Synthesis, characterization and biological evaluation of cyclic peptides: Viscumamide, yunnanin A and evolidine

Article abstract of DOI:10.1515/znb-2005-1217

Three biologically active cyclic peptides, viscumamide, yunnanin A and evolidine were synthesized and the structures were established on the basis of analytical, IR, NMR and mass spectral data. These newly synthesized cyclic peptides were evaluated for an

Full text of DOI:10.1515/znb-2005-1217

Synthesis, Characterization and Biological Evaluation of Cyclic Peptides:
Viscumamide, Yunnanin A and Evolidine
Boja Poojarya 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. 60b, 1313 – 1320 (2005); received May 26, 2004
Three biologically active cyclic peptides, viscumamide, yunnanin A and evolidine were synthe-
sized and the structures were established on the basis of analytical, IR, NMR and mass spectral data.
These newly synthesized cyclic peptides were evaluated for antimicrobial and pharmacological ac-
tivities. Evolidine showed better growth inhibition against bacterial strains than yunnanin A and vis-
cumamide. The anti-inflammatory activity of viscumamide is moderate and the remaining two cyclic
peptides are found to be less active. But, all the three cyclic peptides showed weak anthelmintic ac-
tivity.
Key words: Cyclic Peptides, Viscumamide, Yunnanin A, Evolidine, Antimicrobial Activity
Introduction
spectroscopy and chemical degradations. As a part
of the continuing studies in search of new bioactive
In the past two decades, a wide variety of naturally cyclic peptides from higher plants, these authors have
occurring bioactive cyclic peptides have been isolated also isolated 16 novel cyclic peptides belonging to
from plants, marine sponges and tunicates [1]. The Caryophyllaceae family including Yunnanin A-C from
wide spread increase of bacterial resistance towards the roots of Stellaria yunnanensis [11]. Some of the
conventional antibiotics encouraged the exploration of peptides showed tyrosinase inhibitory and cell growth
novel antimicrobial molecules with unexploited mech- inhibitory activities. The structures of these peptides
anisms of action. Initially discovered as a defensive were elucidated by extensive 2D NMR spectrometry,
system in invertebrates and vertebrates, antimicrobial chemical degradation and ESI MS methods. To deter-
peptides are attracting increased interest as potential mine the importance of the specific functional groups
therapeutics [2 – 4]. Unlike classical antibiotics, which for the overall conformation of cyclic peptides and to
must penetrate the target cell, the principle mode of ac- understand the relationships between chemical com-
tion of peptides involves perturbation and permeability position and three dimensional structure, conforma-
of the cell membrane. This mechanism confers activ- tional analysis of cyclic heptapeptides, yunnanins A
ity towards a broad spectrum of microbial cells, but and C were undertaken using X-ray crystallography,
is also responsible for undesired lytic activity against high field NMR and MD calculations. The solid and
mammalian cells such as erythrocytes [5 – 7].
solution state conformations of isolated yunnanin A
were also studied by Morita et al. [12] using X-ray
diffraction and NMR techniques. Yunnanin A occur-
ring in the roots of Stellaria yunnanensis was effi-
ciently synthesized using a combination of solid and
solution phase techniques [13]. The structural analy-
sis on the synthesized peptide showed that the syn-
thetic sample exhibited a configurational pattern at the
proline peptide linkages identical to the natural prod-
uct. The influence of different coupling reagents and
metal ions in the promotion of the cyclization of linear
A cyclic pentapeptide, viscumamide was isolated
from Viscum album Linn. var. coloratum Ohwi by
Okumura and Sakurai [8] and the structure cyclo(-Leu-
Ile-Leu-Ile-Leu-) was assigned to this substance on the
basis of gas chromatographicstudies on the products of
its partial hydrolysis. They also attempted the synthe-
sis of this cyclic peptide [9]. Itokawa et al. [10] isolated
two novel cyclic peptides, named yunnanins A and B
from Stellaria yunnanensis franch (Caryophyllaceae).
The structures of these peptides were elucidated from
c
0932–0776 / 05 / 1200–1313 $ 06.00 ꢀ 2005 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
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B. Poojary – S. L. Belagali · Synthesis, Characterization and Biological Evaluation of Cyclic Peptides
segment during the synthesis of yunnanin A was also
studied [14– 16]. Another cyclic heptapeptide [cyclo(-
Ser-Phe-Leu-Pro-Val-Asn-Leu-)]was isolated by East-
wood et al. [17] from the leaves of Evodia xanthoxy-
loides and was named evolidine. The structure of this
peptide was deduced from NMR studies [18]. The
structure of evolidine was also confirmed by chemi-
cal degradation of the natural product and by synthe-
sis [19]. The cyclic nature and the amino acid com-
position of evolidine were also investigated [20, 21].
Many authors also studied the conformation of evoli-
dine by proton magnetic resonance and computational
studies [22 – 25]. The structure assigned to the evoli-
dine was also confirmed by its synthesis [26, 27].
As a part of our ongoing study on the synthetic and
biological aspects of cyclic peptides of biological in-
terest [28], an attempt was made towards the synthesis
of viscumamide, a cyclic pentapeptide and two cyclic
heptapeptides, yunnanin A and evolidine. The synthe-
sized peptides were further subjected to antibacterial
and pharmaceutical activity studies.
