Synthesis of a new cyclic peptide, pseudostellarin G

Article abstract of DOI:10.1515/znb-2004-0711

A new biologically active cyclic peptide, Pseudostellarin G was synthesized and the structure was established on the basis of analytical, IR, NMR and mass spectral data. The newly synthesized compound was screened for its antimicrobial and pharmacological

Full text of DOI:10.1515/znb-2004-0711

Synthesis of a New Cyclic Peptide, Pseudostellarin G
Boja Poojarya, K. Harish Kumar, and S. L. Belagalib
a Department of Post-Graduate Studies and Research in Chemistry, Mangalore University,
Mangalagangotri-574 199, Mangalore, India
b Department of Environmental Sciences, University of Mysore, Mysore-575 006, India
Reprint requests to Dr. B. Poojary. Fax: 91 824 2287 367. E-mail:bojapoojary@yahoo.com
Z. Naturforsch. 59b, 817 – 820 (2004); received January 10, 2004
A new biologically active cyclic peptide, Pseudostellarin G was synthesized and the structure was
established on the basis of analytical, IR, NMR and mass spectral data. The newly synthesized com-
pound was screened for its antimicrobial and pharmacological activities.
Key words: Cyclic Peptides, Pseudostellarin G, Antimicrobial Activity, Pharmacological Activity
Introduction
thesized product was further subjected to antibacterial
and pharmaceutical activity studies.
In the past two decades, a wide variety of natu-
rally occurring bioactive cyclic peptides have been iso-
lated from plants, marine sponges and tunicates [1].
Recently a large number of these cyclic peptides are
emerging as an important class of organic compounds
due to their unique structure and biological activities.
The wide spread increase of bacterial resistance to-
wards conventional antibiotics encourages the explo-
ration of novel antimicrobial molecules with unex-
ploited mechanisms. Initially discovered as a defen-
sive system in invertebrates and vertebrates, antimicro-
bial peptides are attracting increase interest as potential
therapeutics [2 – 4]. Unlike classical antibiotics, which
must penetrate the target cell, the principle mode of
action of peptides involves perturbation and permeal-
ization 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
Pseudostellarin G is cyclo(-Pro-Phe-Ser-Phe-Gly-
Pro-Leu-Ala-), a cyclic octa-peptide. In order to carry
out the total synthesis of this cyclic peptide, four
dipeptide units Boc-Phe-Pro-OMe (1), Boc-Ser-Phe-
OMe (2), Boc-Gly-Pro-OMe (3) and Boc-Leu-Ala-
OMe (4) were prepared by coupling Boc-amino acids
with the respective amino acid ester hydrochlorides
using DCC, HOBt and N-methyl morpholine accord-
ing to Bodanszky procedure [10] with suitable mod-
ifications [11]. The ester group of dipeptide (1) was
removed with LiOH and the Boc-group of dipeptide
(2) was removed with trifluoroacetic acid. Both the
deprotected units were coupled to get the tetrapep-
tide, Boc-Pro-Phe-Ser-Phe-OMe (5). The remaining
two dipeptides (3 and 4) were also coupled similarly
to obtain the another tetrapeptide, Boc-Gly-Pro-Leu-
Ala-OMe (6). These tetrapeptides are the coupled af-
ter proper deprotection using DCC, HOBt and NMM
Recently, Itakawa et al. [8] isolated a new biologi-
cally active cyclic peptide Pseudostellarin G from the to get the octapeptide, Boc-Pro-Phe-Ser-Phe-Gly-Pro-
rots of Pseudostellaria heterophylla and the structure Leu-Ala-OMe (7). Finally, the cyclization of the lin-
was elucidated by extensive NMR, chemical and enzy-
matic degradations and mass spectrometric analysis.
ear segment was carried out by the p-nitrophenyl ester
method [12] as depicted in Scheme 1. The intermedi-
In continuation of our research work of synthesiz- ates and final product were purified by column chro-
ing natural cyclic peptides of biological interest [9], matography using dichloromethane-methanol system
an attempt was made towards the synthesis of Pseu- and recrystallized from EtOAc-n-hexane. The newly
dostellarin G to prove its identity with naturally iso- synthesized compounds was analyzed for C,H,N and
1
lated one. Keeping in view of significant biological ac- the structure was confirmed by IR, H NMR and mass
tivities exhibited by various cyclic peptides, the syn- spectral data. The characteristic IR and NMR spectra
c
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B. Poojary et al. · Synthesis of a New Cyclic Peptide, Pseudostellarin G
Table 1. Antimicrobial activity data of Pseudostellarin G.
