Cyclic analogs of insect oostatic peptides: Synthesis, biological activity, and NMR study

Article abstract of DOI:10.1006/bioo.2001.1210

Cyclic peptides 2a-2c, derived from the sequence of the C-terminal shortened analogs of the oostatic decapeptide H-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-OH (1a), were synthesized and assayed on their effect in a reproduction of the flesh fly Neobellieria bullata. The cyclization of the N-terminal linear tetra- and pentapeptides 1b and 1c to the cyclotetra- and cyclopentapeptides 2b and 2c decreased the oostatic activity by one order of magnitude. The cyclodecapeptide 2a, which emerged spontaneously during the pentapeptide cyclization, was quite inactive. Comparative ¹H and ¹³C NMR study on a conformation of the cyclopeptides 2a-2c, and their linear precursors 1b and 1c revealed that a space structure of the cyclic analogues 2b and 2c is too restricted to adopt a biological conformation necessary for receptor binding and therefore only minor oostatic activity is observed after their application. The lack of the oostatic activity in the case of the more flexible dimeric analogue 2a is ascribed to the size of its molecule and its overall shape that is not compatible with a receptor binding.

Full text of DOI:10.1006/bioo.2001.1210

Bioorganic Chemistry 29, 282–292 (2001)
doi:10.1006/bioo.2001.1210, available online at http://www.idealibrary.com on
Cyclic Analogs of Insect Oostatic Peptides:
Synthesis, Biological Activity, and NMR Study
Jan Hlava´cˇek,* Milosˇ Budeˇsˇ´ınsky´,* Blanka Bennettova´,† Jan Marˇ´ık,* and
Richard Tykva*
*Institute of Organic Chemistry and Biochemistry, Academy of Sciences,
166 10 Prague 6, Czech Republic; and †Institute of Entomology,
ˇ
Academy of Sciences, 370 05 Ceske´ Budeˇjovice, Czech Republic
Received January 17, 2001
Cyclic peptides 2a–2c, derived from the sequence of the C-terminal shortened analogs of
the oostatic decapeptide H-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-OH (1a), were synthe-
sized and assayed on their effect in a reproduction of the flesh fly Neobellieria bullata. The
cyclization of the N-terminal linear tetra- and pentapeptides 1b and 1c to the cyclotetra- and
cyclopentapeptides 2b and 2c decreased the oostatic activity by one order of magnitude. The
cyclodecapeptide 2a, which emerged spontaneously during the pentapeptide cyclization, was
1
quite inactive. Comparative H and 13C NMR study on a conformation of the cyclopeptides
2a–2c, and their linear precursors 1b and 1c revealed that a space structure of the cyclic
analogues 2b and 2c is too restricted to adopt a biological conformation necessary for receptor
binding and therefore only minor oostatic activity is observed after their application. The lack
of the oostatic activity in the case of the more flexible dimeric analogue 2a is ascribed to the
size of its molecule and its overall shape that is not compatible with a receptor binding.
᭧
2001 Academic Press
Key Words: solid phase; solution synthesis; oostatic peptide; Neobellieria bullata; reproduc-
1
tion; cyclopeptide; H and 13C NMR study.
INTRODUCTION
Decapeptide H-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-OH (1a), isolated from
mosquito Aedes aegypti by Borovsky et al. (1,2), was postulated to affect oogenesis
through modulation of ovarian ecdysteroid synthesis, gut trypsin biosynthesis in the
midgut of blood-sucking insects, or egg development neurosecretory hormone release
in the Diptera and to influence a juvenile hormone action on ovarian yolk uptake in
some Hemiptera. The peptide was designed to function as a signal that terminates
vitellogenesis (3,4).
In our previous studies, we investigated the effect of this decapeptide together with
effect of its C-terminal truncated analogues (5,6) on a reproduction (development of
eggs in ovaria and hatchability of larvae) of our insect model Sarcophaga (now
Neobellieria) bullata. We found that the N-terminal tetra- and pentapeptide sequences
282
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All rights of reproduction in any form reserved.

