bullata in vitro, the radioactivity
incorporation from the labeled pentapeptides with either
D-aspartic acid or D-alanine was significantly delayed. As
compared to the parent pentapeptide, also the degradation
of both the D-amino acid-containing analogs mentioned
above proceeded at a significantly lower rate. The
decreased intake of radioactivity, the lower degradation
and finally also the high oostatic effect may be ascribed to
the decreased enzymatic degradation of the peptide bonds
neighboring the D-amino acid residues in the corresponding
Keywords D-amino acids Á Oostatic peptide synthesis Á
3H labeling Á Oostatic activity in Neobellieria bullata Á
3H incorporation Á Peptide degradation Á NMR study
Abbreviations
AA
Amino acid analysis
Acetic acid
AcOH
ACN
APT
Acetonitrile
Attached proton test
Correlation spectroscopy
N,N-diisopropylcarbodiimide
Dichloromethane
COSY
DIC
DCM
DIEA
DMF
N,N-diisopropylethylamine
N,N-dimethylformamide
DMSO-d6 Dimethylsulfoxide-d6
ESI MS
FAB MS
Fmoc
Electro-spray ionization mass spectrometry
Fast atom bombardment mass spectrometry
[(Fluoren-1-yl-methoxy]carbonyl
4-(2-hydroxyethyl)-1-piperazine-
ethanesulfonic acid
The nomenclature and symbols of the amino acids follow the
Recommendations of the IUPAC/IUB Joint Commission on
Biochemical Nomenclature. Eur J Biochem 138:9–37 (1984).
HEPES
´ˇ
ˇˇ´
´
´
J. Hlavacek (&) Á R. Tykva Á M. Budesınsky Á J. Slaninova
Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic,
HMBC
HOBt
Heteronuclear multiple bond correlation
1-Hydroxybenzotriazole
´
Flemingovo nam. 2, 166 10 Prague 6, Czech Republic
e-mail: honzah@uochb.cas.cz
HSQC
NOE
Heteronuclear single quantum correlation
Nuclear Overhauser effect
Rotating nuclear Overhauser effect
spectroscopy
ROESY
ˇ
´
´
´
´
J. Holık Á V. Vlasakova Á B. Cerny
Institute of Experimental Botany, Academy of Sciences
of the Czech Republic, 142 20 Prague 4, Czech Republic
tBu
tert-butyl
TFA
TIS
Trifluoroacetic acid
´
B. Bennettova
Institute of Entomology, Academy of Sciences of the Czech
Tri-isopropylsilane
ˇ
´ ˇ
Republic, 370 05 Ceske Budejovice, Czech Republic
TOCSY
Total correlation spectroscopy
123

´ˇ
J. Hlavacek et al.
1716
Introduction
studies on the selected pentapeptides 1c and 1e in the ovaries
of the N. bullata, using their tritiated derivatives 3b and 3c,
which we prepared from the corresponding precursors 2b and
2c, having dehydroproline (DPro) in position 3. The results
were further correlated with the data obtained from the NMR
measurements in DMSO-d6.
In our long-term study, we have been dealing with oostatic
peptides structurally derived from the H-Tyr-Asp-Pro-Ala-
Pro6-OH decapeptide, which was isolated from a mosquito
Aedes aegypti and described by Borovsky et al. 1990. This
decapeptide was named TMOF—trypsin modulating oostatic
factor—for its ability to regulate and terminate trypsin bio-
synthesis in the last phase of insect development.
Materials and methods
We have found that TMOF also exhibits a mild oostatic
effect in the flesh fly Neobellieria bullata and that its
´ˇ
shortened analogs (Hlavacek et al. 1997, 1998, 2004) have
General methodologies
this effect strongly enhanced (decreasing the hatchability
of eggs and changing their structure, Fig. 1).
We subsequently used the 3H-labeled form (3a) (see
Table 1) of the most active pentapeptide 1a in studies on its
degradation and radioactivity accumulation in organs
(ovaries, hemolymph) of the N. bullata (Tykva et al. 1999;
The protected amino acids were purchased from Bachem
(Bubendorf, Switzerland) and the 2-chlorotrityl chloride
resin (1.3 mmol/g) from Calbiochem-Novabiochem AG
(La¨ufelfingen, Switzerland). The pentapeptide sequences
with D-amino acids were assembled on the 2-chlorotrityl
chloride resin using the AVPS-2 automatic peptide syn-
thesizer (IOCB, ASCR, v.v.i., Prague, Czech Republic).
The non-labeled truncated fragments of the pentapeptide
sequence as the standards for the HPLC study have been
prepared and described in our previous paper (Tykva et al.
2007). The samples for amino acid analysis were hydro-
lyzed with 6 M HCl containing 3% of phenol at 110°C for
20 h. The amino acid analyses were performed on a Bio-
chrom 20 instrument (Pharmacia, Sweden). The molecular
weights of the peptides were determined using a FAB MS
(Micromass, Manchester, UK). For the HPLC of the non-
labeled pentapeptides, a Spectra Physics instrument with an
SP 8800 pump, an SP 4290 integrator and a Thermo Sep-
aration Products Spectra 100 UV detector were used. The
´
´ˇ
Slaninova et al. 2004, 2007; Hlavacek et al. 2007). We
found that the radioactivity incorporates into the organs in
minutes and the pentapeptide is quickly degraded to tyro-
sine and the H-Asp-Pro-Ala-Pro-OH tetrapeptide in the
ovaries and to proline and the H-Tyr-Asp-Pro-Ala-OH
tetrapeptide in the hemolymph. The tetrapeptides further
split into corresponding dipeptides with a final cleavage to
a proline molecule.
