First Total Synthesis and Biological Potential of a Heptacyclopeptide of Plant Origin

Article abstract of DOI:10.1002/cjoc.201600419

Synthesis of a natural glycine-rich heptacyclopeptide - mahafacyclin A (7) was accomplished by solution-phase technique of peptide synthesis via coupling of tetrapeptide unit Boc-L-Thr-L-Ile-L-Leu-Gly-OH with tripeptide unit L-Val-L-Phe-Gly-OMe followed b

Full text of DOI:10.1002/cjoc.201600419

FULL PAPER
DOI: 10.1002/cjoc.201600419
First Total Synthesis and Biological Potential of
a Heptacyclopeptide of Plant Origin
Rajiv Dahiya*,a and Sunil Singhb
a Laboratory of Peptide Research and Development, School of Pharmacy, Faculty of Medical Sciences, The Univer-
sity of the West Indies, St. Augustine,
Trinidad & Tobago, West Indies
b Research Scholar, Department of Pharmacy, Mewar University, Gangrar, Chittorgarh, Rajasthan, India
Synthesis of a natural glycine-rich heptacyclopeptide - mahafacyclin A (7) was accomplished by solution-phase
technique of peptide synthesis via coupling of tetrapeptide unit Boc-L-Thr-L-Ile-L-Leu-Gly-OH with tripeptide unit
L-Val-L-Phe-Gly-OMe followed by cyclization of linear heptapeptide fragment. Structure of the newly synthesized
cyclopolypeptide was confirmed by means of chemical, spectroscopic analyses and subjected to antibacterial, anti-
fungal and anthelmintic activity studies. Bioactivity results showed potent antifungal and anthelmintic activities of
the synthesized peptide against dermatophytes T. mentagrophytes, M. audouinii and earthworm species M. kon-
kanensis, P. corethruses and E. eugeniea.
Keywords Jatropha mahafalensis, peptide synthesis, cyclopolypeptide, natural product, pharmacological activity
Introduction
yield, present investigation was directed toward solu-
tion-phase synthesis, structure elucidation and screening
of glycine-rich heptacyclopeptide - mahafacyclin A (7)
for antimicrobial and anthelmintic potential.
Medicinal plants have been used to treat health dis-
orders and to prevent diseases since time immemorial.[1]
Recently, the World Health Organization has estimated
that 80% of people worldwide rely on herbal medicines
for some part of their primary health care. Natural
products from medicinal plants provide unlimited op-
portunities for new drug leads because of the unmatched
availability of chemical diversity.[2] Plant-derived cy-
clopeptides have complex structures with modified
and/or unusual amino acid moieties and are concerned
with a number of bioactivities including antifungal ac-
tivity,[3] insecticidal activity,[4] tyrosinase inhibitory ac-
tivity,[5] anthelmintic activity,[6] anti-inflammatory ac-
tivity,[7,8] vasorelaxant activity,[9,10] estrogen-like activi-
ty,[11] antimalarial activity,[12] and anticancer activi-
ty.[13,14] A natural cyclic heptapeptide-mahafacyclin A
with -bulge characteristics, has been isolated from
Jatropha mahafalensis latex and its structure was eluci-
dated by a combination of chemical degradation,
LSIMS data and 2D NMR experiments.[15] Minute
quantities of this bioactive cyclopeptide obtained from
natural resources (328 mg from 250 g of dry latex of J.
mahafalensis) restricted scientists to investigate its bio-
logical profile in detail.
Experimental
Material and methods
All the reactions requiring anhydrous conditions
were conducted in flame dried apparatus. Melting point
was determined by open capillary method and was un-
corrected. L-Amino acids, dicyclohexylcarbodiimide
(DCC), N,N′-diisopropylcarbodiimide (DIPC), N-(3-di-
methylaminopropyl)-N′-ethylcarbodiimide hydrochlo-
ride (EDC•HCl), trifluoroacetic acid (TFA), penta-
fluorophenol (pfp), N-methylmorpholine (NMM), tri-
ethylamine (TEA), di-tert-butylpyrocarbonate (Boc2O)
and pyridine (C5H5N) were obtained from Spectrochem
Limited/Sigma-Aldrich (Mumbai, India). IR spectra
were recorded on a Shimadzu 8700 FTIR spectropho-
1
tometer and H/13C NMR spectra were taken on a
Bruker AC NMR spectrometer (300 MHz) using deu-
terated methanol as solvent and TMS as internal stand-
ard. A JMS-DX 303 Mass spectrometer operating at 70
eV by FABMS was utilized to record mass spectrum.
Optical rotation was measured on a automatic polar-
imeter using sodium lamp. Elemental analyses of all
compounds were performed on Vario EL III elemental
analyzer and purity of all synthesized peptide deriva-
Keeping in view broad spectrum of bioactivities ex-
hibited by plant-derived cyclopolypeptides[16-21] and an
effort to obtain a potent bioactive compound in good
*
E-mail: rajiv.dahiya@sta.uwi.edu, drrajivdahiya@gmail.com; Tel.: 1-868-4935655, 0091-96302-29885
Received July 17, 2016; accepted September 4, 2016; published online October 24, 2016.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201600419 or from the author.