CYCLIC ANALOGS OF INSECT OOSTATIC PEPTIDES
283
1b and 1c (6) exhibited an accelerated effect and a more pronounced oostatic activity
than the original decapeptide.
H - Tyr - Asp - Pro - Ala - X - OH
X ϭ (Pro)6
X ϭ 0
[1a]
[1b]
[1c]
X ϭ Pro
In this paper we want to report on the synthesis and the insect development inhibitory
activity of the cyclotetra- and cyclopentapeptides 2b and 2c derived from their linear
precursors 1b and 1c and also on those of the cyclodecapeptide 2a, that was detected
and separated after cyclopentapeptide synthesis as the only by-product in a ratio 1:1.
cyclo(Tyr-Asp-Pro-Ala-Pro-Tyr-Asp-Pro-Ala-Pro)
cyclo(Tyr-Asp-Pro-Ala)
[2a]
[2b]
[2c]
cyclo(Tyr-Asp-Pro-Ala-Pro)
Design of the cyclic peptides was also based on some preliminary calculations
using MOPAC 7.01 program (PM 3 method) which suggested a pseudocyclic confor-
mation in the linear tetrapeptide 1b constrained by the proximity of the Ala4 carboxyl
terminus and either Tyr1 and (or) Asp2 amino groups (Fig. 1) or Tyr1 hydroxyl group
(Fig. 2).
A discussion on relationships between structure of the cyclic peptides 2a–2c and
their oostatic activity was carried out using results of morphological and histological
1
assay and of detailed H and 13C NMR study, including linear precursors 1b and 1c.
MATERIAL AND METHODS
TPTU1 was purchased from Senn Chemicals International (Gentilly, France) and
protected amino acids were purchased from Bachem (Bubendorf, Switzerland) or
were prepared in our laboratory following general protocols (7) and were checked
1 Abbreviations used: ACN, acetonitrile; AcOH, acetic acid; COSY, 2D-NMR correlation spectroscopy;
DIC, N,N-diisopropylcarbodiimide; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide;
Et2O, diethyl ether; Fmoc, fluorenylmethyloxycarbonyl; HMQC, heteronuclear multiple quantum coher-
ence; HOBt, 1-hydroxybenzotriazole; MeOH, methanol; Me2SO, dimethyl sulfoxide; FAB MS, fast atom
bombardment mass spectroscopy; NOE, Nuclear Overhauser Effect; Pic, picric acid; ROESY, 2D-NOE in
rotating frame; tBu, tert-butyl; TFA, trifluoroacetic acid; TOCSY, total correlation 2D-NMR spectroscopy;
TPTU, 2-(2-pyridon-1-yl)-1,1,3,3-tetramethyl uronium tetrafluoroborate. The nomenclature and symbols
of amino acids follow published recommendations of the IUPAC/IUB Joint Commission on Biochemical
Nomenclature (Nomenclature and Symbolism for Amino Acids and Peptides (1984) Eur. J. Biochem.
138, 9–37).