In this study, we have focused our search on the oostatic
effects of pentapeptides 1b–1g (Table 1), containing
D-amino-acidresiduesineitheroneorallofthepositionsofthe
parent oostatic pentapeptide structure (1a). Furthermore, we
performed degradation and radioactivity accumulation
Fig. 1 The oostatic effect of
the H-Tyr-Asp-Pro-Ala-Pro-OH
pentapeptide (5P, 1a in Table 1)
on the flesh fly Neobelliera
bullata. The monolayered
follicular epithelium (a) divides,
forming a multilayered structure
(b) and filling the entire egg
chamber (c), which degenerates
and is resorbed (d)
123

Oostatic peptides containing D-amino acids
1717
Table 1 Structures of oostatic pentapeptide analogs
Pept. no.
Structure
Pept. no.
Structure
1a
1b
1c
1d
1e
1f
H-Tyr-Asp-Pro-Ala-Pro-OH (5P)
H-D-Tyr-Asp-Pro-Ala-Pro-OH
H-Tyr-D-Asp-Pro-Ala-Pro-OH
H-Tyr-Asp-D-Pro-Ala-Pro-OH
H-Tyr-Asp-Pro-D-Ala-Pro-OH
H-Tyr-Asp-Pro-Ala-D-Pro-OH
H-D-Tyr-D-Asp-D-Pro-D-Ala-D-Pro-OH
2a
2b
2c
3a
3b
3c
H-Tyr-Asp-DPro-Ala-Pro-OH
H-Tyr-D-Asp-DPro-Ala-Pro-OH
H-Tyr-Asp-DPro-D-Ala-Pro-OH
H-Tyr-Asp-[3H]Pro-Ala-Pro-OH
H-Tyr-D-Asp-[3H]Pro-Ala-Pro-OH
H-Tyr-Asp-[3H]Pro-D-Ala-Pro-OH
1g
compounds were purified by a preparative HPLC on a
25 9 2.2 cm column, 10 lm Vydac RP-18 (The Separa-
tions Group, Hesperia, CA, USA), with a flow rate of 7 ml/
min, detection at 220 nm using a 0–100% gradient of ACN
in 0.05% aqueous TFA, for 120 min, unless otherwise
stated. The analytical HPLC was carried out on a
15 9 0.4 cm column, 5 lm Supelco (Supelco, Bellefonte,
PA, USA) with a flow rate of 1 ml/min, detection at
220 nm, using a 5–100 % gradient of ACN in 0.05 aqueous
TFA, for 40 min. The NMR spectra were recorded on a
Bruker AVANCE-600 spectrometer (Bruker BioSpin
GmbH, Rheinstetten, Germany).
and 2c (9.34) were recorded, using 15 9 0.4 cm, 5 lm
Supelco column at a flow rate of 1 ml/min and a detection
at 220 nm with a 5–100 % gradient of ACN in 0.05
aqueous TFA, for 40 min. For 1b–1g (C26H35N5O9, 561.1),
MS (m/z): 562.1 (M ? H)? and for the 2b and 2c
(C26H35N5O9, 559.1), MS (m/z): 560.1 (M ? H)? were
determined with the FAB technique (Micromass, Man-
chester, UK). A correct amino acid composition was found
for all of the analogs prepared.
3
Preparation of the H-labeled pentapeptides 3b, 3c
The tritiated pentapeptides 3b, 3c were obtained according
´ˇ
to the procedure used for 3a preparation (Hlavacek et al.
Preparation of the pentapeptides 1b–1g and 2b, 2c
2007; Tykva et al. 2007), typically: the pentapeptide 2b or
2c was dissolved in N, N-dimethylacetamide, with a PdO/
BaSO4 catalyst (10% Pd) (Kuhn et al. 1955) being added,
and this mixture was stirred in the original apparatus
The Fmoc-Pro-OH and Fmoc-D-Pro-OH were loaded on
the 2-chlorotrityl chloride resin (4 and 2 g, respectively)
´ˇ
with a substitution of 0.43 mmol/g (Hlavacek et al. 1998),
ˇ
(Hanus et al. 1971; Cerny et al. 1981) with tritium gas
ˇ
´
using the described procedure (Barlos et al.1991). The
loaded resins were divided into six portions and, following
a Fmoc deprotection by a 20% piperidine/DMF mixture
(10 ml) for 20 and 30 min, each of the portions was sep-
arately acylated according to the given pentapeptide
sequence with a fourfold excess (1.72 mmol) of a Fmoc-
derivative of Ala or D-Ala (0.54 g), of Pro, D-Pro (0.58 g)
or 3,4-DPro (0.57 g), of Asp(OtBu) or D-Asp(OtBu)
(0.71 g) and of Tyr(tBu) or D-Tyr(tBu) (0.79 g) using a
DIC (0.24 g; 1.89 mmol) and HOBt (0.25 g; 1.89 mmol)
as the coupling reagents. After the last Fmoc deprotection
as described above, the detachment of the peptides from
the resin and the side-chain deprotection were performed
simultaneously by treatment with a 50% TFA-5% anisole–
5% TIS mixture in DCM for 2 h. The solutions were fil-
tered from the resin and evaporated to dryness. The resi-
dues were triturated with tert-butyl-methylether, then
dissolved in 1 M AcOH, and the solutions were lyophi-
lized. The pentapeptides 1b–1g and 2b, 2c were purified by
a preparative HPLC using the conditions described in the
General methodologies section. The analytical HPLC
retention times in minutes for the peaks of 1b (8.45), 1c
(8.58), 1d (8.62), 1e (8.07), 1f (8.13), 1 g (8.58), 2b (9.41)
3
containing 70% of H2 at a pressure of 80 kPa, at room
temperature for 2 h (Evans 1974). After solvent evapora-
tion, the residue was suspended in water (0.5 ml) and the
catalyst removed by centrifugation and washed using
0.5 ml of water. The combined supernatants were freeze-
dried. The lyophilizate was purified by HPLC on a
25 9 0.4 cm column, 5 lm LiChrospher WP-300 RP-18
(Merck, Germany), with a flow rate of 0.6 ml/min, by an
isocratic elution by a mixture of ACN in 0.05% aqueous
TFA (ACN: 9%), the detection of products—by UV
210 nm and radioactivity. UV spectrophotometry (275 nm)
was used for the determination of the mass of the products.