1158
© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2016, 34, 1158—1164

First Total Synthesis and Biological Potential of a Heptacyclopeptide of Plant Origin
tives was checked by TLC on precoated silica gel G
plates.
mL/1.61 mL, 0.021 mol) was added. Then, whole con-
tents were kept at 0 ℃ for 7 d. The reaction mixture
was washed with 10% NaHCO3 (25 mL×3) and 5%
HCl (25 mL×2) solutions. The organic layer was dried
over anhydrous Na2SO4 and crude cyclized compound
was recrystallized from CH2Cl2/n-hexane to obtain pure
product cyclo (L-threonyl-L-isoleucyl-L-leucyl-glycyl-
L-valyl-L-phenylalanyl-glycyl) (7). Physical characteri-
zation data and elemental analysis data for all the newly
synthesized di/tri/tetra/heptapeptide intermediates and
cyclic products 1－7 are given in Table 1 and Table 2.
Preparation of linear di/tri/tetrapeptide units (1－5)
To a solution of L-amino acid methyl ester hydro-
chloride/dipeptide methyl ester (0.01 mol) in chloro-
form (20 mL), TEA (2.8 mL, 0.021 mol) was added at
0 ℃ and the reaction mixture was stirred for 10 min.
Boc-L-amino acid/Boc-dipeptide (0.01 mol) was dis-
solved in CHCl3 (20 mL) followed by addition of
DCC/DIPC/EDC•HCl (2.1 g/1.26 g/1.92 g, 0.01 mol)
and HOBt (1.34 g, 0.01 mol). The resulting mixture was
added to the above solution with constant shaking and
stirring was continued for 24 h. The reaction mixture
was filtered and the residue was washed with CHCl3 (20
mL) and added to the filtrate. The filtrate was washed
with 5% NaHCO3 and saturated NaCl solutions. The
organic layer was dried over anhydrous Na2SO4, filtered
and evaporated in vacuum. The crude product was re-
crystallized from a mixture of chloroform and petrole-
um ether (b.p. 40－60 ℃) followed by cooling at 0 ℃
to get the title compounds 1－5.
1
1: H NMR (CDCl3) δ: 6.59 (br s, 1H, NH, Thr),
6.45 (br s, 1H, NH, Ile), 4.54 (t, J＝3.95 Hz, 1H, H-α,
Thr), 4.42 (br s, 1H, OH, Thr), 4.17 (t, J＝8.65 Hz, 1H,
H-α, Ile), 3.69－3.64 (m, 1H, H-, Thr), 3.49 (s, 3H,
OCH3), 2.04－1.98 (m, 1H, H-, Ile), 1.69－1.64 (m,
2H, H-γ, Ile), 1.52 (s, 9H, tert-Butyl), 1.26 (d, J＝4.9
Hz, 3H, H-γ, Thr), 0.95 (t, J＝7.75 Hz, 3H, H-δ, Ile),
0.87 (d, J＝5.9 Hz, 3H, H-γ', Ile); IR (CHCl3) v: 3129,
3121 (N－H str, amide), 2969－2962, 2919 (C－H str,
CH3 and CH2), 1744 (C＝O str, ester), 1640, 1632 (C＝
O str, amide), 1539, 1532 (N－H def, amide), 1422,
1329 (O－H def), 1386, 1368 (C－H def, tert-Butyl),
1272 (C－O str, ester) cm1.
Preparation of linear heptapeptide unit and its cy-
clized form (6, 7)
1
2: H NMR (CDCl3) δ: 6.78 (br s, 1H, NH, Gly),
Boc-tetrapeptide, Boc-L-Thr-L-Ile-L-Leu-Gly-OH
(5.02 g, 0.01 mol) was dissolved in 30 mL of THF and
2.23 mL (0.021 mol) of NMM was added at 0 ℃ and
the resulting mixture was stirred for 15 min. Tripeptide
methyl ester, L-Val-L-Phe-Gly-OMe (3.35 g, 0.01 mol)
was dissolved in 30 mL of THF and DCC/DIPC/EDC•
HCl (2.1 g/1.26 g/1.92 g, 0.01 mol) and HOBt (1.34 g,
0.01 mol) were added to the above mixture with stirring.
Stirring was continued for 36 h, after which the reaction
mixture was filtered and the filtrate was washed with 30
mL each of 5% NaHCO3 and saturated NaCl solutions.