Results and Discussion
Retrosynthetically, we disconnected viscumamide
into two dipeptide units of Boc-Leu-Ile-OMe (1) and
a single amino acid unit, H-Leu-OMe·HCl (2). The
required dipeptides were prepared by coupling Boc-
L-amino acids with the respective L-amino acid ester
hydrochlorides using DCC, HOBt and N-methyl mor-
pholine according to the Bodanszky procedure [29]
with suitable modifications [30]. The ester group of
the dipeptide 1 was removed with LiOH and the Boc-
group was removed with trifluoroacetic acid. Both
the deprotected units were coupled to obtain the
tetrapeptide, Boc-Leu-Ile-Leu-Ile-OMe (3). The re-
sulting tetrapeptide was then condensed with H-Leu-
OMe·HCl (2) to obtain the pentapeptide, Boc-Leu-Ile-
Leu-Ile-Leu-OMe (4). The methyl ester group of 4
was deprotected with LiOH and p-nitrophenyl es-
ter group (pnp) was introduced by treatment with p-
nitrophenol in the presence of DCC. The Boc-group of
resulting Boc-Leu-Ile-Leu-Ile-Leu-Opnpwas removed
by treatment with trifluoroacetic acid in chloroform.
The solution of the Boc-deprotected pentapeptide p-
nitrophenyl ester was diluted with chloroform and al-
lowed to cyclize in the presence of pyridine [31] to ob-
tain viscumamide (5) as depicted in Scheme 1.
Scheme 1. a = LiOH, THF:H2O(1:1), RT/1 h; b = TFA,
CHCl3, RT/1 h; c = DCC, NMM, HOBt, DCM, RT/36 h;
d = p-nitrophenol(pnp), DCC, CHCl3, RT/12 h; e = pyridine,
CHCl3, 10 days/0 ◦C.
dipeptide units, Boc-Gly-Gly-OMe (6), Boc-Pro-Phe-
OMe (7), Boc-Pro-Gly-OMe (8) and a single amino
acid unit H-Tyr-OMe·HCl (9). The dipeptides 6 and 7
were coupled after proper deprotection to get the
tetrapeptide, Boc-Gly-Gly-Pro-Phe-OMe (10). The re-
sulting tetrapeptide was then condensed with another
dipeptide unit (8), after proper deprotection to yield
the hexapeptide unit, Boc-Gly-Gly-Pro-Phe-Pro-Gly-
OMe (11). It was then coupled with H-Tyr-OMe·HCl
to obtain the heptapeptide, Boc-Gly-Gly-Pro-Phe-Pro-
Gly-Tyr-OMe (12). Finally, the linear segment was
cyclized to yield yunnanin A (13) as depicted in
Scheme 2. In the same way, evolidine,[cyclo(-Leu-
Ser-Phe-Leu-Pro-Val-Asn-)], was also disconnected
into three dipeptide units of Boc-Phe-Leu-OMe (14),
Boc-Pro-Val-OMe (15), Boc-Leu-Ser-OMe (16), and
a single amino acid unit, Boc-Asn(bzh)-OH (17). The
tetrapeptide unit, Boc-Phe-Leu-Pro-Val-OMe (18) was
prepared as per the above procedure by condensing
the dipeptides 14 and 15 after proper deprtection. The
The synthesis of yunnanin A, cyclo(-Gly-Gly-
Pro-Phe-Pro-Gly-Tyr-) was carried out from three
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B. Poojary – S. L. Belagali · Synthesis, Characterization and Biological Evaluation of Cyclic Peptides
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Scheme 2. a = LiOH, THF:H2O(1:1), RT/1 h; b = TFA,
CHCl3, RT/1 h; c = DCC, NMM, HOBt, DCM, RT/36 h;
d = p-nitrophenol(pnp), DCC, CHCl3, RT/12 h; e = pyridine,
CHCl3, 10 days/0 ◦C.
Scheme 3. a = LiOH, THF:H2O(1:1), RT/1 h; b = TFA,
CHCl3, RT/1 h; c = DCC, NMM, HOBt, DCM, RT/36 h;
d = p-nitrophenol(pnp), DCC, CHCl3, RT/12 h; e = pyridine,
CHCl3, 10 days/0 ◦C.
tetrapeptide 18 was then condensed similarly with the
remaining dipeptide unit 16 to yield the hexapeptide,
Boc-Phe-Leu-Pro-Val-Leu-Ser-OMe (19). The benz-
hydryl(bzh) protection of the carboxamido group to
yield Boc-Asn(bzh)-OH (17) was achieved by treating
the Boc-asparagine with benzhydrolin acetic acid [29].
The Boc-group of the hexapeptide 19 was removed and
then condensed with 17 to obtain Boc-Asn(bzh)-Phe-
Leu-Pro-Val-Leu-Ser-OMe (20), the linear segment of
evolidine. Finally, the cyclization of the linear segment
(Scheme 3) was carried out by the p-nitrophenyl ester
method to obtain evolidine 21.
methanol system and recrystallized from EtOAc-
petroleum ether. The newly synthesized compounds
were analyzed for C,H,N and the structures were con-
firmed by IR, NMR and mass spectral data. The char-
acteristic IR and NMR spectra of all the intermediate
compounds were analyzed. The characteristic IR ab-
sorption bands of –CO-NH- moiety were present in
the cyclized product. The NMR spectra of all cyclized
products clearly indicate the presence of all respective
amino acid moieties. Furthermore, the mass spectra
of the cyclic peptides viscumamide, yunnanin A and
evolidine showed the [M++H] peak at m/z 566, 676
and 771 which are consistent with their molecular for-
The intermediate and final products were puri-
fied by column chromatography using a chloroform-
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B. Poojary – S. L. Belagali · Synthesis, Characterization and Biological Evaluation of Cyclic Peptides
Table 1. Antimicrobial activities of
viscumamide (5), yunnanin A (13),
and evolidine (21).
