Synthetic and biological activity studies on a new cyclic pentapeptide, cyclonitroproctolin

Article abstract of DOI:10.1515/znb-2005-1216

The synthesis of a new biologically active cyclic pentapeptide, cyclonitroproctolin, cyclo(-Arg (NO₂)-Tyr-Leu-Pro-Thr-) by solution phase synthesis is described. The structure of the synthetic peptide was characterized by IR, NMR, FAB mass and

Full text of DOI:10.1515/znb-2005-1216

Synthetic and Biological Activity Studies on a New Cyclic Pentapeptide,
Cyclonitroproctolin
Boja Poojarya and Shiddappa L. Belagalib
a Department of Chemistry, Mangalore University, Mangalagangotri-574 199, India
b Department of Environmental Sciences, Mysore University, Mysore-575 005, India
Reprint requests to Dr. B. Poojary. Fax: 91 824 2287 367. E-mail: bojapoojary@yahoo.com
Z. Naturforsch. 60b, 1308 – 1312 (2005); received November 10, 2004
The synthesis of a new biologically active cyclic pentapeptide, cyclonitroproctolin, cyclo(-Arg
(NO2)-Tyr-Leu-Pro-Thr-) by solution phase synthesis is described. The structure of the synthetic
peptide was characterized by IR, NMR, FAB mass and analytical data. The newly synthesized com-
pound was screened for its antimicrobial and pharmacological activities.
Key words: Cyclonitroproctolin, Antimicrobial Activity, Pharmacological Activity, p-Nitrophenyl
Ester Method
Introduction
In continuation of our research work of synthesizing
cyclic peptides of biological interest [13], it was con-
templated to synthesize nitroproctolin, H-Arg(NO2)-
Tyr-Leu-Pro-Thr-OH (6) and then cyclize it to yield
cyclonitroproctolin (7) using the p-nitrophenyl ester
method. The synthesized product was further evaluated
for antimicrobial, antiinflammatory and anthelmintic
activities.
It is reported in the literature that a wide variety
of naturally occurring bioactive cyclic peptides have
been isolated from plants, marine sponges and tuni-
cates [1] in the past two decades. Recently a large
number of these cyclic peptides have been emerging
as an important class of organic compounds due to
their unique structures and biological activities. The
wide spread increase of bacterial resistance towards
conventional antibiotics encourages the exploration of
novel antimicrobial molecules with unexploited mech-
anisms. Initially discovered as a defensive system in
invertebrates and vertebrates, antimicrobial peptides
are attracting increased interest as potential therapeu-
tics [2 – 4]. Unlike classical antibiotics, which must
penetrate the target cell, the principle mode of ac-
tion of peptides involves perturbation and permeabi-
lization of the cell membrane. This mechanism con-
fers activity towards a broad spectrum of microbial
cells, but is also responsible for undesired lytic ac-
tivity against mammalian cells such as erythrocytes
[5 – 7].
Results and Discussion
In order to carry out the total synthesis of cycloni-
troproctolin, two dipeptide units Boc-Tyr-Leu-OMe
(1) and Boc-Pro-Thr-OMe (2) were prepared by cou-
pling corresponding Boc-L-amino acids with the re-
spective L-amino acid methyl ester hydrochlorides us-
ing DCC, HOBt and N-methyl morpholine according
to Bodanszky procedure [16] with suitable modifica-
tions [14]. The required Boc-amino acids [14] and
amino acid ester hydrochlorides [15] were prepared
by the reported procedures. The protected amino acid
unit, Boc-Arg(NO2)-OH (3) was also prepared [17].
