Synthesis, cytotoxic and antimicrobial screening of a proline-rich cyclopolypeptide

Article abstract of DOI:10.1248/cpb.57.214

Present study describes the first total synthesis of a cyclic heptapeptide, stylisin 1 (8) via coupling of tetrapeptide Boc-L-tyrosinyl-L-prolyl-L-leucyl- L-proline-OH and tripeptide L-phenylalanyl-L-isoleucyl-L-proline- OMe followed by cyclization of lin

Full text of DOI:10.1248/cpb.57.214

214
Notes
Chem. Pharm. Bull. 57(2) 214—217 (2009)
Vol. 57, No. 2
Synthesis, Cytotoxic and Antimicrobial Screening of a Proline-Rich
Cyclopolypeptide
b
Rajiv DAHIYA, ,a Akhilesh KUMAR,b and Rajul GUPTA
*
a Department of Pharmaceutical Chemistry, NRI Institute of Pharmacy; Bhopal–462026, Madhya Pradesh, India: and
b Department of Pharmaceutical Chemistry, Rajiv Academy for Pharmacy; Mathura–281 001, Uttar Pradesh, India.
Received October 3, 2008; accepted November 23, 2008; published online November 28, 2008
Present study describes the first total synthesis of a cyclic heptapeptide, stylisin 1 (8) via coupling of
tetrapeptide Boc-L-tyrosinyl-L-prolyl-L-leucyl-L-proline-OH and tripeptide L-phenylalanyl-L-isoleucyl-L-proline-
OMe followed by cyclization of linear heptapeptide segment. Structure elucidation of synthesized cyclopeptide
was done on basis of detailed spectral as well as elemental analysis. From the results of pharmacological screen-
ing, it was concluded that cyclopeptide 8 possessed moderate cytotoxicity against Dalton’s lymphoma ascites
(DLA) and Ehrlich’s ascites carcinoma (EAC) cell lines with IC50 (inhibitory concentration, 50%) values of 10.6
and 14.6 mM. Furthermore, cyclopeptide 8 exhibited moderate to good antimicrobial activity against Gram ꢀve
(negative) bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa, dermatophytes and Candida albicans
with minimum inhibitory concentration (MIC) of 6 mg/ml.
Key words total synthesis; cyclopolypeptide; marine sponge; stylisin 1; cytotoxicity; antimicrobial activity
Marine environment especially sponges are well known to Dipeptide 1 after deprotection at carboxyl end, was coupled
produce a wide variety of biomedically useful agents that with dipeptide 2 deprotected at amino terminal, to get the
may act as lead compounds for drug development.1—5) tetrapeptide unit Boc-L-tyrosinyl-L-prolyl-L-leucyl-L-proline-
Among them, cyclopeptides having unique structures and OMe (5). Similarly, ester group of dipeptide 3 was removed
a wide pharmacological profile6) are emerged as vital or- by alkaline hydrolysis with LiOH and deprotected peptide
ganic congeners which may prove better candidates to over- was coupled with compound 4 to get the tripeptide unit Boc-
come the problem of widespread increase of resistance to- L-phenylalanyl-L-isoleucyl-L-proline-OMe (6). After removal
wards conventional drugs. Marine sponge-derived natural of ester and Boc groups of tetrapeptide 5 and tripeptide 6,
cyclopolypeptides possess numerous pharmacological activi- deprotected peptide units were coupled with each other to get
ties including antimicrobial,7—9) anti-inflammatory,10) anti- linear heptapeptide unit Boc-L-tyrosinyl-L-prolyl-L-leucyl-
human immunodeficiency virus (HIV),11,12) nematocidal,13) L-prolyl-L-phenylalanyl-L-isoleucyl-L-proline-OMe (7). The
cytotoxic,14,15) antifouling,16) antitubercular,17) histone de- ester group of linear fragment was removed using LiOH and
acetylase, tryptase, thrombin, protein phophatases 1, 2A and p-nitrophenyl or pentafluorophenyl (pnp or pfp) ester groups
superoxide generation inhibitory properties.18—22) A natural were introduced. The Boc-group was removed using trifluo-
cyclic heptapeptide, stylisin 1 has been isolated from the Ja- roacetic acid (CF3COOH) and deprotected linear fragment
maican sponge Stylissa caribica and its structure was eluci- was now cyclized by keeping the whole contents at 0 °C (7 d)
dated on basis of correlation spectroscopy (COSY), het- in presence of catalytic amount of triethylamine (TEA)/N-
eronuclear multiple quantum coherence (HMQC), heteronu- methylmorpholine (NMM)/pyridine to get cyclic product 8
clear multiple bond coherence (HMBC) and nuclear over- (Chart 1).