The organic layer was dried over anhydrous Na2SO4,
filtered and evaporated in vacuum. The crude product
was recrystallized from a mixture of chloroform and
petroleum ether (b.p. 40－60 ℃) followed by cooling
at 0 ℃ to get Boc-L-Thr-L-Ile-L-Leu-Gly-L-Val-L-
Phe-Gly-OMe 6 as yellowish semisolid mass. Linear
heptapeptide unit 6 (4.1 g, 0.005 mol) was deprotected
at carboxyl terminal using lithium hydroxide (LiOH,
0.18 g, 0.0075 mol) to obtain Boc-L-Thr-L-Ile-L-Leu-
Gly-L-Val-L-Phe-Gly-OH. To a solution of the depro-
tected heptapeptide (4.03 g, 0.005 mol) in CHCl3 (50
mL), pentafluorophenol (1.23 g, 0.0067 mol) and DCC
(1.06 g, 0.005 mol) were added followed by stirring at
r.t. for 12 h. Filtrate of the above reaction mixture was
washed with 10% NaHCO3 (20 mL×3) and 5% HCl
(20 mL×2) solutions to obtain corresponding penta-
fluorophenyl ester Boc-L-Thr-L-Ile-L-Leu-Gly-L-Val-
L-Phe-Gly-Opfp. Boc-group of resulting unit (3.89 g,
0.004 mol) was removed using TFA (0.91 g, 0.008 mol)
to get deprotected product L-Thr-L-Ile-L-Leu-Gly-
L-Val-L-Phe-Gly-Opfp which was dissolved in CHCl3
(25 mL) and TEA or NMM or pyridine (2.8 mL/2.21
6.05 (br s, 1H, NH, Leu), 4.25 (q, 1H, H-α, Leu), 4.09
(d, J＝5.15 Hz, 2H, H-α, Gly), 3.65 (s, 3H, OCH3), 1.93
(t, J＝7.95 Hz, 2H, H-, Leu), 1.56 (s, 9H, tert-Butyl),
1.52－1.47 (m, 1H, H-γ, Leu), 1.02 (d, J＝6.25 Hz, 6H,
H-δ, Leu); IR (KBr) v: 3126 (N－H str, amide), 2965－
2961, 2925－2519 (C－H str, CH3 and CH2), 1742 (C＝
O str, ester), 1642, 1635 (C＝O str, amide), 1536, 1531
(N－H def, amide), 1389, 1368 (C－H def, tert-Butyl),
1269 (C－O str, ester) cm1.
3: 1H NMR (CDCl3) δ: 7.12 (t, J＝5.65 Hz, 1H, H-p,
Phe), 7.02－6.97 (m, 2H, H-m, Phe), 6.85 (dd, J＝7.2,
4.1 Hz, 2H, H-o, Phe), 6.75 (br s, 1H, NH, Phe), 6.43
(br s, 1H, NH, Val), 4.35 (q, J＝5.6 Hz, 1H, H-α, Phe),
4.28 (t, J＝5.85 Hz, 1H, H-α, Val), 3.54 (s, 3H, OCH3),
2.95 (d, J＝4.8 Hz, 2H, H-, Phe), 1.87－1.82 (m, 1H,
H-, Val), 1.54 (s, 9H, tert-Butyl), 1.07 (d, J＝4.6 Hz,
6H, H-γ, Val); IR (CHCl3) v: 3124 (N－H str, amide),
3069－3062 (C－H str, aromatic ring), 2923 (C－H str,
CH2), 1744 (C＝O str, ester), 1640, 1632 (C＝O str,
amide), 1562, 1435 (skeletal bands), 1536, 1532 (N－H
def, amide), 1385, 1362 (C－H def, iso-Propyl), 1388,
1369 (C－H def, tert-Butyl), 1271 (C－O str, ester),
716, 689 (C－H def, oop, aromatic ring) cm1.
1
4: H NMR (CDCl3) δ: 8.89 (br s, 1H, NH, Leu),
7.35 (br s, 1H, NH, Ile), 6.58 (br s, 1H, NH, Thr), 6.51
(br s, 1H, NH, Gly), 4.55 (t, J＝8.6 Hz, 1H, H-α, Ile),
4.43 (br s, 1H, OH, Thr), 4.37 (t, J＝3.9 Hz, 1H, H-α,
Thr), 4.05 (d, J＝5.2 Hz, 2H, H-α, Gly), 3.78 (q, 1H,
H-α, Leu), 3.71－3.65 (m, 1H, H-, Thr), 3.61 (s, 3H,
OCH3), 2.07－2.02 (m, 1H, H-, Ile), 1.75 (t, J＝7.9 Hz,
2H, H-, Leu), 1.67－1.62 (m, 2H, H-γ, Ile), 1.55 (s, 9H,
tert-Butyl), 1.51－1.45 (m, 1H, H-γ, Leu), 1.27 (d, J＝
4.85 Hz, 3H, H-γ, Thr), 1.04 (d, J＝5.85 Hz, 3H, H-γ',
Chin. J. Chem. 2016, 34, 1158—1164
© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
1159

Dahiya and Singh
FULL PAPER
Ile), 1.01 (d, J＝6.2 Hz, 6H, H-δ, Leu), 0.94 (t, J＝7.8
Hz, 3H, H-δ, Ile); IR (CHCl3) v: 3128, 3125－3122
(N－H str, amide), 2969－2962, 2925, 2518 (C－H str,
CH3 and CH2), 1743 (C＝O str, ester), 1643－1637
(C＝O str, amide), 1539－1535, 1531 (N－H def, am-
ide), 1424, 1325 (O－H def), 1388, 1366 (C－H def,
tert-Butyl), 1270 (C－O str, ester) cm1.
1435 (skeletal bands), 1538－1533, 1529 (N－H def,
amide), 1426, 1322 (O－H def), 1389, 1366 (C－H def,
tert-Butyl), 1383, 1362 (C－H def, iso-Propyl), 1269
(C－O str, ester), 717, 689 (C－H def, oop, aromatic
ring) cm1.