Radioactivity was estimated by a liquid scintillation mea-
surement. The purity was established by HPLC. Table 2
summarizes the conditions of labeling and characteristics
of the labeled peptides.
Insect treatment
Females of Neobellieria bullata, Diptera (an approximate
weight of 90 mg), raised at 25°C, with 12 h light/12 h dark
cycles were used in the experiments. The larvae were
123

´ˇ
J. Hlavacek et al.
1718
Table 2 Conditions of radiolabeling and characteristics of 3H labeled peptides H-Tyr-D-Asp-[3H]Pro-Ala-Pro-OH (3b) and H-Tyr-Asp-
[3H]Pro-D-Ala-Pro-OH (3c)
Peptidea
Precursor
(mg)
Catalyst
(mg)
Yield
(mg) (Gbq)
Spec. activity
(Tbq/mmol)
Radiochrom.
purity (%)
3b
3c
a
2b; 0.4
2c; 1.0
3.8
4.3
0.25 (0.69)
0.59 (1.72)
1.53
1.64
[97
[97
In N, N-dimethylacetamide solution with tritium gas containing 70% of 3H2, at 80 kPa pressure at room temperature for 2 h, 10% PdO/BaSO4
catalyst
raised on beef liver covered with sawdust (pupariation
Bouin’s solution, embedded in Paraplast (Sigma-Aldrich
s.r.o., Czech Republic), and 5 lm sections were cut and
stained with Mallory’s or Mayer’s hematoxyline. The
sections were checked for changes in the follicular epi-
thelium and the conditions of its nuclei, the yolk deposi-
tion, and its structure in the oocyte as well as the
appearance of nutritive cells and their nuclei. These
changes and their frequency were correlated with the
intervals after each peptide application. The severity of the
changes was assessed, as was the percentage of patholog-
ical changes in the egg chambers of the ovariole (Table 3).
medium). The pupae were separated from the medium and
placed into nylon cages before emergence. The flies were
fed sugar and water for three days and from day 4 beef
liver, which later served as an oviposition medium.
Peptides were injected in Ringer’s solution (10 nmol in
5 lL/female) into the upper part of the thorax of the Et2O-
anesthesized 24–48 h-old flies. The treated female flies
were placed into cages with untreated males and their
ovaries were dissected 2, 4, 8 and 16 days later and
examined for morphological and histological changes. The
hatchability of the eggs in the uterus and the appearance of
the eggs were evaluated in the first and second gonotrophic
cycles. Each experimental group consisted of 50 flies.
In vitro application of radioactively labeled peptides
For the in vitro experiments, twelve dissected pairs of ovaries
of identical stage and appearance from the 4-day-old females
were placed into a solution of tritiated pentapeptide (1 lCi
(37 kBq) in 5 lL of the physiological solution) in small
embryo dishes at room temperature. Because of the different
molar radioactivities of the corresponding compounds
(Table 2), different amounts of each of the peptides were
used: 3a (16.88 ng), 3b (13.57 ng) and 3c (12.66 ng), based
on the pentapeptides’ molecular weight, 561.1. At time
intervals of 5 min and 120 min, the ovaries were removed,
washed twice in the physiological solution and two at one
trial placed into individual scintillation vials and covered
with 1 ml of NCS II tissue solubilizer, Amersham Interna-
tional. After one day, 10 ml of EcoLite liquid scintillator
(ICN Biochemicals Inc.) were added and the radioactivity
was determined in the Beckman 6500 spectrometer. The
highest and lowest values of the sets were eliminated and
the ten remaining samples were used for an evaluation of the
mean values and their standard deviation. From three inde-
pendent experiments, the total SD was calculated from 15
to 25%. Simultaneously, incubations for the selected time
period were done for metabolite determination, and the
samples were frozen until analyzed.
Morphological assessment of the status of the ovaries
The dissected ovaries were spread on a microscopic slide in
a physiological solution and microscopically examined for
yolk deposition and oocyte growth. The different stages of
yolk deposition were classified according to the scale (King
1970). The oocyte development was scored from 1 to 5,
with 1 being no yolk in the oocyte, 2, 3 and 4 being oocyte
with yolk filling up to a quarter, half and three-quarters of
the egg chamber, respectively. Stage 5 indicates mature
eggs ready to be deposited in the uterus. In the radioactivity
incorporation assay, the small = 5th–6th and large =
11th–12th of the 14 developmental stages of yolk deposition
in oocytes were estimated.