Compound
Diameter of zone of inhibition
Antibacterial Antifungal
activity studies
P.aer E.coli B.sub S.aur C.alb A.niger
Pseudostellarin G 8
Penicillin
Greseofulvin
–
12
–
14
12
–
–
18
–
16
18
–
09
–
20
10
–
20
‘–’ indicates no activity.
Table 2. Antiinflammatory activity data of Pseudostellari G.
Compound
Increase in
paw volume SE (ml)
after 3 hr
Inhibition of
oedema (%)
after 3 hr
24.45
Pseudostellarin G 8
Ibuprofen
0.68 0.04
0.55 0.03
38.89
Control
0.90 0.04
–
‘–’ indicates no activity.
Table 3. Anthelmintic activity data of Pseudostellari G.
Compound
Conc of the Mean paralyzing Mean death time
compd (mg) time SE (min)
SE (min)
74.30 1.10
64.05 1.08
55.20 2.00
32.01 1.10
–
Pseudostellarin G
Mebendazole
Control
100
200
100
200
–
42.15 1.49
30.12 2.06
18.01 2.01
12.55 1.02
–
S. aureus. The anti-inflammatory activity was carried
out according to the method of Winter et al. [14] using
Ibuprofen as the standard and the results are presented
in Table 3. The antiinflammatory data reveals that the
compound is moderately active. The anthelmintic ac-
tivity was carried out against the earthworms (pon-
toscotex corethruses) according to Garg’s method [15]
(Table 4) using Mebendazole as standard drug. The
compound is found to be less active as compared to
the standard.
Scheme 1.
of all the intermediate compounds were analyzed. The
characteristic IR absorption bands of –CO-NH- moi-
ety were present in the cyclized product. H NMR and
1
13C NMR spectra of all the cyclized product clearly in-
dicates the presence of all respective amino acid moi-
eties. Further more, the mass spectra of Pseudostellarin
G showed the [M++H] peak at m/z 817 which is con-
sistent with the molecular formula C42H56N8O9.
Experimental Section
Biological Activity Studies
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 CHCl3-d6/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.
The four dipeptides, Boc-Pro-Phe-OMe (1), Boc-Ser-Phe-
OMe (2), Boc-Gly-Pro-OMe (3) and Boc-Leu-Ala-OMe (4)
were prepared and coupled, after proper deprotection using
LiOH and trifluoroacetic acid according to Bodanszky proce-
The synthesized cyclic peptide, Pseudostellarin G
was also screened for its antibacterial, antifungal, anti-
inflammatory and anthelmintic activity. The antibacte-
rial and antifungal activity are carried out against four
bacterial (S. aureus, B. subtilis, P. aeruginosa and E.
coli) and two fungal strains (C. albicans and A. niger).
These activity studies were carried out according to
disc diffusion method [13]. Penicillin and Griseoful-
vin were used as standards against bacteria and fungal
strains at 10 and 25 µg/disc respectively. The results
summarized in Table 2 indicates that the compound
is active against only the bacterial strains E. coli and dure with suitable modifications [11] to get two tetrapeptides,
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B. Poojary et al. · Synthesis of a New Cyclic Peptide, Pseudostellarin G
819
Boc-Pro-Phe-Ser-Phe-OMe (5) and Boc-Gly-Pro-Leu-Ala- N-CH2), 3.2 – 3.0 (m, 4H, β-CH2), 2.2 – 1.8 (m, 8H, -CH2-
OMe (6). The resulting tetrapetides were then condensed to CH2-), 1.7 – 1.6 (m, 2H, -CH2), 1.5 (d, 3H, J = 7.5 Hz, CH3),
obtain the linear segment of Pseudostellarin G, Boc-Pro-Phe-
Ser-Phe-Gly-Pro-Leu-Ala-OMe (7) according to the proce-
dure used for tetrapeptides.