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HLAVACEK ET AL.
284
for their purity by TLC, HPLC, elemental analysis and mass spectrometry. 2-Chlorotri-
tylchloride resin (1.3 mmol/g) was purchased from Calbiochem-Novabiochem AG
(Switzerland).
General Methods
Progress in peptide synthesis was monitored by the Kaiser (8) and bromophenol
Pic
blue (9) tests. Analytical electrophoresis E5.7 was carried out in a moist chamber
on Whatman 3MM paper (20 V/cm) in a pyridine-acetate buffer (pH 5.7) for 60 min.
The compounds were visualized by the ninhydrin or chlorine-KI-o-tolidine detection,
(10). Optical rotations were measured on a Perkin–Elmer 141 MCA polarimeter at
22ЊC and [␣]D values are given in 10Ϫ1 deg cm2 gϪ1. Solvents were evaporated in
vacuo on a rotary evaporator (bath temperature 30ЊC); DMF was evaporated at 30ЊC
and 150 Pa. The samples for amino acid analysis were hydrolyzed with 6 M HCl
containing 3% of phenol at 110ЊC for 20 h. The amino acid analyses were performed
on Biochrom 20 instrument (Pharmacia, Sweden). Molecular weights of the peptides
were determined using mass spectroscopy with FAB technique (Micromass, Manches-
ter, England). For HPLC a Spectra Physics instrument with an SP 8800 pump, an SP
4290 integrator and Thermo Separation Products Spectra 100 UV detector were used.
The compounds were purified by semipreparative HPLC on the 25 ϫ 1 cm column,
10 ␮m Vydac RP-18 (The Separations Group, Hesperia CA), flow rate 3 ml/min,
detection at 220 nm using gradient 0–100% ACN in 0.05% aqueous TFA within 60
min. The analytical HPLC was carried out on the 25 ϫ 0.4 cm column, 5 ␮m
LiChrospher WP-300 RP-18 (Merck, Darmstadt, Germany), flow rate 1 ml/min,
detection at 220 nm, using 0–100% gradient of ACN in 0.05 aqueous TFA within
60 min.
Syntheses
Solid-Phase Synthesis of Linear Precursors of Cyclopeptides 2b and 2c. Fmoc-
Ala-OH (0.85 g; 2.7 mmol) or Fmoc-Pro-OH (0.91 g; 2.7 mmol) was loaded on 2-
chlorotritylchloride resin (1 g) with the substitution 0.43 mmol/g of the resin (11).
The loading of the Fmoc-amino acids on the resin was calculated as the average of
the values determined by (a) weight increase, (b) measurement of the dibenzofulvene–
piperidine complex absorption after cleavage by 5% piperidine in DCM—DMF 1:1
for 10 min and 20% piperidine in DMF for 15 min, and (c) AAA. After the Fmoc
deprotection, the H-Ala-resin was sequentially acylated with three equivalents of
Fmoc-Pro-OH (0.44 g), Fmoc-Asp(OtBu)-OH (0.54 g) and Fmoc-Tyr(tBu)-OH (0.60
g) activated with HOBt (0.19 g; 1.4 mmol) - DIC (0.16 g; 1.3 mmol) mixture in
DMF (30 ml). The H-Pro-resin was similarly acylated with Fmoc-Ala-OH (0.41 g),
Fmoc-Pro-OH (0.44 g), Fmoc-Asp(OtBu)-OH (0.54 g), and Fmoc-Tyr(tBu)-OH (0.60
g). The Fmoc groups were removed by 20% piperidine in DMF (2x 20 ml), 10 and
30 min. After the last Fmoc deprotection, peptide-resins were washed with MeOH,
dried in desiccator and stirred with an AcOH (20 ml)–CH2Cl2 (60 ml) mixture for
2x 90 min at room temperature to detach the partially protected peptides off the resin
which was filtered off. The solutions were concentrated by evaporation of CH2Cl2,
diluted with water and were freeze dried yielding the side-chain protected derivative
of the 1b, H-Tyr(tBu)-Asp(OtBu)-Pro-Ala-OH [0.23 g; 0.39 mmol; HPLC purity