Diameter of zone of inhibition (mm)
Antibacterial activity studies Antifungal activity studies
Compound
P.aer E.coli
B.sub
12
S.aur C.alb
A.niger
10
Viscumamide (5)
Yunnanin A (13)
Evolidine (21)
Penicillin
10
08
12
12
–
08
11
–
08
–
10
08
10
–
14
08
10
18
–
18
18
–
14
–
20
12
–
Greseofulvin
20
Table 2. Antiinflammatory activities of viscumamide (5),
yunnanin A (13), and evolidine (21).
mulae, C30H55N5O5, C34H41N7O8 and C38H58N8O9,
respectively.
Increase in
paw volume after
3 h S E (ml)
0.68 0.01
0.86 0.02
0.73 0.04
Percentage of
inhibition of
oedema after 3 h S E
Compound
Biological Activity Studies
Viscumamide (5)
Yunnanin A (13)
Evolidine (21)
Ibuprofen
24.44
9.09
18.88
40.50
–
The synthesized cyclic peptides, viscumamide (5),
yunnanin A (13) and evolidine (21) were also screened
for their antibacterial, antifungal, antiinflammatory
and anthelmintic activity. The antibacterial and anti-
fungal activities were determined 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 the disc
diffusion method [32]. Penicillin and griseofulvin were
used as standards against bacteria and fungal strains
at 10 and 25 µg/disc respectively. The results are sum-
marized in Table 1. The antibacterial screening results
indicate that viscumamide and yunnanin A are less
active against all tested bacterial and fungal strains.
However, evolidine is as active as the standard drug
against the bacterial strains P. aeruginosai and S. au-
reus but only moderately active against the remaining
two bacterial strains. It is also less active against fungal
strains. The anti-inflammatory activity tests were car-
ried out according to the method of Winter et al. [33]
using Ibuprofen as the standard and the results are pre-
sented in Table 2. The anti-inflammatory data reveal
that viscumamide is moderately active, but evolidine
and yunnanin A are inactive as compared to the stan-
dard drug used for screening studies. The anthelmintic
activity was carried out against the earthworms (Pon-
toscotex corethruses) according to Garg’s method [34]
(Table 3) using mebendazole as standard drug. All the
three cyclic peptides are found to be less active when
compared to the standard.
0.55 0.03
0.90 0.02
Control
Table 3. Anthelmintic activities of viscumamide (5), yun-
nanin A (13), and evolidine (21).
Compound
Conc of the Mean paralyzing Mean death time
Compd (mg) time (min) S E (min) S E
Viscumamide (5)
Yunnanin A (13)
Evolidine (21)
Mebendazole
Control
100
200
100
200
100
200
100
200
–
60.00 1.58
51.50 2.02
82.51 1.05
74.00 1.10
99.84 2.10
89.75 1.90
18.01 2.01
12.55 1.02
–
136.44 2.08
120.04 1.60
122.0
102.6
1.02
1.09
130.81 2.04
115.32 2.03
55.20 2.00
32.01 1.10
–
ture. Hence the synthetic cyclic peptides are screened
for these activities. The screening data of these com-
pounds in the present study showed some promising
results.
Experimental Section
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/DMSO-d6 on a 300 MHz spec-
trophotometer using TMS as an internal standard. The mass
spectra were recorded on a FAB mass spectrometer. TLC
checked the progresses of the reactions on silica gel G plates
and the products were purified by silica gel column chro-
matography.
The synthesis of a cyclic pentapeptide and two
cyclic heptapeptides was achieved in fairly good yields
in a simple solution phase method. The yunnanins and
evolidine were found to possess tyrosinase and growth
inhibitory activities. But, the antibacterial, antifungal,
anti-inflammatory and anthelmintic activity studies on
Procedure for the preparation of Boc-Asn(bzh)-OH (12)
Boc-asparagine (232 g, 10 mmol) was dissolved in acetic
acid (25 ml) and benzhydrol (1.84 g, 10 mmol) was added
these cyclic peptides are not reported in the litera- with stirring at R.T. To the resulting mixture 0.05 ml of
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B. Poojary – S. L. Belagali · Synthesis, Characterization and Biological Evaluation of Cyclic Peptides
1317
concentrated sulphuric acid was added and allowed to stand Deprotection of the amino group of Boc-dipeptide-methyl
overnight. The reaction mixture was poured into water ester
(75 ml). The precipitate formed was filtered. It was dissolved
in ethyl acetate, washed with water (20 ml), dried over an-
The protected dipeptide (1 mmol) was dissolved in chlo-
roform (15 ml) and treated with trifluoroacetic acid (0.228 g,
hydrous sodium sulphate. And ethyl acetate was removed by
2 mmol). The solution was stirred at R.T. for an hour, washed
distillation under reduced pressure to obtain Boc-Asn(bzh)-
with saturated NaHCO3 solution (5 ml). The organic layer
OH. M. p. 145-146 ◦C (Lit. [23] 145 – 146 ◦C); yield 72.4%.
was dried over anhydrous Na2SO4 and evaporated in vacuum
to obtain dipetide methyl ester. The product was purified by
recrystallization from chloroform and petroleum ether.