1.45 (s, 9H, C (CH3)3), 1.3 – 1.1 (m, 1H, γ-CH), 0.95 (d,
6H, J = 6.5 Hz, -C(CH3)2). – C48H68N8O12 (949.11): calcd.
C 60.74, H 7.22, N 11.81; found C 60.64, H 7.02, N 11.74.
Boc-Pro-Phe-Ser-Phe-OMe (5)
Pseudostellarin G (8)
IR(CHCl3): ν=3610 (br.s, O-H str.), 3420 (br.s, N-H str.),
3050 (m, =C-H str.), 2940 (s, C-H str.), 2770 (s), 1710 (s,
C=O str. ester), 1685 (s, C=O str. amide), 1670 (s, C=O
str. amide), 1650 (s, C=O str. amide), 1610 (s), 1600 (m),
1520 (s), 1500 (m), 1450 (s), 1370 (s), 1330 (s), 1230 (s),
1170 (s), 1020 (s), 1000 (s), 670 (s) cm−1. – 1H NMR
(300 MHz, CDCl3): δ = 8.6 (br.s, 1H, NH), 8.3 (br.s, 1H,
NH), 7.9 (br.s, 1H, NH), 7.5 – 7.0 (m, 10H, Ar-H), 5.2 (s, 1H,
α-OH), 4.8 – 4.6 (m, 2H, α-CH), 4.5 – 4.3 (m, 2H, α-CH),
4.1 – 4.0 (m, 2H, β-CH2), 3.7 (s, 3H, O-CH3), 3.6 – 3.4 (m,
2H, -N-CH2), 3.2 – 3.0 (m, 4H, β-CH2), 2.2 – 1.6 (m, 4H, -
CH2-CH2-), 1.4 (s, 9H, C (CH3)3. – C32H42N4O8 (610.7):
calcd. C 62.94, H 6.93, N 9.17; found C 62.70, H 6.85,
N 9.13.
To the solution of Boc-octapeptide p-nitrophenyl es-
ter (1.2 mmol) in chloroform (15 ml), trifluoroacetic acid
(0.274 g, 2.4 mmol) was added, stirred for 1 h at room
temperature and washed with 10% sodium bicarbonate so-
lution. The organic layer was dried over anhydrous sodium
sulpate. To the resulting Boc-deprotected peptide-pnp ester
in THF (15 ml), pyridine (1.4 ml, 2 mmol) was added and
kept at 4 ◦C for seven 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 sulphate. THF and pyridine were distilled under re-
duced pressure to get Pseudostellarin G. The crude product
was purified by silica gel column chromatography using the
dichloromethane-methanol system and finally recrystallized
from EtOAc-n-hexane.
Boc-Gly-Pro-Leu-Ala-OMe (6)
M. p. 267 ◦C dec. (Lit. [8] 268 ◦C dec). - IR(CHCl3):
ν = 3600 (br.s, O-H str.), 3420 (br.s, N-H str.), 3050 (m,
=C-H str.), 2950 (s, C-H str.), 2820 (s, C-H str.),1690 (s,
C=O str. amide), 1680 (s, C=O str. amide), 1650 (s, C=O
str. amide), 1610 (s, C=C str.), 1605 (s, N-H def.), 1445 (s,
C-H def.), 1050 (s, C-H def.), 915 (s, C-H def.) cm−1. –
1H NMR (300 MHz, DMSO-d6): δ = 8.6 − 8.4 (br.s, 4H,
NH), 7.8 – 7.6 (br.s, 2H, NH), 7.3 – 6.9 (m, 10H, Ar-H), 5.1
(s, 1H, -OH), 4.9 – 4.7 (m, 4H, α-CH), 4.6 – 4.4 (m, 3H, α-
CH), 4.2 – 3.9 (m, 4H, α-CH2 and β-CH2), 3.6 – 3.4 (m, 4H,
N-CH2), 3.3 – 3.1 (m, 4H, β-CH2), 2.2 – 1.8 (m, 8H, -CH2-