7: 1H NMR (CDCl3) δ: 9.29 (br s, 1H, NH, Ile), 8.09
(br s, 1H, NH, Thr), 7.98 (br s, 1H, NH, Val), 7.82 (br s,
1H, NH, Leu), 7.62 (br s, 1H, OH, Thr), 7.58 (br s, 1H,
NH, Phe), 7.19－7.15 (m, 2H, H-m, Phe), 7.09 (br s, 1H,
NH, Gly-2), 7.02 (br s, 1H, NH, Gly-1), 6.99 (t, J＝5.7
Hz, 1H, H-p, Phe), 6.87 (dd, J＝7.15, 4.2 Hz, 2H, H-o,
Phe), 6.54 (t, J＝5.9 Hz, 1H, H-α, Val), 6.27 (q, 1H, H-α,
Leu), 5.76 (q, J＝5.6 Hz, 1H, H-α, Phe), 5.68 (t, J＝3.9
Hz, 1H, H-α, Thr), 5.37 (d, J＝5.15 Hz, 2H, H-α, Gly-2),
5.28 (t, J＝8.6 Hz, 1H, H-α, Ile), 5.23 (d, J＝5.2 Hz, 2H,
H-α, Gly-1), 3.84－3.79 (m, 1H, H-, Thr), 2.43 (d, J＝
4.8 Hz, 2H, H-, Phe), 1.89－1.85 (m, 1H, H-, Ile),
1.75－1.71 (m, 1H, H-, Val), 1.69 (t, J＝7.9 Hz, 2H,
H-, Leu), 1.67－1.63 (m, 1H, H-, Ile), 1.42 (d, J＝
4.85 Hz, 3H, H-γ, Thr), 1.18 (d, J＝4.55 Hz, 6H, H-,
Val), 1.02 (d, J＝6.2 Hz, 6H, H-δ, Leu), 0.98 (d, J＝
5.85 Hz, 3H, H-γ', Ile), 0.94 (t, J＝7.8 Hz, 3H, H-δ, Ile),
0.84－0.79 (m, 2H, H-γ, Leu); 13C NMR (CDCl3) δ:
174.9, 173.4 (2C, C＝O, Ile and Val), 171.6, 169.9 (2C,
C＝O, Gly-2 and Gly-1), 169.2, 164.4 (2C, C＝O, Thr
and Leu), 162.8 (C＝O, Phe), 138.0 (C-, Phe), 131.5
(2C, C-o, Phe), 129.8 (2C, C-m, Phe), 126.9 (C-p, Phe),
66.2 (C-, Thr), 60.7, 59.4 (2C, C-α, Ile and Val), 58.0,
54.4 (2C, C-α, Thr and Phe), 49.1 (C-α, Leu), 44.0, 42.3
(2C, C-α, Gly-2 and Gly-1), 39.9 (C-, Phe), 37.4, 35.0
(2C, C-α, Leu and Ile), 33.2 (C-, Val), 25.8, 23.6 (2C,
C-γ, Leu and Ile), 23.1 (2C, C-δ, Leu), 22.3 (C-, Thr),
18.5 (2C, C-γ, Val), 17.4 (C-γ', Ile), 9.9 (C-, Ile); IR
(KBr) v: 3128－3125, 3122, 3119 (N－H str, amide),
3066 (C－H str, aromatic ring), 2967, 2963－2958,
2925, 2917 (C－H str, CH3 and CH2), 1647, 1639,
1632－1627 (C＝O str, amide), 1569, 1437 (skeletal
bands), 1537, 1532, 1529－1525 (N－H def, amide),
1425, 1326 (O － H def), 1382, 1361 (C － H def,
iso-propyl), 719, 686 (C－H def, oop, aromatic ring)
cm1; FABMS m/z (%): 688.8 [(M＋H)＋, 100], 660.8
[(688.8-CO)＋, 11], 631.7 [(H-Thr-Ile-Leu-Gly-Val-Phe)＋,
60], 603.7 [(631.7-CO)＋, 15], 589.7 [(H-Phe-Gly-Thr-
Ile-Leu-Gly)＋, 44], 587.7 [(H-Ile-Leu-Gly-Val-Phe-Gly)＋,
58], 575.6 [(H-Leu-Gly-Val-Phe-Gly-Thr)＋, 41], 561.7
[(589.7–CO)＋, 12], 559.7 [(587.7-CO)＋, 10], 547.6
1
5: H NMR (CDCl3) δ: 7.60 (br s, 1H, NH, Phe),
7.24－7.19 (m, 2H, H-m, Phe), 6.99 (t, J＝5.7 Hz, 1H,
H-p, Phe), 6.83 (dd, J＝7.15, 4.1 Hz, 2H, H-o, Phe),
6.52 (br s, 1H, NH, Gly), 6.40 (br s, 1H, NH, Val), 4.54
(q, J＝5.55 Hz, 1H, H-α, Phe), 4.20 (t, J＝5.9 Hz, 1H,
H-α, Val), 4.05 (d, J＝5.15 Hz, 2H, H-α, Gly), 3.67 (s,
3H, OCH3), 2.77 (d, J＝4.8 Hz, 2H, H-, Phe), 1.89－
1.84 (m, 1H, H-, Val), 1.56 (s, 9H, tert-Butyl), 1.05 (d,
J＝4.55 Hz, 6H, H-γ, Val); IR (CHCl3) v: 3126, 3122
(N－H str, amide), 3065, 3059 (C－H str, aromatic ring),
2926－2921 (C－H str, CH2), 1742 (C＝O str, ester),
1643－1639, 1633 (C＝O str, amide), 1565, 1432 (skel-
etal bands), 1535, 1529 (N－H def, amide), 1384, 1363
(C－H def, iso-Propyl), 1389, 1367 (C－H def, tert-
Butyl), 1268 (C－O str, ester), 714, 687 (C－H def, oop,
aromatic ring) cm1.