General procedure for the preparation of dipeptides
Amino acid methyl ester hydrochloride (10 mmol) was
Preparation of tetrapeptides
taken in dichloromethane (20 ml) and triethylamine (4 ml,
28.7 mmol) was added at 0 ◦C. The reaction mixture
The Boc-dipeptide (1 mmol) and the dipeptide methyl es-
was stirred for 15 min and Boc-amino acid (10 mmol) in
dichloromethane (20 ml) was added. DCC (2.09 g, 10 mmol)
and HOBt (2.34 g, 12 mmol) were added with stirring. Af-
ter 24 h, the reaction mixture was filtered and the residue
was washed with dichloromethane (30 ml) and added to the
filtrate. The filtrate was washed with 5% HCl (20 ml), 5%
NaHCO3 (20 ml), and saturated NaCl (20 ml) solutions. The
organic layer was dried over anhydrous Na2SO4, filtered and
evaporated in vacuum. To remove trces of the dicyclohexy-
lurea, the product was dissolved in minimum amount of chlo-
roform and cooled to 0 ◦C. The recrystallized dicyclohexy-
lurea was removed by filtration. To the filtrate petroleum
ether was added at 0 ◦C to recrystallize the pure product.
ter (1 mmol) were dissolved in dichloromethane (15 ml) and
trieth◦ylamine (0.4 ml) was added. The solution was cooled
to 0 C and DCC (0.216 g, 1 mmol) and HOBt (0.153 g,
1 mmol) were added and stirred for 24 h. The separated di-
cyclohexylurea was removed by filtration. The filtrate was
washed with 10% citric acid solution (2 ml), again with
5% NaHCO3 solutin (2 ml) and water (2 ml). The organic
layer was dried over anhydrous Na2SO4, filtered and evapo-
rated to dryness in vacuum. The residue was triturated with
hexane, filtered, washed with hexane and dried. The sam-
ple was purified by silica gel column chromatography using
dichloromethane-methanol system and finally recrystallized
from chloroform and petroleum ether.
Boc-Leu-Ile-OMe (1)
Boc-Leu-Ile-Leu-Ile-OMe (3)
IR (CHCl3): ν = 3400 (br.s, N-H str.), 3270 (br.s, N-H
str.), 2960 (s, C-H str.), 2940 (s, C-H str.), 1750 (s, C=O
str. ester), 1690 (s, C=O str. amide), 1660 (s, C=O str.
amide), 1520 (m), 1430 (s), 1310 (s), 1155 (s), 1010 (s),
860 (s) cm−1. – 1H NMR (300 MHz, CDCl3): δ = 6.4 (br.s,
1H, NH), 5.8 (br.s, 3H, NH), 4.6 – 4.3 (m, 4H, α-CH), 3.7 (s,
3H, O-CH3), 2.1 – 1.6 (m, 6H, β-CH2 and β-CH), 1.45 (s,
9H, C(CH3)3), 1.3 – 1.1 (m, 6H, γ-CH2 and γ-CH), 1.05
(doublet overlapped with a triplet, 24H, -(CH3)2 and –CH3),
– C30H56N4O7(568): calcd. C 63.30, H 9.85, N 9.85; found
C 63.42, H 9.78, N 10.00.
IR (CHCl3): ν = 3340 (br.s, N-H str.), 3250 (br.s, N-H
str.), 2940 (s, C-H str.), 1760 (s, C=O str. ester), 1685 (s, C=O
str. amide), 1520 (s), 1450 (s), 1370 (s), 1250 (s), 1140 (s),
1070 (s), 1000 (s), 860 (s) cm−1. – 1H NMR (300 MHz,
CDCl3): δ = 8.65 (br.s, 1H, NH), 5.7 – 5.5 (br.s, 1H, -NH),
4.4 – 4.3 (m,1H, α-CH), 4.2 – 4.1 (m,1H, α-CH), 3.7 (s, 3H,
O-CH3), 2.0 – 1.7 (m, 3H, β-CH2 and β-CH), 1.4 (s, 9H,
C(CH3)3), 1.3 – 1.1 (m, 3H, γ-CH2 and γ-CH), 0.95 (dou-
blet overlapped with a triplet, 12H, -C(CH3)2 and –CH3). –
C18H34N2O5 (358): calcd. C 60.28, H 9.49, N 7.81; found
C 60.38, H 9.42, N 7.93.
Boc-Gly-Gly-Pro-Phe-OMe (10)
General procedure for the preparation of tetrapeptides
IR (CHCl3): ν = 3310 (br.s, N-H str.), 3010 (m, C-H str.),
2950 (s, C-H str.), 2850 (s, C-H str.), 1720 (s, C=O str. ester),
1690 (s, C=O str. amide), 1670 (s, C=O str. amide), 1650 (s,
Deprotection of the carboxyl group of Boc-dipeptide-methyl
ester
To a solution of the Boc-dipeptide-methyl ester (1 mmol) C=O str. amide), 1510 (s), 1310 (m), 1220 (s), 1140 (s),
in tetrahydrofuran:water(1:1) (36 ml◦), lithium hydroxide 1080 (s), 970 (s) cm−1. – 1H NMR (300 MHz, CDCl3):
(0.041 g, 1.5 mmol) was added at 0 C. The mixture was δ = 8.0 (br.s, 2H, NH), 7.8 (br.s, 1H, NH), 7.4 – 7.1 (m, 5H,
stirred for 1 h at R.T. and then acidified to ph 3.5 with Ar-H), 4.5 – 4.3 (m, 2H, α-CH), 4.2 – 3.9 (m, 4H, α-CH2),
1N H2SO4. The aqueous layer was extracted with ether 3.7 (s, 3H, -O-CH3), 3.6 – 3.4 (m, 2H, -N-CH2), 3.3 – 3.1 (m,
(3×15 ml). The combined organic extracts were dried over 2H, β-CH2), 2.2 – 1.8 (m, 4H, -CH2-CH2-), 1.45 (s, 9H,
anhydrous Na2SO4 and evaporated in vacuum to yield Boc- C(CH3)3). – C24H34N4O7 (490): calcd. C 58.88, H 6.94,
dipeptide.