CYCLIC ANALOGS OF INSECT OOSTATIC PEPTIDES
285
81.6% and that of the 1c, H-Tyr(tBu)-Asp(OtBu)-Pro-Ala-Pro-OH [0.26 g; 0.38 mmol;
HPLC purity 86% (Table 1).
Cyclo(Tyr-Asp-Pro-Ala) (2b). H-Tyr(tBu)-Asp(OtBu)-Pro-Ala-OH (0.15 g; 0.26
mmol) in DMF (125 ml) was added dropwise to solution of HOBt (0.1 g; 0.75 mmol)
and TPTU (0.2 g; 0.67 mmol) in DMF (250 ml) at room temperature during 6 h at
pH 7 adjusted with DIEA. The DMF was evaporated to dryness, the residue was
dissolved in EtOAc and the solution was washed with 1 N NaHCO3, 20% citric acid,
brine (2x 20 ml of each) and was dried over Na2SO4. The EtOAc was evaporated to
give 0.14 g of the side-chain protected derivative of cyclopeptide 2b (HPLC peak
retention time was 30.57 min). This product was treated with a TFA (4.5 ml)–anisole
(0.5 ml) mixture at room temperature for 1 h, the TFA was evaporated, the residue
triturated with Et2O, and dissolved in 20% AcOH. The solution was freeze dried
yielding the cyclopeptide 2b (90 mg), which was purified by preparative HPLC to
1
afford 70 mg of pure peptide (Table 1); the H and 13C NMR—see Tables 2 and 3.
Cyclo(Tyr-Asp-Pro-Ala-Pro) (2c) and cyclo(Tyr-Asp-Pro-Ala-Pro)2 (2a). The reac-
tion and purification conditions described for compound 2b were used in the cycliza-
tion of H-Tyr(tBu)-Asp(OtBu)-Pro-Ala-Pro-OH (0.11 g; 0.17 mmol) in DMF (80 ml)
with HOBt (0.08 g; 0.6 mmol) and TPTU (0.11 g; 0.37 mmol) in DMF (150 ml).
The side-chain protected cyclopentapeptide (HPLC peak retention time was 31.45
min) and its cyclodecapeptide dimer (HPLC peak retention time was 44.90 min) were
detected in the reaction mixture in the ratio 1:1. A treatment of this mixture (0.1 g)
with TFA (4.5 ml)-anisole (0.5 ml) and the HPLC purification described for the
compound 2b yielded pure cyclopeptides 2c (40 mg) and 2a (30 mg) (see Table 1);
1
the H and 13C NMR—see Tables 2 and 3.
Biological Activity Assay
Peptides were separately injected in Ringer solution (1 ␮g/1 ␮l, 5 ␮l per female)
into the upper part of the thorax of Et2O-anestetized, 24-h old females of Neobellieria
bullata (Diptera). Treated female flies were placed into cages with untreated males
and dissected at regular time intervals (on days 1, 2, 8, and 15 after application). The
ovaries were checked for signs of resorption and hatchability of eggs in the uterus
during the first gonotrophic cycle. The ovaries undergoing the second gonotrophic
cycle were examined as well. Histological preparations made of suspect ovaries of
both gonotrophic cycles were prepared by standard procedure. They were stained
with Mallory’s or Mayer’s hematoxyline.
NMR Spectra Measurement
NMR spectra were measured on Varian UNITY-500 spectrometer (1H at 500 MHz,
13C at 125.7 MHz) in Me2SO-d6. Complete assignment of all proton signals was
derived from COSY, TOCSY, and ROESY experiments. The amino acid residues were
identified according to their characteristic spin-systems, scalar-coupling connectivity
patterns derived from COSY and TOCSY spectra, and NOE contacts (aromatic pro-
tons). Sequential assignment of the amino acid residues was carried out from the
analysis of ROESY spectrum (mainly NH(i ϩ 1)-H␣ (i) NOE contacts). Proton
chemical shifts and coupling constants are given in Table 2. “Attached proton test”
13C NMR spectra allowed distinguishing C, CH, CH2, and CH3 carbon signals. Their