Hatchability assessment
The uterus content was examined before the time of the
egg/larva deposition, and hatchability was expressed as the
number of developed larvae in the uterus. The untreated
flies deposited in the uterus 98–110 eggs, in which the
segmented larvae were 95–98%. This corresponds to a
hatchability of 95–98%.
Extraction for the metabolite determination
Histological approach, assessment of the changes
An ice-cold solution (0.4 mL) of Complete Mini protease
inhibitor cocktail (Roche Applied Sciences) was added
(one tablet dissolved in 3.5 mL of 50 mM HEPES buffer,
Selected ovaries, in which visible changes in the mor-
phology of the egg chambers were detected, were fixed in
123

Oostatic peptides containing D-amino acids
1719
Table 3 Larval hatching and resorption of oocytes after peptide administration
Peptide
Hatchabilitya
(First gonotrophic cycle, %)
Resorptionb (Second
gonotrophic cycle, %)
H-Tyr-Asp-Pro-Ala-Pro-OH (5P), 1a
H-D-Tyr-Asp-Pro-Ala-Pro-OH, 1b
H-Tyr-D-Asp-Pro-Ala-Pro-OH, 1c
H-Tyr-Asp-D-Pro-Ala-Pro-OH, 1d
H-Tyr-Asp-Pro-D-Ala-Pro-OH, 1e
H-Tyr-Asp-Pro-Ala-D-Pro-OH, 1f
all-D-(H-Tyr-Asp-Pro-Ala-Pro-OH), 1g
a
20
40
50
30
30
40
20
20
99
80
10
30
40
88
Hatchability in the first gonotrophic cycle is given only as in the second gonotrophic cycle no hatchability was observed
b
The difference to 100% is equal to yolk content in egg. 100% resorption means no egg yolk. Resorption influences the embryogenesis, which
results in zero hatchability
pH 7.6) to the frozen, pooled ovaries in an Eppendorf tube
and the content was homogenized for 1 min using a teflon
pestel. After centrifugation, the supernatant was removed
and either immediately analyzed by radio-HPLC or frozen
and analyzed later.
Analysis of the metabolites of pentapeptides 3a–3c
(Figs. 2, 3, 4)
All of the radio-HPLC analyses were performed using the
Waters liquid chromatograph (Waters, USA). A program-
mable UV detector was connected online to a radiometric
flow-through detection system (Beckman 171, http://www.
beckman.com). The LiChroCART stainless steel analytical
column (250 9 4 mm; Merck, http://www.merck.com.),
packed with a LiChrosphere WP-300, with a particle size
of 5 lm was used. The column was protected with a guard
column (4 9 4 mm), packed with a LiChrosphere 100
RP-18, using a particle size of 5 lm (Merck). The mobile
Fig. 3 The radio-HPLC (full line) of the pentapeptide 3c after a
25-min incubation with the ovaries of the flesh fly Neobellieria
bullata and the UV-HPLC (dotted line) of the corresponding non-
labeled fragment standards
Fig. 4 The incorporation of radioactivity into the ovaries of the flesh
fly Neobellieria bullata after incubation with the H-Tyr-Asp-[3H]Pro-
Ala-Pro-OH (3a), H-Tyr-D-Asp-[3H]Pro-Ala-Pro-OH (3b) and
H-Tyr-Asp-[3H]Pro-D-Ala-Pro-OH (3c) pentapeptides. The ‘‘small’’
and ‘‘large’’ mean the 5th–6th and 11th–12th, respectively, of the 14
developmental stages of the oocytes based on the deposition of the
yolk (King 1970)
Fig. 2 The radio-HPLC (full line) of the pentapeptide 3b after a
25-min incubation with the ovaries of the flesh fly Neobellieria
bullata and the UV-HPLC (dotted line) of the corresponding non-
labeled fragment standards
123

´ˇ
J. Hlavacek et al.
1720
phase was composed of the aqueous phase (0.035% TFA in
redistilled water) and the organic phase (0.05% in ACN).
After passing through the UV detector, the eluent was con-
tinuously mixed with the Ready Safe liquid scintillator
(Beckman Coulter, USA) in a ratio of 1:2.5 (v/v) in an online
mixer. The mixture ran through a 500-lL detection cell. The
radiometric detector threshold was set at 0.02%. The UV
detector was set at 230 nm with 0.05 AUFS. The separation
was performed at ambient temperature using a 30-min linear
gradient from 0 to 30% of the organic phase using a flow rate
of 0.8 mL/min and continuous degassing with helium. A
sample volume of 20–80 lL was used. The area of each peak
was evaluated as the ratio of its counting rate (cpm) to the
totally measured sum of the counting rate in all of the peaks of
the appropriate radiochromatogram (relative concentration
crel in percentage). The stability of the radiolabeled penta-
peptides was checked before each experiment.
of the amide NH protons were obtained from the 1D-proton
spectra measured at 27, 37 and 47°C.
Results and discussion
The assay of the oostatic activity of the pentapeptides 1b–
1g with D-amino acids in the peptide chain has shown a
significant decrease of hatchability in the first gonotrophic
cycle, which was comparable with that of the parent pen-
tapeptide 1a, and no hatchability in the second gonotrophic
cycle (Table 3).
The morphological changes in ovaries correspond to a
large resorption of the oocytes of treated flies after the
application of the pentapeptides 1b, 1c and 1g. Even if the
pentapeptides 1d–1f exhibit low resorption, this effect still
interferes with the embryogenesis, so that hatching is
impossible. Thus, the changes in the female reproductive
system of N. bullata, after the application of the penta-
peptides assayed, are degenerative and correspond to the
histological pictures shown in Fig. 1. The strong oostatic
activity of the all D-amino acids containing peptide 1g
speaks in favor of the suggestion that the effect of oostatic
peptides in N. bullata is complex and proceeds without
involvement of a specific receptor (Bennettova et al. 2010).