1
6: H NMR (CDCl3) δ: 8.88 (br s, 1H, NH, Leu),
8.29 (br s, 1H, NH, Gly-1), 7.99 (br s, 1H, NH, Val),
7.92 (br s, 1H, NH, Phe), 7.37 (br s, 1H, NH, Ile),
7.20－7.15 (m, 2H, H-m, Phe), 6.98 (t, J＝5.65 Hz, 1H,
H-p, Phe), 6.84 (dd, J＝7.2 Hz, 4.15 Hz, 2H, H-o, Phe),
6.56 (br s, 1H, NH, Thr), 6.49 (br s, 1H, NH, Gly-2),
4.53 (t, J＝8.55 Hz, 1H, H-α, Ile), 4.45 (br s, 1H, OH,
Thr), 4.38 (t, J＝3.85 Hz, 1H, H-α, Thr), 4.08 (d, J＝
5.15 Hz, 2H, H-α, Gly-1), 4.04 (d, J＝5.2 Hz, 2H, H-α,
Gly-2), 3.92 (q, 1H, H-α, Leu), 3.85 (q, J＝5.55 Hz, 1H,
H-α, Phe), 3.78 (t, J＝5.85 Hz, 1H, H-α, Val), 3.72－
3.67 (m, 1H, H-, Thr), 3.63 (s, 3H, OCH3), 2.98 (d,
J＝4.75 Hz, 2H, H-, Phe), 2.09－2.04 (m, 1H, H-,
Ile), 1.82 (t, J＝7.85 Hz, 2H, H-, Leu), 1.66－1.61 (m,
2H, H-γ, Ile), 1.59 (s, 9H, tert-Butyl), 1.56－1.51 (m,
1H, H-, Val), 1.50－1.44 (m, 1H, H-γ, Leu), 1.29 (d,
J＝4.9 Hz, 3H, H-γ, Thr), 1.06 (d, J＝5.9 Hz, 3H, H-γ',
Ile), 0.99 (d, J＝6.15 Hz, 6H, H-δ, Leu), 0.96 (d, J＝4.6
Hz, 6H, H-γ, Val), 0.93 (t, J＝7.75 Hz, 3H, H-, Ile);
13C NMR (CDCl3) δ: 174.5, 172.8 (2C, C＝O, Ile and
Thr), 170.6, 169.1 (2C, C＝O, Phe and Leu), 168.2,
166.9 (2C, C＝O, Gly-2 and Val), 166.5, 149.2 (2C, C
＝O, Gly-1, Boc), 138.4 (C-γ, Phe), 130.2 (2C, C-o,
Phe), 127.7 (2C, C-m, Phe), 127.1 (C-p, Phe), 79.7 (C-α,
Boc), 69.4 (C-, Thr), 62.1, 58.5 (2C, C-α, Thr and Val),
53.8 (C-α, Phe), 51.9 (OCH3), 49.8, 48.1 (2C, C-α, Ile
and Leu), 41.3, 39.4 (2C, C-α, Gly-2 and Gly-1), 38.8,
36.7 (2C, C-, Leu and Phe), 35.5, 33.2 (2C, C-, Ile
and Val), 29.4 (3C, C-, Boc), 26.6, 23.2 (2C, C-γ, Ile
and Leu), 22.6 (2C, C-δ, Leu), 20.5 (C-γ, Thr), 18.8 (2C,
C-γ, Val), 16.9 (C-γ', Ile), 10.3 (C-δ, Ile); IR (CHCl3) v:
3129－3126, 3123, 3119 (N－H str, amide), 3069, 3056
(C－H str, aromatic ring), 2969, 2965－2959, 2924－
2916 (C－H str, CH3 and CH2), 1741 (C＝O str, ester),
1643－1638, 1634－1629 (C＝O str, amide), 1568,
[(575.6-CO)＋, 11], 532.6 [(H-Phe-Gly-Thr-Ile-Leu)＋,
＋
66], 530.6 [(H-Ile-Leu-Gly-Val-Phe) , 73], 504.6
[(532.6CO)＋, 14], 502.6 [(530.6CO)＋, 12], 484.6
[(H-Thr-Ile-Leu-Gly-Val) ＋ , 70], 474.5 [(H-Leu-Gly-
Val-Phe-Gly) ＋, 62], 456.6 [(484.6CO)＋, 11], 446.5
[(474.5CO)＋, 17], 419.5 [(H-Phe-Gly-Thr-Ile)＋, 54],
417.5 [(H-Leu-Gly-Val-Phe)＋, 36], 391.5 [(419.5CO)＋,
11], 389.5 [(417.5CO)＋, 10], 385.4 [(H-Thr-Ile-Leu-
Gly)＋, 49], 383.5 [(H-Ile-Leu-Gly-Val)＋, 43], 357.4
[(385.4CO)＋, 10], 355.4 [(383.5CO)＋, 14], 306.3
[(H-Phe-Gly-Thr)＋, 61], 284.3 [(H-Ile-Leu-Gly)＋, 52],
278.3 [(306.3CO)＋, 11], 270.3 [(H-Leu-Gly-Val)＋, 32],
1160
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First Total Synthesis and Biological Potential of a Heptacyclopeptide of Plant Origin
242.3 [(270.3CO)＋, 10], 205.2 [(H-Phe-Gly)＋, 28],
177.2 [(205.2CO)＋, 15], 171.2 [(H-Leu-Gly)＋, 22],
148.2 [(H-Phe)＋, 19], 143.2 [(171.2CO)＋, 11], 120.2
[Phe immonium ion (C8H10N)＋, 24], 114.2 [(H-Leu)＋,
11], 91.1 [(C7H7)＋, 15], 86.1 [Leu/Ile immonium ion
(C5H12N)＋, 25], 77.1 [(C6H5)＋, 11], 74.1 [(Thr immo-
nium ion C3H8NO)＋, 15], 72.1 [Val immonium ion
(C4H10N)＋, 17], 57.1 [(C4H9)＋, 8], 45.1 [(C2H5O)＋, 11],
43.1 [(C3H7)＋, 11], 30.0 [(Gly immonium ion CH4N)＋,
16], 29.1 [(C2H5)＋, 9], 15.0 [(CH3)＋, 11].