´ ˇ
HLAVACEK ET AL.
286
1
structural assignment (except C ϭ O signals) was done using H, 13C-correlated
HMQC spectra—for data see Table 3.
RESULTS AND DISCUSSION
The synthesis of cyclopeptides 2a–2c was carried out by a cyclization of their
linear, tBu side-chain protected, tetra- and pentapeptide precursors followed by the
side-chain deprotection. The linear precursors were prepared on a solid support with
2-chlorotrityl linker (11,12), which is particularly suited for the synthesis of the
peptides containing C-terminal proline using corresponding N␣-Fmoc-amino acids.
The extreme steric hindrance of the trityl group effectively suppresses a formation
of dioxopiperazine at the dipeptide step and a premature cleavage of the peptide from
the resin. Prior to each synthetic step mediated by DIC and HOBt as coupling reagents
in DMF, the N␣-Fmoc protecting group was removed by 20% piperidine in DMF.
After the last Fmoc-deprotection, the peptides were cleaved off the resin quantitatively,
with an AcOH-CH2Cl2 mixture 1:3. The cyclization was carried out in high DMF
dilution with TPTU-HOBt coupling reagents in the presence of DIEA. At this step,
the dimerization of the linear pentapeptide occurred yielding a mixture of the side-
chain protected cyclopenta- and cyclodecapeptides. Finally, the tBu protecting groups
TABLE 1
Analytical Data on Protected Linear Peptides and Cyclopeptides 2a–2c
AAAd
e
Formulab
E5.7
Pic
Compound HPLCa
M.W./(M+ ϩ 1)
[␣]D
Tyr
Asp
Pro
Ala
c
H-Tyr(tBu)Asp(OtBu)
ProAla-OH
C29H44N4O8
576.7/577.3
ND
1.00
1.08
0.92
1.00
24.97f
H-Tyr(tBu)Asp(OtBu)
ProAlaPro-OH
C34H51N5O9
673.8/674.4
ND
0.98
0.92
0.92
0.94
1.07
0.99
0.99
0.97
1.85
1.03
2.03
1.98
1.00
1.00
1.00
1.00
26.23f
c(TyrAspProAla)
2b
C21H26N4O7
446.4/447.1
Ϫ 86.5Њ
(c 0.06)
0.26
0.21
0.19
13.72f
c(TyrAspProAlaPro)
2c
15.81
c(TyrAspProAlaPro)2
2a
22.34
C26H33N5O8
543.6/544.2
Ϫ112.8Њ
(c 0.12)
C52H66N10O16
1087.2/1087.7
Ϫ138.8Њ
(c 0.07)
a Retention time in minutes; 250ϫ4-mm column RP-18, 5 ␮m (Lichrospher WP-300, Merck, Darmstadt,
Germany), flow rate 1 ml/min, detection at 220 nm. Spectra Physics 8800 pump with an integrator SP
4290 and a UV/VIS detector TSPS 100.
b Determined with FAB MS technique (Micromas, Manchester, England).
c Measured in 1 M AcOH solution on a Perkin–Elmer 141 MCA polarimeter at 22ЊC. ND, not determined.
d Amino acid analyses were performed on a Biochrom 20 (Pharmacia, Sweden).
e Analytical electrophoresis was carried out in a moist chamber on a Whatman No. 3MM paper (20
V/cm) in a pyridine-acetate buffer (pH 5.7) for 60 min.
f 0–100% gradient of ACN in 0.05% aqueous TFA within 60 min.

CYCLIC ANALOGS OF INSECT OOSTATIC PEPTIDES
287
were removed with a TFA-anisole 9:1 mixture and the cyclopeptides 2a–2c were
separated, purified by preparative HPLC and characterized by analytical HPLC, mass
spectroscopy, amino acid analysis (Table 1) and NMR spectroscopy (Tables 2 and 3).
Morphology and Histology
In comparison with their linear precursors 1b and 1c (see Ref. 6) the cyclopeptides
2b–2c exhibited diminished oostatic activity even though the morphological and
histological observations revealed minor changes in ovarian development. Morpholog-
ically affected egg chambers had irregular appearance and their shape was distorted.
Histological evaluation revealed a division of nucleolus of the follicular cell followed
by the nuclear and cell division, and the migration of such modified cells into yolk
as well as into a region of nutritive cells causing their desintegration. In this way,
TABLE 2
1H NMR Parameters of Compounds 1b–2c in Me2SO-d6
Residue
Tyr1
Parameter
1b
1c
2ba
2c
2a
NH
J(A␣)
␣H
J(␣,␤)
␤H2
J(␤1,␤2)
H-2,6
H-3,5
OH
b
b
3.91
8.08
b
3.91
6.75
b
3.92
7.45
10.0
4.54
5.2; 10.0
2.81; 2.62
13.6
7.90
7.0
4.03
5.2; 7.2
2.95; 2.83
14.2
5.8; 7.4
2.95; 2.82
14.2
3.6; 9.8
2.94; 2.70
14.2
3.6; 10.0
3.03; 2.96
13.6
6.99
6.69
9.41
8.90
6.99
6.69
9.38
8.90
7.04
6.69
8.95
8.06
6.89
6.64
9.23
8.36
6.92
6.64
9.19
7.65
Asp2
NH
J(A␣)
␣H
7.8
4.83
7.8
4.83
b
4.73
8.0
4.69
7.6
4.76
J(␣,␤)
␤H2
J(␤1,␤2)
COOH
␣H
6.9; 6.8
2.77; 2.48
16.8
6.4; 7.2
2.76; 2.46
16.9
12.47
4.31
7.6; 5.2
2.88; 2.33
16.0
9.4; 3.6
2.63; 2.43
16.0
12.25
4.34
11.2; 3.6
2.73; 2.34
16.2
12.25
4.44
b
4.33
b
4.24
Pro3
Ala4
Pro5
␤H2
␥H2
␦H2
1.75–2.00
1.75–2.00
3.56; 3.38
—
1.95
1.83
3.58; 3.42
—
2.09; 1.95
1.79; 1.64
3.44 (2H)
—
2.09; 1.46
1.66; 1.38
3.42 (2H)
—
1.93
1.83
3.69; 3.54
—
␺-(CH2X)
NH
J(A␣)
␣H
J(␣,␤)
␤-CH3
␣H
7.99
7.4
4.24
7.3
1.26
—
7.96
7.5
4.47
7.0
1.17
4.21
7.96
b
4.20
6.7
1.06
—
6.37
6.8
4.22
6.4
1.11
4.23
7.70
7.2
4.47
7.0
1.20
3.93
␤H2
␥H2
␦H2
—
—
—
1.90; 1.85
2.13
3.60; 3.48
—
—
—
1.98; 2.11
1.87; 1.75
3.59; 3.42
1.93
1.83; 1.59
3.45; 3.38
a Data were obtained at 80ЊC.
b The value of parameter could not be determined.