The ester group of the dipeptide 1 was removed with
LiOH and the Boc-group of the dipeptide 2 was re-
moved with trifluoro acetic acid (TFA). Both the de-
protected units were coupled to obtain Boc-Tyr-Leu-
Pro-Thr-OMe (4), which was then coupled with Boc-
Arg(NO2)-OH (3) after appropriate deprotection to
obtain Boc-Arg(NO2)-Tyr-Leu-Pro-Thr-OMe (5). The
guanidino group of arginine was protected by nitration
to prevent the side reaction. Finally, cyclization of lin-
In 1975, Brown and his group reported the isolation
of a myotropic substance from the cockroach, Perpla-
centa americana which was identified as H-Arg-Tyr-
Leu-Pro-Thr-OH [8, 9]. The synthesis of proctolin was
also undertaken by Brown et al. [10]. Further studies
on this compound have shown that proctolin functions
as an excitatory neuromuscular transmitter in insect
visceral muscle [11, 12].
c
0932–0776 / 05 / 1200–1308 $ 06.00 ꢀ 2005 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
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B. Poojary – S. L. Belagali · Cyclonitroproctolin
1309
Scheme 1. a = DCC, NMM,
HOBt, DCM, RT/36 h; b = LiOH,
THF:H2O(1:1), RT/1 h; c = TFA,
CHCl3, RT/1 h; d = p-nitrophenol
(pnp), DCC, CHCl3, RT/12 h; e =
pyridine, CHCl3, 10 days/0 ◦C.
ear pentapeptide 5 was carried out by the p-nitrophenyl clization of linear segment. Characteristic IR absorp-
ester method [18] to obtain cyclonitroproctolin7 as de- tion band of nitro group showed the presence of nitro-
picted in Scheme 1.
The Boc and methyl ester protecting groups of 5
were also removed to yield nitroproctolin (6). The in-
protected arginine intact in the title compound. The
FAB mass spectrum showed the (M+ +1) peak at m/z
676, which is consistent with the molecular formula,
termediates and final products were purified by column C30H45N9O9.
chromatography using a dichloromethane-methanol
system and recrystallized from EtOAc-n-hexane.
Biological Activity Studies
The structures assigned to the intermediates and the
final products are in good agreement with elemental
Cyclonitroproctolin was screened for its antibacte-
1
analyses, IR, H NMR and mass spectral data. The rial, antifungal, anti-inflammatory and anthelmintic ac-
characteristic IR absorption bands of –CO-NH- and tivity. The antibacterial and antifungal activity were
–NO2 moiety were present in the cyclized product. carried out against four bacterial (S. aureus, B. subtilis,
1H NMR spectrum of the cyclized product clearly in- P. aeruginosa and E. coli) and two fungal strains(C. al-
dicated the presence of all respective amino acid moi- bicans and A. niger). These activity studies were car-
eties. The absence of peaks corresponding to the p- ried out by the disc diffusion method [18]. Penicillin
1
nitrophenyl ester group in the IR, H NMR and mass and griseofulvin were used as standards against bac-
spectra of the cyclic peptide clearly confirmed the cy- teria and fungal strains at 50 µg/disc respectively. To
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1310
B. Poojary – S. L. Belagali · Cyclonitroproctolin
Table 1. Antibacterial and antifungal activity data of nitro-
proctolin and cyclonitroproctolin.
thesized by solution phase method. The antimicrobial
results indicated the better activity of synthetic cyclic
peptide than its linear analogue and it proved to be a
good antimicrobial agent. Evaluation of antiinflamma-
tory activity of it revealed good activity as compared
to the standard drug, Ibuprofen.
Compound
Diameter of zone of inhibition
Antibacterial
activity data
Antifungal
activity data
P.aer E.coli B.sub S.aur C.alb A.niger
Nitroproctolin (6)
Cyclonitroproctolin (7) 20
Penicillin
Griseofulvin
14
12
14
12
–
18
20
18
–
12
16
18
–
18
20
–
12
18
–
12
–
Experimental Section
20
20
Melting points were taken in open capillary and are
uncorrected. IR spectra (in CHCl3) were recorded on
a Perkin-Elmer infrared spectrophotometer. NMR spectra
were recorded in CDCl3/DMSO-d6 on a 300 MHz spec-
trophotometer using TMS as an internal standard. The mass
spectra were recorded on a FAB mass spectrometer. The pro-
gresses of the reactions were checked by TLC on silica gel
G plates and the products were purified by silica gel column
chromatography.
Table 2. Antiinflammatory activity data of cyclonitroproct-
olin.