was used as standard drug. The results of anthelmintic
screening are tabulated in Table 3.
Antibacterial and antifungal screening Antimi-
crobial activity studies for linear heptapeptide and hep-
tacyclopeptide 6 and 7 were performed against Gram-
positive bacteria Bacillus subtilis, Staphylococcus aure-
us, Gram-negative bacteria Pseudomonas aeruginosa,
Klebsiella pneumonia, dermatophytes Microsporum
audouinii, Trichophyton mentagrophytes, diamorphic
fungi Candida albicans and other fungal strains, in-
cluding Aspergillus niger at 2003.1 μg/mL concentra-
tion by using modified Kirby-Bauer disc diffusion
method.[23] MIC values of test compounds were deter-
mined by tube dilution technique. The Petri plates inoc-
ulated with bacterial cultures were incubated at 37 ℃
for 18 h and those inoculated with fungal cultures were
incubated at 37 ℃ for 48 h. Gatifloxacin and griseo-
fulvin were used as reference drugs and DMF/DMSO
were used as control. The results of antibacterial and
antifungal studies are presented in Table 4 and Table 5.
Experimental details of the biological activity studies
are described in our previously published reports.[24,25]
Table 1 Physical characterization data for 1－7
c
Compd.
Physical state
Semisolid mass
White solida
[α]D /() Rff Yield/%
1
2
3
4
5
6
7
＋11.7 0.72
–44.9d 0.86
–107.2 0.67
＋82.9 0.58
–51.6d 0.80
–56.7d 0.62
69
77
85
80
72
78
84
Semisolid mass
Semisolid mass
Viscous mass
Semisolid mass
Pale yellow solidb
–78.4e 0.53g
a
b
d
e
m.p. 87－88 ℃; 93－95 ℃ (d); c c, 0.5 in MeOH; c, 0.25 in MeOH; c,
0.1 in MeOH; (CHCl3/MeOH, V∶V＝9∶1); g (CHCl3/MeOH , V∶V＝7∶3).
f
Results and Discussion
Biological activity studies
An outline of the synthetic strategy for heptacyclo-
peptide 7 is illustrated in Scheme 1.
Anthelmintic screening Newly synthesized linear
heptapeptide and heptacyclopeptide 6 and 7 were sub-
jected to anthelmintic activity studies against three
earthworm species Megascoplex konkanensis, Pontos-
cotex corethruses and Eudrilus eugeniea at 2 mg/mL
concentration using Garg’s method.[22] Tween 80 (0.5%)
in distilled water was used as control and mebendazole
To prepare the target molecule, it was split into three
dipeptide units viz. Boc-L-Thr-L-Ile-OMe (1), Boc-L-
Leu-Gly-OMe (2), Boc-L-Val-L-Phe-OMe (3) and an
amino acid unit Gly-OMe.HCl. The dipeptides were
prepared by coupling Boc-amino acid viz. Boc-L-Thr-
OH, Boc-L-Leu-OH, Boc-L-Val-OH with respective
Table 2 Elemental analysis data for 1－7
Compd.
Mol. formula (Mr)
C16H30N2O6 (346)
C14H26N2O5 (302)
C20H30N2O5 (378)
C24H44N4O8 (516)
C22H33N3O6 (435)
C40H65N7O11 (819)
C34H53N7O8 (687)
C (Calcd/found)/%
55.47 (55.49)
55.61 (55.58)
63.47 (63.45)
55.80 (55.79)
60.67 (60.65)
58.59 (58.57)
59.37 (59.39)
H (Calcd/found) /%
8.73 (8.72)
8.67 (8.65)
7.99 (7.96)
8.58 (8.60)
7.64 (7.65)
7.99 (8.01)
7.77 (7.78)
N (Calcd/found) /%
8.09 (8.10)
1
2
3
4
5
6
7
9.26 (9.25)
7.40 (7.39)
10.84 (10.85)
9.65 (9.68)
11.96 (11.95)
14.25 (14.23)
Table 3 Anthelmintic activity data for 6 and 7a
Earthworm species
P. core.
Compd.