´ ˇ
288
HLAVACEK ET AL.
TABLE 3
13C NMR Chemical Shifts of Compounds 1b–2c in Me2SO-d6
Residue
Tyr1
Carbon
1b
1c
2ba
2c
2a
CO
C␣
C␤
171.68b
53.54
36.16
124.68
130.67
115.43
156.79
171.05b
47.66
36.13
174.16b
168.50b
59.37
29.12
24.22
46.78
—
170.76b
53.53
36.15
124.66
130.67
115.43
156.79
170.38b
47.69
171.95b
57.70
36.55
127.66
129.58
115.12
155.83
171.71b
48.75
36.09
ϳ171.0b
170.86b
60.33
31.16
21.12
45.60
—
170.63b
55.24
37.74
126.83
130.25
115.09
156.21
170.46b
48.04
171.95b
55.79
35.89
128.55
130.05
115.14
155.99
171.83b
48.42
C1
C2, C6
C3, C5
C4
CO
C␣
C␤
COOH
CO
C␣
C␤
Asp2
Pro3
36.09
36.01
34.53
173.44b
168.59b
59.48
172.15b
170.42b
61.46
172.08b
170.92b
61.59
29.20
24.72
46.38
31.58
22.10
46.72
28.78
25.16
46.69
C␥
C␦
␺-(CH2X)
CO
—
—
—
Ala4
Pro5
167.88b
47.64
17.25
—
167.68b
46.86
170.70b
46.95
16.22
—
170.39b
46.82
170.12b
47.15
C␣
C␤
CO
C␣
16.90
18.19
17.18
171.59b
58.68
169.70b
60.98
168.37b
60.04
—
—
C␤
—
28.75
—
31.30
27.96
C␥
—
24.32
—
21.89
24.44
C␦
—
46.20
—
46.60
46.65
a Data were obtained at 80ЊC.
b The chemical shift values may be mutually interchanged.
the whole inner space of the egg chamber was filled up by those proliferating cells.
Finally, resorption of such affected egg chamber occurred. Approximately only 10%
of egg chambers was resorbed after cyclopeptides application and the changes appeared
mostly in the development of the second gonotrophic cycle in 15 days assay. The
dimeric cyclopeptide 2a, however, showed no signs of the biological effect.
NMR Study
After the bioassay observation has shown only low oostatic activity of the cyclopep-
tides 2b and 2c and no activity of the 2a, we started to be suspicious about results
of the space-structure computation performed with linear peptide 1b, which suggested
the pseudocyclic conformation constrained by the proximity of the Tyr1 or Asp2 and
the Ala4 residues (Figs. 1 and 2). We tried to find an explanation for such diminished
activity using NMR spectroscopy. This study was carried out with the series of
peptides containing also corresponding linear tetra- and pentapeptides 1b and 1c,
which were reported to exhibit strong oostatic activity when injected into fly

CYCLIC ANALOGS OF INSECT OOSTATIC PEPTIDES
289
FIG. 1. Calculated conformation of H-Tyr-Asp-Pro-Ala-OH with H-bond between amino Tyr1 and
(or) Asp2 and carboxyl Ala4 groups (MOPAC 7.01).
FIG. 2. Calculated conformation of H-Tyr-Asp-Pro-Ala-OH with H-bond between hydroxyl Tyr1 and
carboxyl Ala4 groups (MOPAC 7.01).