N 11.43; found C 58.88, H 6.88, N 11.53.
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B. Poojary – S. L. Belagali · Synthesis, Characterization and Biological Evaluation of Cyclic Peptides
Boc-Phe-Leu-Pro-Val-OMe (18)
3.4 – 3.2 (m, 6H, N-CH2 and β-CH2), 2.3 – 2.0 (m, 8H,
-CH2-CH2), 1.45 (s, 9H, -C(CH3)3). – C31H44N6O9 (644):
calcd. C 57.76, H 6.83, N 13.04; found C 57.86, H 6.95,
N 12.96.
IR (CHCl3): ν = 3327 (br.s, N-H str.), 3030 (m = C-H
str.), 2970 (s, C-H str.), 2851 (s, C-H str.), 1720 (br.s, C=O
str. ester), 1680 (s, C=O str. amide), 1660 (s, C=O str. amide),
1622 (s, N-H def.), 1574 (s), 1510 (s), 1430 (s), 1310 (s),
1240 (s), 1150 (s), 892 (s) cm−1. – 1H NMR (300 MHz,
CDCl3): δ = 8.2 (br.s, 1H, NH), 7.9 (br.s, 1H, NH), 7.5 –
7.3 (m, 5H, Ar-H), 6.9 (br.s, 1H, NH), 4.6 – 4.35 (m, 2H,
α-CH), 4.3 – 4.1 (m, 2H, α-CH), 3.75 (s, 3H, O-CH3), 3.6 –
3.3 (m, 2H, -N-CH2), 3.1 – 2.8 (m, 2H, β-CH2), 2.4 – 1.8 (m,
5H, -CH2-CH2- & β-CH), 1.45 (s, 9H, C(CH3)3), 1.3 –
1.2 (1H, m, γ-CH), 1.0 (d, J = 6.5 Hz, 12H, C(CH3)2). –
C31H48N4O7 (588): calcd. C 63.27, H 8.16, N 9.52; found
C 63.20, H 8.28, N 9.63.
Procedure for the preparation of hexapeptide, Boc-Leu-Ser-
Phe-Leu-Pro-Val- OMe (19)
The methyl ester group of the dipeptide, Boc-Leu-Ser-
OMe (8) and the Boc group of the tetrapeptide, Boc-Phe-
Leu-Pro-Val-OMe (18) were removed using aqueous LiOH
and CF3CO2H respectively. The resulting deprotected frag-
ments were then condensed to get the hexapeptide 19 ac-
cording to the procedure employed for the preparation of
tetrapeptides.
IR (CHCl3): ν = 3600 (br.s, O-H str.), 3330 (br.s, N-H
str.), 3060 (m,=C-H str.), 3010 (s, =C-H str.), 2940 (s, C-H
str.), 2880 (s, C-H str.), 1740 (s, C=O str. ester), 1695 (s,
C=O str. amide), 1680 (s, C=O str. amide), 1660 (s, C=O
str. amide), 1602 (s, N-H def.), 1574 (s), 1520 (s, C-N str.),
1310 (s, C-H def.), 892 (s, C-H def.) cm−1. – 1H NMR
(300 MHz, CDCl3): δ = 8.3 (br.s, 2H, NH), 7.9 (br.s, 1H,
NH), 7.4 – 7.1 (m, 5H, Ar-H), 6.8 (br.s, 2H, N-H), 5.1 (s,
1H, O-H), 4.7 – 4.6 (m, 1H, α-CH), 4.5 – 4.4 (m, 4H, α-CH),
4.3 – 4.0 (m, 3H, α-CH and β-CH2), 3.75 (s, 3H, -OCH3),
3.6 – 3.3 (m, 2H, N-CH2), 3.2 – 2.8 (m, 2H, β-CH2), 2.4 –
2.0 (m, 4H, -CH2-CH2), 1.9 – 1.6 (m, 5H, β-CH2 and β-CH),
1.45 (s, 9H, -C(CH3)3), 1.3 – 1.15 (m, 2H, γ-CH), 0.95 (d,
J = 6.5 Hz, 18H, -C(CH3)2). – C40H64N6O10 (788): calcd.
C 60.91, H 8.12, N 10.66; found C 60.98, H 8.2, N 10.77.
Procedure for the preparation of pentapeptide, Boc-Leu-Ile-
Leu-Ile-Leu-OMe (4)
The methyl ester group of Boc-Leu-Ile-Leu-Ile-OMe (3)
was removed using aqueous LiOH and then condensed with
H-Leu-OMe·HCl to obtain the pentapetide 4 according to the
procedure employed for the preparation of tetrapeptides.
IR (CHCl3): ν = 3450 (br.s, N-H str.), 3300 (br.s, N-H
str.), 2980 (s, C-H str.), 2890 (s, C-H str.), 1760 (s, C=O str.
ester), 1685 (s, C=O str. amide), 1670 (s, C=O str. amide),
1530 (m), 1420 (s), 1300 (s), 1160 (s), 1000 (br.s), 870 (s),
720 (s) cm−1. – 1H NMR (300 MHz, CDCl3): δ = 6.6
(br.s, 1H, NH), 6.0 (br.s, 1H, NH), 5.7 (br.s, 3H, NH), 4.6 –
4.4 (m, 2H, α-CH), 4.3 – 4.1(m, 3H, α-CH), 3.65 (s, 3H,
OCH3), 2.2 – 1.8 (m, 8H, β-CH2 and β-CH), 1.45 (s, 9H,
C(CH3)3), 1.3 – 1.1 (m, 7H, γ-CH2 and γ-CH), 0.90 (dou-
blet overlapped with a triplet, 30H, -(CH3)2 and –CH3). –
C36H67N5O8 (697): calcd. C 61.90, H 9.68, N 10.10; found
C 61.82, H 9.60, N 9.94.