Compound
Increase in
paw volume after
3 h S E [ml]
0.55 0.03
Percentage inhibition
of oedema after
3 h S E
39.56
Cyclonitroproctolin (7)
Ibuprofen
0.55 0.04
40.50
Control
0.91 0.02
–
For the protection of amino group of amino acids, di-
tertiary butyl pyrocarbonate(Boc-O-Boc) was used. The car-
boxy group of amino acids was protected by esterification.
Bodanszky procedure with modification was used for the
synthesis of peptides. Boc-group was removed by stirring the
Boc-amino acid/peptide (1 mmol) with CF3CO2H (2 mmol)
in CHCl3 (15 ml) for 1 h at r. t. and the ester group was
removed by stirring the amino acid/peptide methyl ester
(1 mmol) with LiOH (1.5 mmol) in (1:1) THF:H2O (16 ml)
for 1 h at r. t. The p-nitrophenyl ester method was employed
for cyclization of the linear segment 5.
Table 3. Anthelmintic activity data of cyclonitroproctolin.
Compound
Conc. of the Mean paralyzing Mean death time
Compd (mg) time(min) S E
(min) S E
99.24 2.03
89.94 2.15
54.40 1.08
31.04 1.09
–
Cyclonitro-
proctolin (7)
Mebendazole
100
200
100
200
–
65.30 2.01
58.60 2.05
17.44 0.09
12.14 1.02
–
Control
compare and contrast, the antibacterial and antifungal
activity of linear and cyclic nitroproctolin was carried
out at 50 µg/disc. The results summarized in Table 1
indicate that the cyclized product shows good activ-
ity in comparison to the linear segment. The results
also indicate that compound shows moderate to ex-
cellent activity against the bacterial and fungal strains
tested as compared with the standards used. The anti-
inflammatory activity was carried out according to the
method of Winter et al. [19] using Ibuprofen as the
standard and the results are presented in Table 2. The
anti-inflammatory data reveal that the compound pos-
sesses good anti-inflammatory activity in comparison
with the standard test compound. The anthelmintic ac-
tivity was carried out against the earthworms (Pon-
toscolex corethruses) according to Garg’s method [20]
using Mebendazole as standard drug (Table 3). The
compound is found to be moderately active as com-
pared to the standard.
Boc-tyrosinyl-leucine methyl ester (1)
IR(CHCl3): ν = 3500 (br.s, O-H str.), 3200 (br.s, N-H
str.), 3050 (m,=C-H str.), 2980 (m, C-H str.), 2820 (m, C-H
str.),1710 (s, C=O str. ester), 1670 (s, C=O str. amide), 1610
(m, C=C str.), 1445 (s, C-H def.), 1420 (m, C-N str.), 1395
(s, N-H def.), 1200 (s, C-O str.), 1150 (m, C-O str.), 915 –
850 (m, C-H def.) cm−1. – 1H NMR (300 MHz, CDCl3):
δ = 8.9 (br.s, 1H, -NH), 7.0 (d, 2H, J = 6.5 Hz, Ar-C8H &
-C5H), 6.85 (d, 2H, J = 6.5 Hz, Ar-C2H & -C6H), 6.4 (br.s,
1H, -NH), 6.1 (br.s, 1H, -NH), 4.9 – 4.7 (m, 1H, α-CH),
4.5 – 4.4 (m,1H, α-CH), 3.75 (s, 3H, -OCH3), 3.1 – 3.0 (m,
t
2H, β-CH2), 1.6 – 1.5 (m, 2H, -CH2), 1.4 (s, 9H, Boc),
1.2 – 1.1 (m, 1H, -CH-), 0.95 (d, 6H, J = 6.5 Hz, -CH3). –
C21H32N2O6 (330): calcd. C 61.76, H 7.84, N 6.86; found:
C 61.82, H 7.90, N 6.84.