Rather an intake of the analyzed sequences into the inter-
cellular spaces between follicular cells could be assumed.
This intake increases with continuous yolk deposition even
if the mechanism responsible for the oostatic peptide
The samples were centrifuged for 5 min and an aliquot of
the supernatant was analyzed. The standard unlabeled pep-
´ˇ
tides (Hlavacek et al. 2007) and proline were detected by UV
spectrometry; their retention times were compared to those of
the peaks in the radiochromatogram. The retention times of
the radioactive fractions were corrected to the time delay
between the UV and the radiometric detector (0.55 min). The
precision of the method was expressed as the coefficient of the
variation in percentage, which fluctuated from 1.3 to 7.7%. It
was determined by analyzing five replicates of the same bio-
logical sample within 1 day.
´
transport has yet to have been found (Slaninova et al.
The extraction procedure recovery was evaluated using
pentapeptide 3a calibration solutions of three different
concentrations (42, 150 and 370 kBq/mL) by a comparison
of the extracted and applied radioactivity. The average
recovery was between 86.6 and 97.2% (n = 4) with a
precision range from 3.3 to 5.2 (% coefficient of variation).
The linearity of the radiometric detector response was
verified using 3a calibration solutions in the range of
0.2–10 kBq with an average correlation coefficient of
0.997 (n = 4). The absolute detection limit in the system,
defined by a signal-to-noise ratio of 3, was assigned for 3a
in the range of 85–150 Bq, which corresponds to 59–104
(rounded to 60–100) fmol.
2007).
Moreover, a majority of the effects occurred not in the
first but in the second gonotropic cycle (approximately
sixteen days after eclosion). The second gonotrophic cycle
(the one more affected) starts several days after the peptide
application, and it seems improbable that the peptide would
persist in active concentration so long considering that its
half-life is in the order of tens of minutes.
With the aim of explaining the action of the oostatic
peptides in N. bullata, we have also performed studies on
the degradation and accumulation of tritiated pentapeptides
in the ovaries of this insect. For these studies, we have
chosen the parent pentapeptide 1a (radioactive form 3a
assayed) and its analogs 1c and 1e (radioactive forms 3b
and 3c) containing D-amino acid residues located in the
neighborhood of the tritiated central Pro residue [3H]Pro3.
In these analogs, the D-Asp2 residue is linked by its
N-terminus to a Tyr residue in position 1 and the D-Ala4
residue by its C-terminus to Pro residue in position 5. In
such an arrangement, the degradation of the pentapeptides
3a–3c and the radioactivity accumulation could be evalu-
ated via the whole pentapeptide sequence.
The NMR structure determination
The spectra were measured on a Bruker AVANCE-II
instrument using a cryo-probe (1H at 600.13 MHz; 13C at
150.9 MHz frequency) in DMSO-d6. The series of proton
1D, 2D-COSY, 2D-TOCSY (spin-lock time 90 ms) and
2D-ROESY (mixing time 300 ms) was measured at 27°C
for the complete structural assignment of the proton sig-
nals. The structural assignment of the carbon-13 signals
was achieved by combining the 1D-APT, 2D-H, C-HSQC
and 2D-H,C-HMBC spectra. The temperature coefficients
A slower degradation of the parent peptide 1a in the
ovaries in comparison with the hemolymph (Tykva et al.
123

Oostatic peptides containing D-amino acids
1721
2007) suggests the participation of different enzymatic
systems for the degradation of this peptide in the two
organs. Therefore, our initial study with the D-amino acid
containing analogs followed a degradation and accumula-
tion of the corresponding pentapeptides in the ovaries.
Contrary to the quick degradation of the peptide 1a
(radioactive form 3a assayed) in the ovaries of N. bullata
species in solution because of the population of trans- and
cis-isomers on both the Asp-Pro and Ala-Pro peptide
bonds. The isomers can be distinguished by the NOE
contacts observed in the 2D-ROESY spectra: the NOE
contact between the Ha of Asp or Ala and the Hd of Pro
indicates a trans- X-Pro bond and the NOE contact
between the Ha of Asp or Ala and the Ha of Pro indicates a
cis- X-Pro bond (Fig. 5).
´
(Bennettova et al. 2010), its analogs 1c and 1e (radioactive
forms 3b and 3c), owing to the presence of the D-Asp2 and
D-Ala4 residues, respectively, were degraded much more
slowly. Apparently, the proteolytic enzymes must handle
an opposite orientation of the side-chains, which change
the conformation of the corresponding pentapeptide (see
the discussion of the NMR spectra below). Figure 2 shows
that 120 min after the incubation with the ovaries, the
pentapeptide 1c (radioactive pentapeptide 3b) and its
degradation product Tyr-D-Asp-Pro are still present in the
ovaries as detected by radio-HPLC (full line). Similarly, as
shown in Fig. 3, the pentapeptide 1e (radioactive penta-
peptide 3c) degrades slowly within the same period of time
to Asp-Pro-D-Ala-Pro and Pro-D-Ala-Pro (full line). Using
UV-HPLC, the corresponding peaks of the non-labeled
standards (Tykva et al. 2007) of the degradation products
were detected (dotted lines). However, the degradation of
the pentapeptide 1e (radioactive 3c) to the final [3H]Pro in
position 3 is significantly quicker than that of the penta-
peptide 1c (radioactive 3b).