Procedure for the preparation of heptapeptide, Boc-Gly-Gly-
Pro-Phe-Pro-Gly-Tyr-OMe (12)
The methyl ester group of Boc-Gly-Gly-Pro-Phe-Pro-
Gly-OMe (11) was removed using aqueous LiOH and then
condensed with H-Tyr-OMe·HCl to obtain the heptapetide 12
according to the procedure employed for the preparation of
tetrapeptides.
Procedure for the preparation of hexapeptide, Boc-Gly-Gly-
Pro-Phe-Pro-Gly-OMe (11)
The methyl ester group of Boc-Gly-Gly-Pro-Phe-OMe
(10) and the Boc group of the dipeptide Boc-Pro-Gly-
OMe (8) were removed using aqueous LiOH and CF3CO2H
respectively. The resulting deprotected fragments were then
condensed to get the hexapeptide 11 according to the proce-
dure employed for the preparation of tetrapeptides.
IR (CHCl3): ν = 3600 (br.s, N-H str.), 3315 (br.s, N-H
str.), 3080 (m,=C-H str.), 2970 (s, C-H str.), 2860 (s, C-H
str.), 1725 (s, C=O str. ester), 1690 (s, C=O str. amide),
1685 (s, C=O str. amide), 1680 (s, C=O str. amide), 1675 (s,
C=O str. amide), 1620 (s, N-H def.), 1520 (s, C-N str.),
IR (CHCl3): ν = 3320 (br.s, N-H str.), 3005 (m,=C-H str.), 1320 (s, C-H def.), 1220 (s, C-O str.), 1140 (s, C-O str.),
2960(s, C-H str.), 2840 (s, C-H str.), 1730 (s, C=O str. es- 1080 (s, C-H def.), 970 (s, C-H def.) cm−1. – 1H NMR
ter), 1690 (s, C=O str. amide), 1680 (s, C=O str. amide), (300 MHz, CDCl3): δ = 8.4 (br.s, 4H, NH and OH), 8.1
1670 (s, C=O str. amide), 1620 (s, N-H def.), 1520 (s, (br.s, 2H, NH), 7.4 – 7.1 (m, 7H, Ar-H and Ar1-H), 6.9 (2H,
C-N str.), 1310 (s, C-H def.), 1220 (s, C-O str.), 1170 (s, d, J = 8.0 Hz, Ar1-H), 4.5 – 4.4 (m, 4H, α-CH), 4.2 – 4.0 (m,
C-O str.), 1080 (s, C-H def.), 980 (s, C-H def.) cm−1. – 6H, α-CH2), 3.7 (s, 3H, -OCH3), 3.5 – 3.2 (m, 4H, N-CH2),
1H NMR (300 MHz, CDCl3): δ = 8.3 (br.s, 2H, NH), 8.0 3.1 – 2.8 (m, 4H, β-CH2), 2.2 – 1.9 (m, 8H, -CH2-CH2),
(br.s, 2H, NH), 7.4 – 7.2 (m, 5H, Ar-H), 4.6 – 4.3 (m, 3H, 1.4 (s, 9H, -C(CH3)3). – C40H53N7O11(807): calcd. C 59.48,
α-CH), 4.2 – 4.0 (m, 6H, α-CH2), 3.75 (s, 3H, -OCH3), H 6.57, N 12.14; found C 59.06, H 6.72, N 12.06.
Unauthenticated
Download Date | 1/4/18 11:39 AM

B. Poojary – S. L. Belagali · Synthesis, Characterization and Biological Evaluation of Cyclic Peptides
1319
Procedure for the preparation of heptapeptide, Boc- dichloromethane-methanol system and finally recrystallized
Asn(bzh)-Leu-Ser-Phe-Leu-Pro-Val-OMe (20)
from EtOAc-n-hexane.
Viscumamide (5)
The Boc-group of Boc-Leu-Ser-Phe-Leu-Pro-Val-
OMe (19) was removed using aqueous CF3CO2H and
then condensed with Boc-Asn(bzh)-OH (12) to obtain the
heptapetide 20 according to the procedure employed for the
preparation of tetrapeptides.