CH2-), 1.8 – 1.7 (m, 1H, γ-CH), 1.5 (d, 3H, J = 6.6 Hz,
CH3), 1.3 – 1.1 (m, 2H, β-CH2), 0.9 (d, 6H, J = 6.5 Hz,
-C(CH3)2). – 13C NMR (DMSO-d6): δ = 173.0 (s, C=O),
172.2 (s, C=O), 172.0 (s, C=O), 171.4 (s, C=O), 171.1 (s,
C=O), 170.5 (s, C=O), 138.6 (s, Ar-γ-C), 137.4 (s, Ar1-γ-
C), 129.3 (d, Ar and Ar1-ε-C), 128.8 (d, Ar and Ar1-δ-C),
127.2 (d, Ar-ζ-C), 126.6 (d, Ar1-ζ-C), 61.8 (d, α-CH), 61.2
(d, α-CH), 61.0 (t, β-CH2), 58.4 (d, α-CH), 55.4 (d, α-CH),
53.9 (d, α-CH), 51.3 (d, α-CH), 50.4 (d, α-CH), 48.3 (t, N-
CH2), 46.8 (t, N-CH2), 42.9 (t, α-CH2), 40.0 (t, β-CH2),
39.0 (t, β-CH2), 37.6 (t, β-CH2), 31.6 (t, β-CH2), 29.8 (t,
β-CH2), 25.7 (t, γ-CH), 25.7 (t, γ-CH2), 24.7 (t, γ-CH2),
23.3 (q, -CH3), 21.6 (t, γ-CH2), 20.8 (q, -CH3), 15.8 (q, -
CH3). – FAB mass: m/z = 817 [M++H]. – C42H56N8O9
(817): calcd. C 61.75, H 6.91, N 13.72; found C 61.70,
H 6.89, N 13.68.
IR(CHCl3): ν = 3200 (br.s, N-H str.), 2970 (s, C-H str.),
2800 (s), 1735 (s, C=O str. ester), 1685 (s, C=O str. amide),
1660 (s, C=O str. amide), 1655 (s, C=O str. amide), 1600
(s), 1535 (s), 1440 (s), 1370 (s), 1310 (s), 1265 (s), 1200
(s), 1170 (s), 1030 (s), 870 (s) cm−1. – 1H NMR (300 MHz,
CDCl3): δ = 8.3 (br.s, 2H, NH), 7.8 (br.s, 1H, NH), 4.7 –
4.5 (m, 2H, α-CH), 4.4 – 4.2 (m, 1H, α-CH), 4.1 – 3.9 (m,
2H, β-CH2), 3.7 (s, 3H, O-CH3), 3.6 – 3.4 (m, 2H, -N-CH2),
2.2 – 1.8 (m, 4H, -CH2-CH2-), 1.7 – 1.6 (m, 2H, CH2), 1.4 (s,
9H, C (CH3)3, 1.3 (d, J = 7.0 Hz, 3H, -CH3), 1.2 – 1.1 (1H,
m, γ-CH), 0.9 (d, J = 7.0 Hz, 6H, C (CH3)2). – C22H38N4O7
(470.6): calcd. C 56.15, H 8.14, N 11.90; found C 56.02,
H 8.08, N 11.67.
Boc-Pro-Phe-Ser-Phe-Gly-Pro-Leu-Ala-OMe (7)
IR(CHCl3): ν = 3580 (br.s, O-H str.), 3200 (br.s, N-H
str.), 3060 (m,=C-H str.), 2980 (s, C-H str.), 2800 (s, C-H
str.), 1720 (s, C=O str. ester), 1670 (s, C=O str. amide), 1665
(s, C=O str. amide), 1650 (s, C=O str. amide), 1645 (s, C=O
str. amide), 1605 (s), 1600 (s), 1520 (s), 1500 (s), 1445 (s),
1370 (s), 1280 (s), 1170 (s), 1020 (s), 980 (s), 720 (s) cm−1. –
1H NMR (300 MHz, (CDCl3)): δ = 8.4 (br.s, 2H, NH), 8.0
(br.s, 2H, NH), 7.5 (br.s, 2H, NH), 7.3 – 6.9 (m, 10H, Ar-
H), 5.15 (s, 1H, -OH), 4.8 – 4.6 (m, 4H, α-CH), 4.45 – 4.3
(m, 3H, α-CH), 4.2 – 4.0 (m, 3H, α-CH2 and α-CH), 3.95 –
3.8 (m, 2H, β-CH2), 3.75 (s, 3H, OCH3), 3.6 – 3.4 (m, 4H,
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