hauser enhancement spectroscopy (NOESY) experiments
Structure of the newly synthesized cyclic heptapeptide as
followed by determination of absolute configuration of well as intermediates di/tri/tetrapeptides were confirmed by
amino acids by Marfey’s analysis method.23)
infrared (IR), nuclear magnetic resonance (1H-NMR) and el-
Prompted by the medicinal properties of proline-rich emental analysis. In addition, 13C-NMR and mass spectra
cyclic peptides24—27) as well as to obtain a natural bioactive were recorded for the linear as well as cycloheptapeptide.
peptide in good yield, present investigation was aimed at first
Cytotoxicity Activity The synthesized linear and cyclic
total synthesis of natural peptide, stylisin 1 (8) employing heptapeptides 7, 8 were subjected to short term in vitro cyto-
disconnection strategy. The potential cytotoxic, antibacterial toxicity study29) against Dalton’s lymphoma ascites (DLA)
and antifungal activities of the synthesized peptide were also and Ehrlich’s ascites carcinoma (EAC) cell lines at 62.5—
evaluated.
3.91 mg/ml using 5-fluorouracil (5-FU) as reference com-
Chemistry To carry out the synthesis of stylisin 1 (8), pound. Activity was assessed by determining the percentage
the cyclic heptapeptide molecule was split into three dipep- inhibition of DLA and EAC cells. IC50 values were deter-
tide units Boc-L-tyrosinyl-L-proline-OMe (1), Boc-L-leucyl- mined by graphical extrapolation method. The cytotoxic ac-
L-proline-OMe (2), Boc-L-phenylalanyl-L-isoleucine-OMe tivity data of synthesized compounds 7, 8 is tabulated in
(3) and single amino acid unit L-proline-OMe·HCl (4). The Table 1.
required dipeptide units 1—3 were prepared by coupling of
Antibacterial and Antifungal Activity The synthesized
Boc-amino acids viz. Boc-L-tyrosine-OH, Boc-L-leucine-OH linear and cycloheptapeptides 7, 8 were also evaluated for
and Boc-L-phenylalanine-OH with corresponding amino acid their antimicrobial activity30) against six bacterial strains
methyl ester hydrochlorides such as L-proline-OMe·HCl and Corynebacterium pyogenes, Staphylococcus aureus, Bacillus
L-isoleucine-OMe·HCl using DCC as coupling agent.28) subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa
∗ To whom correspondence should be addressed. e-mail: rajivdahiya77@rediffmail.com
© 2009 Pharmaceutical Society of Japan

February 2009
215
Chart 1. Synthetic Route to Stylisin 1 (8)
Table 1. Cytotoxic Activity Data against Pathogenic Cell Lines
which are formed only if peptide bond between ‘Pro–Phe’
and ‘Pro–Tyr’, breaks. In addition, elemental analysis of 8 af-
forded average values (ꢂ0.03) strictly in accordance to the
molecular composition.
Cell lines
Compd.
DLA cells
EAC cells
Synthesized cyclopeptide 8 possessed moderate cytotoxic
activity against DLA and EAC cell lines with IC50 values of
10.6 and 14.6 mM respectively, in comparison to 5-FU (IC50
values, 37.4 and 90.6 mM). Comparison of antimicrobial
screening data suggested that 8 exhibited good antibacterial
activity against pathogenic microbes K. pneumoniae and P.
aeruginosa with MIC value of 6 mg/ml, in comparison to
7
8
14.8a)
10.6
—
20.3
14.6
—
Control
5-Fluorouracil
37.4
90.6
a) IC50 values in mM.
and Escherichia coli, and five fungal strains Candida albi- standard drug, gatifloxacin. The mechanism of antibacterial
cans, Aspergillus niger, Ganoderma sp., Microsporum au- action of 8 may be interference with the cell membrane func-
douinii and Trichophyton mentagrophytes at 25—6 mg/ml. tions by penetrating the outer membrane of bacteria through
Minimum inhibitory concentration (MIC) values of test com- porin channels. Moreover, 8 displayed moderate level of ac-
pounds were determined by tube dilution technique. The sol- tivity against dermatophytes M. audouinii and T. mentagro-
vents dimethylformamide (DMF) and dimethylsulphoxide phytes, and pathogenic Candida albicans with MIC values of
(DMSO) were used as negative controls, and gatifloxacin and 6 mg/ml. However, 8 displayed no significant activity against
griseofulvin were used as antibacterial and antifungal stan- neither Gram ꢁve (positive) bacteria nor pathogenic Gano-
dards. The antimicrobial activity shown by synthesized com- derma sp. and A. niger.
pounds 7, 8 towards various microbes is recorded in Tables 2
and 3.