The equilibrium population of the individual isomers
(trans-/trans-, cis-/trans-, trans-/cis- and cis-/cis-) observed
in the peptides 1a, 1c and 1e is shown in Table 4. It can be
seen that an isomer with both trans-peptide bonds is pre-
ferred in all of the studied peptides while the isomer with
both cis-peptide bonds has the lowest population. The
presence of D-Asp or D-Ala leads to a significant increase of
the isomer with cis-D-Asp-Pro in 1c (29 %) and cis-D-Ala-
Pro in 1e (27 %), which, along with a lower population of
the trans-/trans isomer (62 % in 1c and 52 % in 1e), can be
responsible for the slower enzymatic degradation of 1c and
1e. The fastest step of degradation (the splitting of the
Tyr-Asp peptide bond—see above) can explain the differ-
ence between the rates of degradation of 1c and 1e.
Since the presence of four isomers significantly com-
plicates the analysis of the NMR spectra, a complete
structural assignment of all of the signals (1H and 13C) was
performed only for the major trans-/trans- isomers. Their
NMR data are summarized in Table 5.
As far as the total accumulation of radioactivity is
concerned, the incubation of N. bullata ovaries with the
labeled pentapeptides 3a–3c showed similarly different
results. The significantly decreased uptake of radioactivity
from peptides 3b and 3c into the ovaries in comparison
with that of the parent peptide 3a (Fig. 4) could be con-
nected with an increased stability of the relevant peptide
bonds in pentapeptides with D-Asp2 and D-Ala4 toward the
corresponding proteolytic enzymes.
For an estimation of the effect of the presence of the
D-residue in position 2 (in 1c) and in position 4 (in 1e) on
the molecular geometry, we have constructed models of 1a,
1c and 1e using extended b-sheet parameters for all of the
residues. The presence of two Pro residues partially distorts
the regular b-sheet chain. We have therefore applied
unrestrained geometry optimization to minimize the energy
of the model structures using the AMBER version of the
Hyperchem package. The calculated structures for 1a, 1c
and 1e using the carbon atoms of the Asp-Pro peptide bond
(C=O of Asp-2 and Ca, Cd of Pro-3 for the structure
overlap) are shown in Fig. 6.
The relatively large difference in the radioactivity
incorporation between the two analogs 3b and 3c could be
explained by the different effect of the D-Asp2 and D-Ala4
residues on the conformation of the N-terminal part of the
pentapeptides. Tyr in position 1 is believed to start a very
quick metabolic cleavage of the parent 5P (1a) in the
3
3
´
ovaries, with the final release of [ H]Pro (Bennettova et al.
2010). Owing to the impaired ability of proteolytic
enzymes to recognize the area around the peptide bond
Tyr-D-Asp2, this cleavage and consequently the penetra-
tion of the released [3H]Pro3 into the ovaries is more
strongly decreased with pentapeptide 3b as compared to 3c.
With the aim of rationalizing the results of the degra-
dation and incorporation studies with the pentapeptides 1a,
1c and 1e, we studied these peptides using NMR spec-
troscopy. The presence of two proline residues in the
peptides 1a, 1c, 1e leads to the appearance of four isomeric
Fig. 5 A schematic diagram for distinguishing the trans- and cis-X-
Pro peptide bonds from the characteristic NOE contacts
123

´ˇ
J. Hlavacek et al.
1722
constants J(N,a) = 7.8–8.6 Hz are in the range of the
random-coil values, the temperature coefficients DdNH/DT
of the Asp2 (-3.8 to -4.2 ppb) indicate a moderate solvent
shielding of the NH, and those of the Ala4 (-5.0 to
-7.4 ppb) suggest the NH exposed to a bulk solvent. There
is no evidence for a strong solvent shielding or an
H-bonding of the peptide NH protons. The observed NOE
contacts are the most important parameters used in the
conformation analysis of the peptides in solution. Their
application for non-rigid molecules is not straightforward,
since the observed NOEs can come from different con-
formations present in solution. The situation in our case is
further complicated by a partial overlap of some signals of
the major and minor isomers that make the correct assign-
ments of the weak NOE crosspeaks in the 2D-ROESY
Table 4 Equilibrium population of isomers with trans- and cis-X-Pro
bonds in peptides 1a, 1c and 1e observed in DMSO-d6 at 27°C
Configuration on peptide bond
1a
1c
1e
Asp-Pro
trans-
cis-
Ala-Pro
trans-
trans-
cis-
(all-L)
71%
11%
10%
8%
(D-Asp)
62%
29%
6%
(D-Ala)
52%
12%
27%
9%
trans-
cis-
cis-
3%
The linear pentapeptides 1a, 1c and 1e are expected to