M. konk.
E. euge.
mpt (mdt)b
mpt (mdt)
mpt (mdt)
6
14.46±0.32c (23.43 ± 0.18)
09.18±0.13 (17.39±0.28)
－
19.26±0.22 (30.14±0.43)
13.44±0.40 (22.04±0.26)
－
14.33±0.40 (24.54±0.41)
11.28±0.19 (21.35±0.26)
－
7
Controld
Stde
13.65±0.42 (22.45±0.36)
17.57±0.47 (29.55±0.19)
13.51±0.43 (24.09±0.60)
a M. konk.: Megascoplex konkanensis, P. core.: Pontoscotex corethruses, E. euge.: Eudrilus eugeniea; b mean paralyzing time (mean death
time) in minutes; c Data are given as mean±S.D. (n＝3); d Tween 80 (0.5%) in distilled water; e Mebendazole.
Chin. J. Chem. 2016, 34, 1158—1164
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Dahiya and Singh
FULL PAPER
Table 4 Antibacterial activity data for 6 and 7a
heptacyclopeptide
cyclo-(L-threonyl-L-isoleucyl-L-
leucyl-glycyl-L-valyl-L-phenylalanyl-glycyl) (7). Fur-
ther, in order to describe the intermolecular forces of
drug receptor interaction as well as transport and distri-
bution of drugs in a quantitative manner, various steric
and lipophilicity parameters are needed to be calculated
for the synthesized linear and cycloheptapeptide.
Diameter of zone of inhibition/mm
Bacterial strains
Compd.
B. sub.
－
S. aur.
13 (25)
17 (25)
－
P. aeru.
11 (6.25)
16 (6.25)
－
K. pneu.
15 (6.25)
18 (6.25)
－
6
7
－
Controlb
Stdc
－
18 (12.5)d
Scheme 1 Synthetic route for heptacyclopeptide 7
27 (6.25)
23 (6.25)
25 (6.25)
a B. sub.: Bacillus subtilis, S. aur.: Staphylococcus aureus, P. aeru.:
Pseudomonas aeruginosa, K. pneu.: Klebsiella pneumonia; b DMF;
c Gatifloxacin; d Values in bracket are MIC values (g/mL).
Table 5 Antifungal activity data for 6 and 7a
Diameter of zone of inhibition/mm
Fungal strains
Compd.
C. alb.
M. audo.
A. niger
－
T. menta.
18 (6.25)
24 (6.25)
－
6
12 (6.25)
15 (6.25)
－
16 (6.25)
22 (6.25)
－
7
－
Controlb
Stdc
－
20 (6.25)d
18 (6.25)
20 (12.5) 19 (6.25)
a C. alb.: Candida albicans, M. audo.: Microsporum audouinii, A.
niger: Aspergillus niger, T. menta.: Trichophyon mentagrophytes;
b
c
d
DMSO; Griseofulvin; Values in bracket are MIC values
(g/mL).
amino acid methyl ester hydrochlorides like L-Ile-OMe•
HCl, Gly-OMe•HCl and L-Phe-OMe•HCl using dicy-
clohexylcarbodiimide (DCC)/N,N'-diisopropylcarbodi-
imide (DIPC)/1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide hydrochloride (EDC•HCl) as coupling agents
and TEA/NMM as base.[26] The ester group of dipeptide
unit 1 was removed using LiOH and deprotected peptide
was coupled with another dipeptide unit 2 deprotected at
amino terminal using trifluoroacetic acid (TFA), using
DIPC as coupling agent and NMM as base, to get the
linear tetrapeptide unit Boc-L-Thr-L-Ile-L-Leu-Gly-
OMe (4).
Similarily, the ester group of dipeptide unit 3 was
removed using LiOH and deprotected peptide was cou-
pled with Gly-OMe•HCl using EDC•HCl as coupling
agent and TEA as base, to get the tripeptide unit
Boc-L-Val-L-Phe-Gly-OMe (5). Now, tetrapeptide unit
4 deprotected at carboxyl end was coupled with tripep-
tide unit 5 deprotected at amino terminal, using DIPC as
coupling agent and NMM as base, to get linear hep-
tapeptide unit Boc-L-Thr-L-Ile-L-Leu-Gly-L-Val-L-
Phe-Gly-OMe (6). Finally, cyclization of the linear
heptapeptide 6 was done by pentafluorophenyl ester
method which involves replacement of methyl ester
with pentafluorophenyl ester which is a better leaving
group, through deprotection at carboxyl terminal using
LiOH and coupling with pentafluorophenol using DCC,
followed by removal of Boc-group using TFA and
keeping the deprotected unit for 7 d at 0 ℃ in the
presence of base (NMM/TEA/pyridine) to obtain the
Disappearance of absorption bands at 1741 and 1269
cm1 and 1389, 1366 cm1 (C＝Ostr and C－O str, me-
1162
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Chin. J. Chem. 2016, 34, 1158—1164

First Total Synthesis and Biological Potential of a Heptacyclopeptide of Plant Origin
Conclusions
thyl ester group and C－H bend, tert-butyl group) in
FT-IR spectrum of 7 clearly indicated cyclization of
linear heptapeptide unit. This fact was further supported
by disappearance of two singlets at δ 1.59 and 3.63,
corresponding to protons of tert-butyl and methyl ester
The first total synthesis of naturally occurring hep-
tacyclopeptide mahafacyclin A (7) was carried out suc-
cessfully in a good yield from the linear precursor via
coupling reactions utilizing carbodiimide chemistry.