In addition, analysis of pharmacological activity data re-
vealed that cycloheptapeptide 8 displayed more bioactivity
against pathogenic microbes and cell lines when compared to
its linear form 7. This is possibly because, cyclization of pep-
Results and Discussion
Synthesis of stylisin 1 (8) was carried out successfully tides reduces the degree of freedom for each constituent
with good yield (ꢀ85%) utilizing three different bases for within the ring and thus substantially leads to reduced flexi-
cyclization of linear heptapeptide fragment. Presence of bility, increased potency and selectivity of cyclic peptides.
Mꢁꢁ1 ion peak at m/z 828 corresponding to the molecular Further, inherent flexibility of linear peptides leads to differ-
formula C45H61N7O8 in mass spectra of 8, along with other ent conformations which can bind to more than one receptor
fragment ion peaks resulting from cleavage at ‘Pro–Leu’ molecules, resulting in undesirable adverse effects.31,32)
amide bond level, showed exact sequence of attachment of
all the seven amino acid moieties in a chain. Presence of Conclusions
peaks at 618, 590 and 261 mu in mass spectrum of 8 further
First total synthesis of natural peptide, stylisin 1 (8) was
indicated fragmentation at ‘Pro–Phe’ and ‘Pro–Tyr’ amide accomplished with good yield via coupling reactions uti-
bond levels. This fact was further confirmed by presence of lizing different carbodiimides. Diisopropylcarbodiimide
additional fragment ion peaks at 521, 148 and 374, 164 mu (DIPC)/TEA coupling method proved to be yield-effective,

216
Vol. 57, No. 2
Table 2. Antibacterial Activity Data against Pathogenic Bacteria
Bacterial strains
Compd.
C. pyogenes
S. aureus
B. subtilis
K. pneumoniae
P. aeruginosa
E. coli
7
8
25a)
25
—
12.5
12.5
—
25
25
—
12.5
6
6
—
6
6
6
—
6
12.5
12.5
—
Control
Gatifloxacin
12.5
6
6
a) MIC values in mg/ml.
Table 3. Antifungal Activity Data against Pathogenic Fungi
Fungal strains
Compd.
C. albicans
A. niger
Ganoderma sp.
M. audouinii
T. mentagrophytes
7
8
6a)
6
—
6
25
25
—
12.5
—
—
—
6
6
6
—
6
6
6
—
6
Control
Griseofulvin
a) MIC values in mg/ml.
in comparison to methods utilizing 1-ethyl-3-(3-dimethyl- 5 °C and then further for 24 h at room temperature. After the completion of
reaction, the reaction mixture was diluted with equal amount of water. The
precipitated solid was filtered, washed with water and recrystallized from a
mixture of chloroform and petroleum ether (bp 40—60 °C) followed by
cooling at 0 °C to get the title compounds.
aminopropyl)carbodiimide hydrochloride (EDC·HCl) and
TEA or NMM, providing 10—12% additional yield. Penta-
fluorophenyl ester was proved to be better for the activation
of acid functionality of linear heptapeptide unit when com-
pared to p-nitrophenol. NMM was found to be a good base
for intramolecular cyclization of linear peptide fragment in
comparison to TEA and pyridine. Synthesized cyclohep-
tapeptide displayed moderate cytotoxicity as well as antimi-
crobial activity against K. pneumoniae and P. aeruginosa,
dermatophytes and C. albicans. In comparison, Gram ꢃve
bacteria were found to be more sensitive than Gram ꢁve
bacteria towards the newly synthesized peptide. Cyclopoly-
peptide 8 showed sufficient promise as antimicrobial and cy-
totoxic agent and should be subjected for further toxicity
tests.
Deprotection of Tetrapeptide Unit at Carboxyl Terminal To a solu-
tion of the tetrapeptide 5 (6.0 g, 0.01 mol) in tetrahydrofuran (THF) : H2O
(1 : 1, 36 ml), 0.36 g (0.015 mol) of LiOH was added at 0 °C. The mixture
was stirred at room temperature for 1 h and then acidified to pH 3.5 with 1 N
H2SO4. The aqueous layer was extracted with Et2O (3ꢄ25 ml). The com-
bined organic extracts were dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The crude product was finally crystallized from
methanol and ether to get pure deprotected compound.