exist in solution as flexible molecules. Table 5 shows a
close similarity of the majority of the proton NMR
parameters in 1a, 1c and 1e. The observed coupling
Table 5 Proton and carbon-13 NMR data of peptides H-Tyr-Asp-Pro-Ala-Pro-OH (1a), H-Tyr-D-Asp-Pro-Ala-Pro-OH (1c) and H-Tyr-Asp-Pro-
D-Ala-Pro-OH (1e) in DMSO at 27°C
Residue
Tyr-1
Parameter
1a
1c
1e
Parameter
1a
1c
1e
NH
8.09
8.11
8.013
CO
167.93
53.56
167.95
53.78
167.82
53.51
J(N,a)
Ha
a
a
a
Ca
3.905
3.92
3.915
Cb
36.18
36.62
36.14
J(a,b)
Hb1, Hb2
J(b1,b2)
H-2,6
H-3,5
OH
5.8; 7.4
2.94; 2.82
14.2
5.8; 8.1
2.92; 2.81
14.2
6.2; *7.0
2.93; 2.82
14.2
C1
124.71
130.70
115.45
156.80
124.88
130.82
115.52
156.82
124.65
130.68
115.40
156.80
C2,C6
C3,C5
C4
6.985
7.04
6.97
6.68
6.70
6.67
9.40
9.43
9.42
Asp-2
NH (DdNH/DT)
J(N,a)
Ha
8.91 (-4.2)
7.8
8.87 (-3.8)
8.6
8.91 (-3.8)
8.0
CO
168.62
47.70
168.17
47.17
168.78
47.43
Ca
4.82
4.89
4.83
Cb
36.10
36.62
36.14
J(a,b)
Hb1, Hb2
J(b1,b2)
COOH
Ha
6.4; 7.2
2.76; 2.46
16.9
8.4; 6.1
2.63; 2.22
16.4
6.8; a
COOH
171.64
171.76
171.99
2.82; 2.50
16.7
12.47
*12.40
4.29
12.46
Pro-3
Ala-4
Pro-5
a
4.30
4.28
CO
Ca
Cb
Cc
170.80
59.49
29.25
24.75
46.88
170.41
46.22
16.92
171.12
59.43
29.46
24.75
47.08
170.80
46.20
17.00
170.40
60.06
29.73
24.10
46.53
170.15
46.02
17.59
H2b
1.96; 1.84
1.84; 1.80
3.56; 3.38
2.01; 1.78
*1.82 (2H)
3.65; 3.58
1.90; 1.79
*1.79 (2H)
3.64; 3.42
H2c
H2d
Cd
CO
Ca
Cb
NH (DdNH/DT)
7.97 (-5.6)
7.5
8.06 (-7.4)
7.3
7.535 (-5.0)
8.4
J(N,a)
Ha
4.46
4.44
4.60
J(a,b)
H3b
7.0
7.0
6.9
1.165
1.19
1.13
Ha
4.21
4.23
4.18
COOH
Ca
173.49
58.70
28.78
24.35
46.40
173.39
58.70
28.77
24.42
46.57
173.46
58.95
28.86
24.61
46.94
H2b
2.13; 1.83
1.90; 1.83
3.59; 3.475
12.47
2.13; 1.87
*1.87 (2H)
3.58; 3.52
*12.40
2.13; 1.825
*1.89 (2H)
3.61; 3.50
12.46
H2c
Cb
H2d
Cc
COOH
Cd
The J-value was not determined
123

Oostatic peptides containing D-amino acids
1723
Fig. 6 The unrestrained geometry optimized structures of the H-Tyr-Asp-Pro-Ala-Pro-OH (1a), H-Tyr-D-Asp-Pro-Ala-Pro-OH (1c) and H-Tyr-
Asp-Pro-D-Ala-Pro-OH (1e) pentapeptides
Fig. 7 A schematic diagram of
the observed inter-residue
NOEs in the H-Tyr-Asp-Pro-
Ala-Pro-OH (1a), H-Tyr-D-Asp-
Pro-Ala-Pro-OH (1c) and
H-Tyr-Asp-Pro-D-Ala-Pro-OH
(1e) pentapeptides
123

´ˇ
J. Hlavacek et al.
1724
spectra difficult. Therefore, we have selected only unam-
biguously assignable inter-residue strong and medium
cross-peaks from the ROESY spectra, which are shown
schematically in Fig. 7. While the NOEs between the
protons of neighboring residues (sequential NOEs) can
usually be observed in all of the even non-structured
flexible peptides, the observations of medium-range (non-
sequential NOEs) are taken as indicators of the presence of
the population of some secondary structure fragments. As
can be seen in Fig. 7, there are only sequential NOEs
identified in 1c but some additional non-sequential NOEs
(between the protons of Tyr1/Pro3 and Asp2/Ala4) in 1a and
1e. We have tried to use all of the NOEs shown in Fig.7 as
distance restraints and apply them on previously unre-
strained optimized structures (Fig. 6). An energy minimi-
zation (AMBER; for all of the NOE restraints, the upper
In general, the enhanced stability of the oostatic analogs
after the introduction of D-amino acid residues is reflected
in the prolonged half-life of the corresponding pentapep-
tides. Thus, more oocytes might be affected during the
period of their development, which enhances the regulatory
potency of the majority of the analogs.
Acknowledgments This research was supported by Research Pro-
ject Z4 055 0506 and the Czech Science Foundation No. 203/06/1272.
References
Barlos K, Chatzi O, Gatos D, Stavropoulos G (1991) 2-Chlorotrityl
chloride resin. Int J Peptide Protein Res 37:513–520
´
´ˇ
Bennettova B, Hlavacek J, Tykva R (2002) Follicular epithelium
changes after application of insect oostatic hormone analogues in
Neobellieria bullata (Diptera). In: Abstract book of VIIth
European Congress of Entomology, October 7–13, 2002 Thes-
saloniki, Greece. Abstract No 10: p. 25. Hellenic Entomological
Society 2002
˚
distance limit was 4 A) provided structures with markedly
higher energy and some torsion angles out of the allowed
regions. It seems obvious that these peptides adopt not only
one preferred conformation in solution but some confor-
mation families contributing to the overall observed NOEs.
The analysis combining molecular dynamics to identify the
conformation families, a detailed analysis of the integrated
NOE values, and back calculations of the relaxation
matrices will be the subject of a future paper.