Boc-prolyl-threonine methyl ester (2)
IR(CHCl3): ν = 3400 (br.s, O-H / N-H str.), 2950 (m, C-H
str.), 2890 (m, C-H str.), 1720 (s, C=O str. ester), 1665 (s,
C=O str. amide), 1610 (m, C=C str.), 1450 (s, C-H def.),
1420 (m, C-N str.), 1405 (s, N-H def.), 1150 (s, C-O str.),
Conclusion
In conclusion, a new biologically active cyclic pen-
tapeptide, cyclonitroproctolin, was successfully syn- 1100 (m, C-O str.) cm−1. – 1H NMR (300 MHz, CDCl3):
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B. Poojary – S. L. Belagali · Cyclonitroproctolin
1311
δ = 6.1 (br.s, 1H, -NH), 5.15 (s, 1H, -OH), 4.9 – 4.4 (m, 2H, General procedure for the synthesis of cyclonitroproctolin
β-CH & α-CH), 4.3 – 4.1 (m,1H, α-CH), 3.7(s, 3H, -OCH3), (6)
3.6 – 3.2 (m, 2H, N-CH2), 2.3 – 1.6 (m, 4H, -CH2- CH2), 1.4
(s, 9H, Boc), 1.1 (d, 3H, J = 6.5 Hz, CH3). -C15H26N2O6
(330): calcd. C 54.54, H 7.87, N 8.48; found C 54.50, H 7.85,
N 8.44.
To the solution of Boc-nitroargininyl-tyrosinyl-leucyl-
prolyl-threonine-p -nitrophenyl ester (1.2 mmol) in chloro-
form (15 ml), trifluoroacetic acid (0.274 g, 2.4 mmol) was
added, stirred for 1 h at r. t. and washed with 10% sodium
bicarbonate solution. The organic layer was dried over an-
hydrous sodium sulphate. To the resulting Boc-deprotected
peptide-p-nitrophenyl ester in tetrahydrofuran (15◦ml), pyri-
dine (1.4 ml, 2 mmol) was added and kept at 4 C for ten
days. The reaction mixture was washed with 10% sodium
bicarbonate solution until the byproduct p-nitrophenol was
removed completely and finally washed with 5% HCl (5 ml).
The organic layer was dried over anhydrous sodium sul-
phate. Tetrahydrofuran and pyridine were distilled under re-
duced pressure to get cyclonitroproctolin. The crude product
was purified by silica gel column chromatography using the
dichloromethane-methanol system and finally recrystallized
from EtOAc-n-hexane.
t
Boc-tyrosinyl-leucyl-prolyl-threonine methyl ester (4)
IR(CHCl3): ν = 3600 (br.s, O-H str.), 3200 (br.s, N-H
str.), 3080 (m, =C-H str.), 2920 (m, C-H str.), 2880 (m, C-H
str.), 1740 (s, C=O str. ester), 1690 (s, C=O str. amide),
1670 (s, C=O str. amide), 1450 (s, C-H def.), 1430 (m, C-N
str.), 1390 (s, N-H def.), 1300 (s, C-O str.), 1250 (m, C-O
1
str.), 950 – 850 (m, C-H def.) cm−1. – H NMR (300 MHz,
CDCl3): δ = 8.2 (br.s, 2H, -NH), 7.6 (br.s, 1H, -NH), 7.2 (d,
2H, J = 6.5 Hz, Ar-C8H & -C5H), 6.95 (d, 2H, J = 6.5 Hz,
Ar-C2H & -C6H), 6.5 (br.s, 1H, -NH), 5.2 (s, 1H, -OH), 4.9 –
4.8 (m, 1H, β-CH), 4.65 – 4.4 (m, 3H, α-CH), 4.3 – 4.0 (m,
2H, α-CH), 3.75 (s, 3H, -OCH3), 3.6 – 3.2 (m, 4H, N-CH2 &
β-CH2), 2.2 – 1.6 (m, 4H, -CH2- CH2), 1.6 – 1.5 (m, 1H,
◦
Pale white solid; m.p. 153 – 155 C. – IR(CHCl3): ν =
t
β-CH2), 1.4 (s, 9H, Boc), 1.3 (d, 3H, J = 6.5 Hz, -CH3),
3600 (br.s, O-H str.), 3325 (br.s, N-H str.), 3020 (m, =C-H
str.), 2920 (s, C-H str.), 2850 (s, C-H str.), 1690 (s, C=O
str. amide), 1680 (s, C=O str. amide), 1660 (s, C=O str.