M. p. 347 – 349 ◦C (Lit. [8] 349.5 – 351 ◦C). – IR (CHCl3):
ν = 3340 (br.s, N-H str.), 3160 (br.s, N-H str.), 2960 (s,
C-H str.), 2820 (s, C-H str.), 1670 (s, C=O str. amide),
1630 (s, C=O str. amide), 1490 (s, N-H def.), 1450 (s, C-H
def.), 1100 (m, C-H def.) cm−1. – 1H NMR (300 MHz,
DMSO-d6): δ = 6.9 (br.s, 3H, NH), 6.1 (br.s, 2H, NH), 4.7 –
4.4 (m,3H, α-CH), 4.3 – 4.1 (m, 2H, α-CH), 2.3 – 1.9 (m,
8H, β-CH2 and β-CH), 1.45 – 1.15 (m, 7H, γ-CH2 and
γ-CH), 0.95 (doublet overlapped with triplet, 30H, -C(CH3)2
and CH3). – 13C NMR (DMSO-d6): δ = 175.2 (s, C=O),
174.6 (s, C=O), 174.4 (s, C=O), 60.5 (d, α-CH), 60.3 (d,
α-CH), 60.2 (d, α-CH), 59.8 (d, α-CH), 59.5 (d, α-CH),
40.6 (t, β-CH2), 40.4 (t, β-CH2), 40.3 (t, β-CH2), 39.7 (t,
β-CH), 39.6 (d, β-CH), 25.7 (d, γ-CH), 25.4 (d, γ-CH),
25.1 (d, γ-CH), 24.9 (t, γ-CH2), 24.7 (t, γ-CH2), 23.3 (q,
-CH3), 23.0 (q, -CH3), 22.7 (q, -CH3), 22.6 (q, -CH3),
22.4 (q, -CH3), 19.5 (q, -CH3), 19.2 (q, -CH3). – FAB
mass: m/z = 566 (m+ + H, 40%), 523 (15%), 509 (45%),
454 (15%), 439 (90%), 410 (15%), 344 (5%), 325 (87%),
296 (34%), 232 (20%), 211 (12%), 184 (17%), 86 (10%),
57 (23%), 43 (25%). – C30H55N5O5 (565): calcd. C 64.29,
H 9.73, N 12.39; found C 64.32, H 9.81, N 12.32.
IR (CHCl3): ν = 3610 (br.s, O-H str.), 3320 (br.s, N-H
str.), 3070 (m,=C-H str.), 3010 (s, =C-H str.), 2940 (s, C-H
str.), 2860 (s, C-H str.), 1730 (s, C=O str. ester), 1695 (s,
C=O str. amide), 1680 (s, C=O str. amide), 1660 (s, C=O str.
amide), 1612 (s, N-H def.), 1574 (s, N-H def.), 1510 (s, C-N
str.), 1310 (s, C-H def.), 992 (s, C-H def.) cm−1. – 1H NMR
(300 MHz, CDCl3): δ = 8.2 (br.s, 3H, NH), 7.9 (br.s, 2H,
NH), 7.5 – 7.1 (m, 15H, Ar-H), 6.8 (br.s, 1H, N-H), 6.0 (br.s,
1H, NH), 5.1 (s, 1H, O-H), 4.7 – 4.4 (m, 6H, α-CH), 4.3 –
4.1 (m, 3H, α-CH), 3.68 (s, 3H, OCH3), 3.6 – 3.3 (m, 4H,
N-CH2 and β-CH2), 3.2 – 2.8 (m, 2H, β-CH2), 2.4 – 2.2 (m,
5H, -CH2-CH2 and Ar2CH), 1.9 – 1.6 (m, 5H, β-CH2 and
β-CH), 1.45 (s, 9H, -C(CH3)3), 1.3 – 1.1 (m, 2H, γ-CH), 1.0
(d, J = 6.5 Hz, 6H, -C(CH3)2), 0.95 (d, J = 6.8 Hz, 12H,
-C(CH3)2). – C57H80N8O12 (1068): calcd. C 64.04, H 7.49,
N 10.49; found C 64.28, H 7.32, N 10.60.
General procedure for the preparation of cyclic peptides, vis-
cumamide (5), yunnanin A (13) and evolidine (21)
Yunnanin A (13)
The methyl ester group of the linear segment of
Boc-(penta/heptapeptide)-OMe was removed using aqueous
LiOH as explained earlier to yield Boc-(penta/heptapeptide)-
OH. The resulting Boc-peptide-OH (1.5 mmol) was dis-
◦
M. p. 235 – 237 C. – IR(CHCl3): ν = 3600 (br.s, O-H
str.), 3400 (br.s, N-H str.), 3025 (m = C-H str.), 2945 (s,
C-H str.), 2855 (s, C-H str.), 1680 (s, C=O str. amide),
1650 (s, C=O str. amide), 1630 (s, C=O str. amide), 1605 (s,
N-H def.), 1520 (s, C-N str.), 1355 (s, C-H def.), 1090 (s,
◦
solved in chloroform (15 ml) at 0 C. Then p-nitrophenol
(0.27 g, 2 mmol) and DCC (0.31 g, 1.5 mmol) were added.
The resulting mixture was stirred at R.T. for 12 h. The
reaction mixture was filtered and the filtrate was washed
with 10% NaHCO3 solution until the excess of p-nitrophenol
was removed and finally washed with 5% HCl (5 ml) to get
Boc-peptide-p-nitrophenyl ester.
C-H def.), 1015 (s, C-H def.), 910 (s, C-H def.) cm−1
.