´
´
´
´
´ˇ
´
Bennettova B, Slaninova J, Vlasakova V, Hlavacek J, Holık J, Tykva
R (2010) J Insect Sci 10:48. http://insectscience.org/10:48
Borovsky D, Carlson DA, Griffin PR, Shabanovicz J, Hunt DF (1990)
Mosquito oostatic factor: a novel decapeptide modulating trypsin-
like enzyme biosynthesis in the midgut. FASEB J 4:3015–3020
ˇ
´
ˇ
Cerny B, Hanus J (1981) Tritium incorporation into solvents under
the conditions of catalytic dehalogenation. J Label Comp
18:947–954
ˇ
ˇ
Hanus J, Veres K (1971) Preparation of tritium labeled D, L-2,
6-diaminopimelic acid. J Label Comp 7:425–430
Conclusion
´ˇ
´
Hlavacek J, Bennettova B, Barth T, Tykva R (1997) Synthesis,
radiolabelling and biological activity of peptide oostatic hor-
mone and its analogs. J Peptide Res 50:153–158
The introduction of D-amino acids into the oostatic peptide
1a retained the oostatic activity and enhanced the resorp-
tion of the developing eggs in N.bullata. As no receptors
´ˇ
´
Hlavacek J, Tykva R, Bennettova B, Barth T (1998) The C-terminus
shortened analogs of the insect peptide oostatic hormone with
accelerated activity. Bioorg Chem 26:131–140
´ˇ
ˇ´
´
Hlavacek J, Marık J, Konvalinka J, Bennettova B, Tykva R (2004)
Synthesis and application of methyleneoxy pseudodipeptide
building blocks in biologically active peptides. Amino Acids
´
have yet been found (Slaninova et al. 2004), an autono-
mous function of the ovaries in the process of active intake
by passing peptides through the inter-spaces of the follic-
ular cells could be proposed. D-amino acid-containing
analogs efficiently interfere with an enzymatic system,
which very effectively degrades the parent pentapeptide 1a
during its transport into the egg chamber. Contrary to the
parent pentapeptide 1a, the D-Asp2- or D-Ala4-containing
isomers 1c and 1e, assayed for their degradation and
radioactivity incorporation into ovaries as the radiolabeled
forms 3b and 3c, were degraded much more slowly owing
to a decrease of the respective peptide bonds cleavage. The
increased stability (with the D-Asp2 pentapeptide 1c pri-
ority) thus also results in a retardation of the incorporation
of the radioactivity of [3H]Pro into the ovaries. The NMR
spectra showed that the presence of D-Asp or D-Ala leads to
a significant increase of the isomer with cis-D-Asp-Pro in
1c (29%) and cis-D-Ala-Pro in 1e (27%), which, along with
the lower population of trans-/trans isomer (62% in 1c and
52% in 1e), can be responsible for the slower enzymatic
degradation of 1c and 1e.
27:19–27
ˇ
´ˇ
´
´
´
Hlavacek J, Cerny B, Bennettova B, Holık J, Tykva R (2007)
Preparation of tritiated oostatic peptides for study of radioactiv-
ity incorporation in flesh fly Neobellieria bullata. Amino Acids
33:489–497
King RC (1970) Ovarian development in Drosophila melanogaster,
pp 33–54. Academic Press, New York
Kuhn R, Haas HJ (1955) Braunes Palladiumoxydhydrat-bariumsulfat
f}ur katalytische Hydrierungen. Angew Chem 67:785
Marık J, Bennettova B, Tykva R, Budesınsky M, Hlavacek J (2001)
Synthesis and effect of shortened oostatic decapeptide (TMOF)
analogs with isosteric structures on reproduction of Neobellieria
bullata. J Peptide Res 57:401–408
ˇ´
´
ˇˇ´
´
´ˇ
ˇ
´
´
´
Slaninova J, Bennetova B, Nazarov ES, Simek P, Holık J, Vlasakova
(2004) Activity and
´
´
ˇ
´ˇ
´
V, Hlavacek J, Cerny B, Tykva
R
mechanism of action of insect oostatic peptides in flesh fly.
Bioorg Chem 32:263–273
´ˇ
ˇ
´
Tykva R, Hlavacek J, Nemec V, Bennettova B (1999) Effect of a
peptide and some of its analogs on the reproduction of
Sarcophaga bullata and Locusta migratoria. In: Del Re AAM,
Brown C, Capri E, Errera G, Evans SP, Trevisan M (eds) Human
and environmental exposure to xenobiotics. La Goliardica
Pavese, Pavia, pp 895–901
123

Oostatic peptides containing D-amino acids
1725
ˇ
Tykva R, Hlavacek J, Vlasakova V, Cerny B, Borovickova L,
´ˇ
´
´
´
´
ˇ
´ˇ
´
´
´
ˇ
´
Tykva R, Hlavacek J, Bennettova B, Slaninova J, Novak J, Nemec V,
ˇ
´
´
´
´
´
´
Cerny B, Vlasakova V, Holık J (2007a) Conditions of elimina-
tion of environmental pollution in insect management. In: Book
of abstracts of the 14th international symposium on environ-
mental pollution and its impact on life in the Mediterranean
region with focus on environment and health, October 10–14,
2007. Sevilla, Spain, p 407
Bennettova B, Holık J, Slaninova J (2007b) Radiochromato-
graphic assay of metabolites of the oostatic peptide labeled in
different positions of the peptide chain. J Chrom B 848:258–263
123