Deprotection of Tripeptide Unit at Amino Terminal Tripeptide 6
(4.9 g, 0.01 mol) was dissolved in CHCl3 (15 ml) and treated with 2.28 g
(0.02 mol) of CF3COOH. The resulting solution was stirred at room temper-
ature for 1 h, washed with saturated NaHCO3 solution (25 ml). The organic
layer was dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The crude product was purified by crystallization from CHCl3 and
petroleum ether (bp 40—60 °C) to get pure deprotected compound.
Experimental
Procedure for the Synthesis of Linear Heptapeptide Unit Compound
Materials and Methods All the reactions requiring anhydrous condi- 6a (3.9 g, 0.01 mol) was dissolved in THF (35 ml). To this solution, TEA
tions were conducted in flame dried apparatus. Melting points were deter- (2.8 ml, 0.021 mol) was added at 0 °C and the resulting mixture was stirred
mined using a Jindal Scientific melting point apparatus (Jindal, Delhi, India)
by open capillary method and are uncorrected. IR spectra were recorded on
FTIR-8400S fourier transform infrared spectrophotometer (Shimadzu,
Kyoto, Japan) using a thin film supported on KBr pellets for solids and
for 15 min. 5.9 g (0.01 mol) of compound 5a was dissolved in THF (35 ml)
and DIPC (1.26 g, 0.01 mol) was added to above mixture with stirring. Stir-
ring was continued for 36 h, after which the reaction mixture was filtered
and the filtrate was washed with 5% NaHCO3 and saturated NaCl solutions
CHCl3 as solvent for intermediate semisolids. 1H- and 13C-NMR spectra (30 ml each). The organic layer was dried over anhydrous Na2SO4, filtered
were recorded on Bruker AC 300 spectrometer at 300 MHz (Brucker, IL, and evaporated in vacuum. The crude product was recrystallized from a
U.S.A.) using CDCl3 or acetone-d6 as solvent and tetramethylsilane as inter- mixture of chloroform and petroleum ether (bp 40—60 °C) followed by
nal standard. Mass spectra were recorded on JMS-DX 303 Mass spectrome- cooling at 0 °C.
ter (Jeol, Tokyo, Japan) operating at 70 eV using fast atom bombardment
Synthesis of the Cyclic Heptapeptide (8) To synthesize stylisin 1 (8),
technique. Elemental analyses of cyclopeptide as well as intermediates were linear heptapeptide unit 7 (4.8 g, 0.005 mol) was deprotected at carboxyl end
performed on Vario EL III elemental analyzer (Elementar Vario EL III,
Hanau, Germany). Optical rotation of the synthesized peptides was meas-
using LiOH (0.18 g, 0.0075 mol) to get Boc-L-tyrosinyl-L-prolyl-L-leucyl-L-
prolyl-L-phenylalanyl-L-isoleucyl-L-proline-OH. The deprotected heptapep-
ured on Optics Technology automatic polarimeter (OpticsTech, Delhi, India) tide unit (4.73 g, 0.005 mol) was now dissolved in CHCl3 (50 ml) at 0 °C. To
in a 2 dm tube at 25 °C using sodium lamp and methanol as solvent. Purity
this solution, 0.0067 mol of p-nitrophenol or pentafluorophenol (0.94 g or
of synthesized cyclopeptide was checked by TLC on precoated silica 1.23 g) and DIPC (0.63 g, 0.005 mol) were added and stirring was done at
gel G plates (Kieselgel 0.25 mm, 60G F254, Merck, Germany) utilizing room temperature for 12 h. The reaction mixture was filtered and the filtrate
CHCl3 : AcOH : H2O as developing solvent and brown spots were detected was washed with 10% NaHCO3 solution (3ꢄ25 ml) and finally washed with
on exposure to iodine vapours in a tightly closed chamber.
5% HCl (2ꢄ30 ml) to get the corresponding p-nitrophenyl or pentaflu-
General Procedure for the Preparation of Linear Tri/Tetrapeptide orophenyl ester Boc-L-tyrosinyl-L-prolyl-L-leucyl-L-prolyl-L-phenylalanyl-
Segments L-Amino acid methyl ester hydrochloride or dipeptide methyl
ester (0.01 mol) was dissolved in DMF (25 ml). To this, TEA or NMM
L-isoleucyl-L-proline-Opnp or Boc-L-tyrosinyl-L-prolyl-L-leucyl-L-prolyl-L-
phenylalanyl-L-isoleucyl-L-proline-Opfp. To this compound (4.27 g or
(0.021 mol) was added at 0 °C and the reaction mixture was stirred for 4.45 g, 0.004 mol) dissolved in CHCl3 (35 ml), CF3COOH (0.91 g, 0.008
15 min. Boc-dipeptide (0.01 mol) in DMF (25 ml) and EDC·HCl or DIPC
(0.01 mol) were added with stirring. Stirring was first done for 1 h at 0—
mol) was added, stirred at room temperature for 1 h and washed with 10%
NaHCO3 solution (2ꢄ25 ml). The organic layer was dried over anhydrous

February 2009
217
Na2SO4 to get L-tyrosinyl-L-prolyl-L-leucyl-L-prolyl-L-phenylalanyl-
L
-
3039—3042 (2002).