–
1H NMR (300 MHz, DMSO-d6): δ = 8.6 (br.s, 2H,
NH), 8.4 (br.s, 2H, NH and OH), 7.9 (br.s, 2H, NH), 7.5 –
7.1 (m, 7H, Ar-H and Ar1-H), 6.9 (d, 2H, J = 8 Hz), 4.6 –
4.4 (m, 4H, α-CH), 4.2 – 4.0 (m, 6H, α-CH2), 3.6 – 3.4 (m,
To the solution of Boc-(penta/heptapeptide)-p-nitrophenyl 4H, N-CH2), 3.2 – 2.8 (m, 4H, β-CH2), 2.2 – 1.8 (m, 8H,
ester (1.2 mmol) in chloroform (25 ml), trifluoroacetic -CH2-CH2-). – 13C NMR (DMSO-d6): δ = 173.7 (s, C=O),
acid (0.274 g, 2.4 mmol) was added, stirred for 1 h 173.1 (s, C=O), 172.6 (s, C=O), 171.1 (s, C=O), 170.8 (s,
at R.T. and washed with 10% sodium bicarbonate solution. C=O), 170.4 (s, C=O), 168.4 (s, C=O), 155.2 (s, Ar1-4-C),
The organic layer was dried over anhydrous sodium sul- 138.6 (s, Ar-1-C), 130.4 (d, Ar1-2,6-C), 129.3 (s, Ar1-1-C),
phate. To the resulting Boc-deprotected peptide-pnp ester in 128.9 (d, Ar-2,6-C), 128.2 (d, Ar-3,5-C), 126.3 (d, Ar-4-C),
THF(25 ml), pyridine(1.4 ml, 2 mmol) was added and kept 114.3 (d, Ar1-3,5-C), 61.9 (d, α-CH), 61.2 (d, α-CH),
at 4 ◦C for seven days. The reaction mixture was washed 60.5 (d, α-CH), 56.7 (d, α-CH), 55.0 (d, α-CH), 50.4 (d,
with 10% sodium bicarbonate solution until the byproduct α-CH), 44.2 (d, α-CH), 61.0 (t, β-CH2), 58.4 (d, α-CH),
p-nitrophenol was removed completely and finally washed 55.4 (d, α-CH), 53.9 (d, α-CH), 51.3 (d, α-CH), 50.4 (d,
with 5% HCl (5 ml). The organic layer was dried over anhy- α-CH), 48.3 (t, δ(N)-CH2), 46.8 (t, δ(N)-CH2), 44.2 (t,
drous sodium sulphate. THF and pyridine were distilled un- α-CH2), 42.9 (t, α-CH2), 40.0 (t, α-CH2), 38.0 (t, β-CH2),
der reduced pressure to get cyclic peptide. The crude product 37.4 (t, β-CH2), 29.8 (t, β-CH2), 28.7 (t, β-CH2), 25.7 (t,
was purified by silica gel column chromatography using the γ-CH2), 25.3 (t, γ-CH2). – FAB mass : m/z = 676 [M+ + H].
Unauthenticated
Download Date | 1/4/18 11:39 AM

1320
B. Poojary – S. L. Belagali · Synthesis, Characterization and Biological Evaluation of Cyclic Peptides
– C34H41N7O8(675): calcd. C 60.45, H 6.07, N 14.52; found β-CH2), 2.3 – 1.9 (m, 4H, -CH2-CH2-), 1.8 – 1.5 (m, 5H,
C 60.72, H 6.15, N 14.38.
β-CH2 and β-CH), 1.4 – 1.2 (m, 2H, γ-CH), 0.95 (d, J =
6.5 Hz, 6H, -C(CH3)2), 0.90 (d, J = 6.8 Hz, 12H, -C(CH3)2).
–
13C NMR(DMSO-d6): δ = 173.0 (s, C=O), 172.7 (s,
Evolidine (16)
C=O), 172.0 (s, C=O), 171.5 (s, C=O), 171.1 (s, C=O),
◦
◦
M.p. 276 – 27 C (Lit. [11] 278 C). – IR (CHCl3): ν = 170.9 (s, C=O), 170.5 (s, C=O), 157.2 (s, C=O), 138.6 (s,
3600 (br.s, O-H str.), 3420 (br.s, N-H str.), 3050 (m,=C-H Ar-1-C), 128.9 (d, Ar-2,6-C), 128.2 (d, Ar-3,5-C), 126.3 (d,
str.), 3010 (m,=C-H str.), 2950 (s, C-H str.), 2825 (s, C-H Ar-4-C), 62.8 (t, β-CH2), 60.5 (d, α-CH), 58.2 (d, α-CH),
str.), 1690 (s, C=O str. amide), 1680 (s, C=O str. amide), 57.1 (d, α-CH), 55.8 (d, α-CH), 54.9 (d, α-CH), 53.3 (d,
1650 (s, C=O str. amide), 1610 (s, N-H def.), 1605 (s, α-CH), 53.1 (d, α-CH), 51.0 (t, β-CH2), 46.9 (t, δ(N)-CH2),
N-H def.), 1510 (s, C-N str.), 1445 (s, C-H def.), 1050 (s, 40.0 (t, β-CH2), 39.4 (t, β-CH2), 37.5 (t, β-CH2), 31.6 (t,
C-H def.), 915 (s, C-H def.) cm−1. – 1H NMR (300 MHz, β-CH2), 29.3 (d, β-CH), 28.8 (t, β-CH2), 25.7 (t, γ-CH),
DMSO-d6): δ = 8.2 (br.s, 3H, NH), 7.8 (br.s, 1H, NH), 25.5 (t, γ-CH), 19.0 (q, -CH3), 18.5 (q, -CH3), 17.8 (q, -
7.4 – 7.1 (m,5H, Ar-H), 6.9 (1H, br.s, NH), 6.8 (br.s, 2H, CH3), 17.6 (q, -CH3), 17.2 (q, -CH3), 17.1 (q, -CH3). –
NH2), 6.0 (br.s, 1H, NH), 5.15 (s, 1H, -OH), 4.6 – 4.4 (m, FAB mass : m/z = 771 [M+ + H]. – C38H58N8O9 (770):
6H, α-CH and β-CH2), 4.3 – 4.0 (m, 3H, α-CH), 3.6 – calcd. C 59.22, H 7.53, N 14.55; found C 59.34, H 7.62,
3.2 (m, 4H, N-CH2 and β-CH2(Asn)), 3.1 – 2.9 (m, 2H, N 14.70.
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Unauthenticated
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