amide), 1610 (s, C=N str.), 1540 (s, NO2), 1450 (s, C-H def.),
1410 (m, C-N str.), 1400 (s, N-H def.), 1380 (s, NO2), 1110
(m, C-O str.), 970 – 915 (m, C-H def.) cm−1. – 1H NMR
(300 MHz, DMSO-d6): δ = 8.2 (br.s, 2H, -NH / OH), 7.6
(br.s, 3H, -NH), 7.0 (d, 2H, J = 6.5 Hz, Ar-C8H & -C5H), 6.9
(d, 2H, J = 6.5 Hz, Ar-C2H & -C6H), 6.4 (br.s, 3H, -NH), 5.2
(s, 1H, -OH), 4.7 – 4.5 (m, 4H, α-CH & β-CH), 4.4 – 4.2 (m,
3H, α-CH), 3.5 – 3.1 (m, 4H, N-CH2 & β-CH2), 2.2 – 1.7 (m,
8H, -CH2-CH2 & -CH2), 1.6 – 1.4 (m, 4H, β-CH2 & -CH2),
1.2 – 1.1 (m, 4H, γ-CH & -CH3), 0.95 (d, 6H, J = 6.5 Hz,
C(CH3)2). – FAB mass: m/z 676 [M++ H]. -C30H45N9O9
(675): calcd. C 53.30, H 6.60, N 18.65; found C 53.38,
H 6.66, N 18.74.
1.2 – 1.1 (m, 1H, -CH-), 0.98 (d, 6H, J = 6.5 Hz, -CH3).
-C30H46N4O9 (606): calcd. C 59.40, H 7.59, N 9.24; found
C 59.40, H 7.59, N 9.24.
Boc-nitroargininyl-tyrosinyl-leucyl-prolyl-threonine methyl
ester (5)
IR(CHCl3): ν = 3600 (br.s, O-H str.), 3320 (br.s, N-H
str.), 3030 (m, =C-H str.), 2930 (m, C-H str.), 2880 (m, C-H
str.), 1750 (s, C=O str. ester), 1690 (br.s, C=O str. amide),
1670 (s, C=O str. amide), 1530 (s, NO2), 1450 (s, C-H def.),
1420 (m, C-N str.), 1400 (s, N-H def.), 1380 (s, NO2), 1250
(s, C-O str.), 1110 (m, C-O str.), 970-870 (m, C-H def.)
cm−1. – 1H NMR (300 MHz, CDCl3): δ = 8.3 (br.s, 2H,
-NH), 7.6 (br.s, 2H, -NH), 7.1 (d, 2H, J = 6.5 Hz, Ar-C8H & -
C5H), 6.8 (d, 2H, J = 6.5 Hz, Ar-C2H & -C6H), 6.4 (br.s, 3H,
-NH), 5.2 (s, 1H, -OH), 4.9 – 4.7 (m, 4H, α-CH & β-CH),
4.6 – 4.4 (m, 3H, α-CH), 3.37 (s, 3H, O-CH3), 3.5 – 3.2 (m,
6H, N-CH2 & β-CH2), 2.2 – 1.8 (m, 4H, -CH2- CH2), 1.65 –
Acknowledgements
t
1.5 (m, 6H, N-CH2 & β-CH2), 1.45 (s, 9H, Boc), 1.3 (d,
The authors are grateful to Ms. Beena Mascarenhas, KMC
3H, J = 6.5 Hz, -CH3), 1.2 – 1.1 (m, 1H, -CH), 0.98 (m, Hospital, Manipal for biological screening of the cyclic pep-
9H, 3 -CH3). – C36H57N9O12 (807): calcd. C 53.50, H 7.00, tides. This work was supported by Mangalore University
N 15.58; found C 53.56, H 7.10, N 15.63.
from UGC minor research grant.
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