DIPC was found to be a good coupling agent in com-
parison to DCC, EDC•HCl and pentafluorophenyl ester
proved to be better for the activation of acid functional-
ity of linear heptapeptide unit. Moreover, NMM proved
to be a good base for intramolecular cyclization of line-
ar heptapeptide fragment in comparison to pyridine and
TEA. The synthesized heptacyclopeptide 7 displayed
potent anthelmintic activity against M. konkanensis, P.
corethruses and E. eugeniea, alongwith remarkable an-
tifungal activity against dermatophytes M. audouinii
and T. mentagrophytes, in comparison to reference drugs
mebendazole and griseofulvin. The newly synthesized
cyclic peptide 7 displayed better activity than its linear
counterpart 6 due to the conformational rigidity of cy-
clic form. The rigidity of cyclic form decreases the en-
tropy term of the Gibbs free energy and therefore, al-
lows the enhanced binding toward target molecules or
receptor selectivity. Further studies are needed to de-
velop SAR of natural/synthetic cycloheptapeptide by
replacing phenylalanine unit of mahafacyclin A with
other units like N-methylphenylalanine, tyrosine, histi-
dine, tryptophan units and indicate effect of modifica-
tion on biological activities. On passing toxicity tests,
newly synthesized heptacyclopeptide 7 may prove a
good candidate for clinical studies and can be a new
anthelmintic and antidermatophyte drug of future.
1
groups, in H NMR spectrum and disappearance of sin-
glets at δ 79.7, 29.4 and 51.9, corresponding to carbon
atoms of tert-butyl and methyl ester groups, in 13C
NMR spectrum of 7. Seven signals between δ 6.54－
5.23 in the proton spectrum of 7 suggested a peptidic
structure for the synthesized product, with these signals
being attributable to the α-protons of all seven amino
acid units and values being in a slightly higher range
1
when compared to natural mahafacyclin A.[15] The H
NMR spectrum of cyclized product showed the pres-
ence of seven broad singlets between δ 9.29－7.58, 7.09
－7.02 corresponding to the imino protons of the iso-
leucine, threonine, valine, leucine, phenylalanine and
two glycine moieties and 13C NMR spectrum of cyclo-
peptide 7 indicated the presence of seven signals be-
tween δ 174.9－162.8 due to carbonyl carbons of seven
amino acids, which were in agreement with δ values for
imino protons and carbonyl carbons of natural ma-
hafacyclin A,[15] indicating similarity of the structure of
the newly synthesized heptacyclopeptide with that of
the natural molecule. Moreover, 1H/13C NMR spectra of
the cyclized product 7 showed characteristic peaks con-
firming the presence of all the 53 protons and 34 carbon
atoms. Presence of pseudomolecular ion peak at m/z
688.8 corresponding to the molecular formula
C34H54N7O8 in mass spectra of 7, along with other
fragment ion peaks resulting from cleavage at ‘Leu-Ile’,
‘Phe-Val’, ‘Thr-Gly’ and ‘Ile-Thr’ amide bond levels,
showed the exact sequence of attachment of all the sev-
en amino acid units in a chain and was in agreement
with protonated ion peak of natural mahafacyclin A.[15]
In addition, elemental analysis data of 7 afforded values
(±0.02) strictly in accordance to the molecular compo-
sition. Anthelmintic activity studies revealed that the
synthesized heptacyclopeptide 7 possessed potent an-
thelmintic activity at 2 mg/mL concentration in tween
80 (0.5%) and distilled water. From the comparison of
anthelmintic activity data, it is observed that cyclopep-
tide 7 displayed more activity than its corresponding
linear precursor 6 against all three earthworm species M.
konkanensis, P. corethruses and E. eugeniea, in com-
parison to standard drug mebendazole (Table 3). More-
over, heptacyclopeptide 7 displayed remarkable bioac-
tivity against dermatophytes M. audouinii and T. men-
tagrophytes with MIC values of 6 μg/mL. Analysis of
antimicrobial activity data indicated that cyclic hep-
tapeptide 7 displayed moderate level of bioactivity
against pathogenic Candida albicans and Gram-nega-
tive bacteria, in comparison to standard drugs griseoful-
vin and gatifloxacin. However, no significant antimi-
crobial activity was observed against Gram-positive
bacteria and Aspergillus niger (Table 4).
Acknowledgement
The authors are thankful to Sophisticated Analytical
Instrumentation Laboratory, School of Pharmaceutical
Sciences, R. G. P. V., Bhopal, Madhya Pradesh, India for
spectral analysis.
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Chin. J. Chem. 2016, 34, 1158—1164