isoleucyl-
L
-proline-Opnp or
L
-tyrosinyl-
L
-prolyl-
L
-leucyl-
L
-prolyl-
L
-
phenyl-
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TEA/NMM/pyridine (2.8 ml or 2.21 ml or 1.61 ml, 0.021 mol) was added.
Then, whole contents were kept at 0 °C for 7 d. The reaction mixture was
washed with 10% NaHCO3 (3ꢄ25 ml) and 5% HCl (2ꢄ30 ml) solutions.
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Cyclo (L-Tyrosinyl-L-prolyl-L-leucyl-L-prolyl-L-phenylalanyl-L-isoleucyl-
L-prolyl) (8): Yield: 3.64 g (88%, NMM), 3.1 g (75%, TEA), 2.86 g (69%,
1
C5H5N). mp 225 °C (dec). Rf, 0.68 (CHCl3 : AcOH : H2O, 3 : 2 : 5). H-NMR
(acetone-d6) d: 0.81 (1H, m), 0.94 (3H, t, Jꢅ7.8 Hz), 0.99 (6H, d, 13) Capon R. J., Ford J., Lacey E., Gill J. H., Heiland K., Friedel T., J. Nat.
Jꢅ6.3 Hz), 1.02 (3H, d, Jꢅ5.85 Hz), 1.37 (3H, m), 1.75 (4H, m), 1.83 (2H,
m), 1.89 (2H, t, Jꢅ7.9 Hz), 2.59 (4H, m), 2.67 (6H, m), 3.23 (2H, t,
Jꢅ7.3 Hz), 3.27 (2H, t, Jꢅ7.25 Hz), 3.32 (2H, t, Jꢅ7.3 Hz), 3.87 (1H, t,
Jꢅ6.85 Hz), 3.92 (1H, t, Jꢅ6.9 Hz), 3.96 (1H, t, Jꢅ6.9 Hz), 4.20 (1H, m),
4.40 (1H, m), 5.13 (1H, m), 5.25 (1H, dd, Jꢅ5.9, 4.25 Hz), 5.97 (1H, br s),
6.80 (2H, dd, Jꢅ8.75, 4.2 Hz), 6.92 (2H, dd, Jꢅ8.6, 5.25 Hz), 6.96 (2H, dd,
Jꢅ8.55, 4.9 Hz), 7.02 (1H, t, Jꢅ6.3 Hz), 7.25 (2H, tt, Jꢅ6.8, 4.45 Hz), 9.05
(1H, br s), 9.22 (1H, br s), 9.72 (1H, br s), 9.88 (1H, br s). 13C-NMR d: 10.2,
17.7, 22.1, 21.5, 22.7, 23.1 (2C), 24.4, 26.2, 33.0, 31.5, 34.7, 39.0, 36.8,
43.5, 42.2, 46.4, 48.3, 49.1, 51.7, 52.1, 55.8, 55.2, 56.9, 58.1, 58.5, 128.0,
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169.3, 169.8, 170.1, 172.5, 173.2, 174.8. IR (KBr) cmꢃ1: 3377, 3128, 3125,
3120, 3059, 3055, 2999, 2995, 2989, 2964, 2927, 2921, 2869, 2847, 2842,
1675, 1671, 1666, 1663, 1646, 1640, 1586, 1582, 1477, 1474, 1538, 1532,
1529, 1382, 1365, 1225, 864, 821, 713, 698. MS m/z: 829, 828 (Mꢁꢁ1),
801, 731, 618, 590, 568, 540, 521, 471, 443, 374, 358, 330, 261, 211, 183,
164, 148, 136, 114, 120, 107, 93, 91, 86, 70, 65, 57, 56, 43, 42, 29, 17, 15.
[a]D ꢃ44.7 (cꢅ0.2, MeOH) (ꢃ44.9 for natural stylisin 1). Anal. Calcd for
C45H61N7O8: C, 65.28; H, 7.43; N, 11.84. Found: C, 65.25; H, 7.43; N,
11.85.
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