´ ˇ
HLAVACEK ET AL.
290
Neobellieria bullata (6)
It is well known that proline-containing peptides can show a presence of two
isomers with trans- and cis-X-Pro peptide bond in solution. Both isomers can be
distinguished by characteristic NOE contacts: trans-isomer has contact between H␣
of X and Ho¯ of Pro residue while cis-isomer exhibits contact between H␣ protons of
X and Pro residue. The existence of isomers with trans- and cis-X-Pro was indeed
observed in linear peptides 1b and 1c in Me2SO-d6. In all cases the isomer with trans-
X-Pro bonds significantly prevails (more than 80%). Although the occurrence of some
NOE contacts in ROESY spectra (e.g., between aromatic protons of Tyr1 and H␣ and
Ho¯ of Pro3 in 1b and 1c (see Fig. 3) indicates certain tendency to the folding, the
peptide chain is general flexible and rapid conformational interconversion occurs.
The ring closure in cyclic peptides 2b and 2c reduces the conformation flexibility
dramatically. The NMR spectra of cyclotetrapeptide 2b in Me2SO-d6 showed extremely
broadened signals at room temperature (observed also in deuterated water and AcOH),
probably due to slow conformation interconversion. The increasing temperature led
to the successive signal narrowing so that the spectra at 80ЊC could be at least partially
analyzed. In contrast, the cyclopentapeptide 2c and its dimeric analogue 2a provided
well-resolved configurationally homogeneous spectra already at room temperature.
While in cyclopentapeptide 2c we found that both X-Pro bonds are cis- (and we
FIG. 3. Interresidual NOE contacts observed in linear tetrapeptide 1b and pentapeptide 1c.

CYCLIC ANALOGS OF INSECT OOSTATIC PEPTIDES
291
FIG. 4. Interresidual NOE contacts observed in cyclopentapeptide 2c.
suppose cis-X-Pro bond also for cyclotetrapeptide 2b) in its dimeric form 2a all X-
Pro bonds adopt trans-configuration. The observed values of temperature coefficients
of NH protons in 2c (Ϫ1.3, Ϫ2.1, and ϩ0.2 ppb/deg in Tyr, Asp and Ala, respectively)
indicate that they are hydrogen-bonded and/or at least significantly shielded from
solvent. A series of inter-residual NOE contacts in 2c (see Fig. 4) indicates a restricted
space structure of the cyclic molecule. Significantly larger temperature coefficients
of NH protons in 2a (Ϫ5.2, Ϫ2.6, and Ϫ1.8 ppb/deg in Tyr, Asp, and Ala, respectively)
correspond to the expected enhanced mobility of larger ring of the cyclodecapeptide 2a.
The biological assay and consequently the NMR studies revealed that a restricted
space structure of cyclopeptides 2b and 2c is not acceptable for bioactive conformation
necessary in receptor binding and therefore only weak oostatic activity was observed
after their application. Linear peptides 1b and 1c showed some population of pseudocy-
clic conformation at their aminoterminal tripeptide part (see NOE contacts in ROESY
spectra between aromatic protons of Tyr1 and H␣ and H␤ of Pro3 residues) conferring
them to interact with corresponding receptor and to initialize the biological effect. In
addition to their certain tendency to the folding, however, the general flexibility of
these peptide chains with rapid conformational interconversion deduced from NMR
spectra seems to be important for proper peptides binding to receptor. Therefore the
reduced conformation flexibility induced by cyclization of linear oostatic peptides
has led to substantial diminution of biological activity. The lack of the oostatic activity
in the case of the more flexible dimeric analogue 2a could be ascribed to the size of
its molecule and its overall shape that is not compatible with a receptor binding.
ACKNOWLEDGMENTS
This research was supported by grants of the Grant Agency of the Czech Republic (203/98/0741, 203/
98/1330) and by Ministry of Education of Czech Republic (LB98233).

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292
HLAVACEK ET AL.
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