Synthetic and pharmacological studies on a natural cyclopeptide from Gypsophila arabica

The present study deals with the synthesis of a novel proline-rich cyclopeptide gypsin B 7 via coupling of tripeptide units Boc-L-Tyr-L-Phe-L-Pro-OH and Gly-L-Leu-L-Pro-OMe utilizing two different carbodiimides, followed by cyclization of linear polypeptide fragment. Structure elucidation of newly synthesized peptide was done on basis of FT-IR, 1 H-NMR, 13 C-NMR, ESI-MS/MS data. From biological evaluation, it was concluded that cyclic hexapeptide 7 exhibited potent antidermatophyte activity against pathogenic Microsporum audouinii and Trichophyton mentagrophytes. Also, good bioactivity against pathogenic Candida albicans and gram-negative bacteria, and moderate antihelmintic activity against three species of earthworms was observed for newly synthesized cyclohexapeptide.


INTRODUCTION
Diverse reports in past literature have proved the potential of roots of plants to produce a range of natural products with different pharmacological activities (Tan and Zhou, 2006).Among these, cyclic peptides have received special attention due to their wide pharmacological profile and may prove better candidates to overcome the problem of resistance towards conventional drugs.A novel cyclic hexapeptide, gypsin B has been isolated from roots of Gypsophila arabica (Caryophyllaceae) and its structure was elucidated from the spectroscopic and chemical evidence.The stereochemistry of all amino acid residues was found to be in the L-configuration by GC analysis (Bruzual De Abreu et al., 2008).Although, syntheses of natural products have remained a challenge for scientists since decades due to diverse complicated moieties present in their structures, yet syntheses of cyclopolypeptides employing solid as well as solution-phase techniques is carried out frequently by various research groups (Pettit elucidated by means of spectral as well as elemental analysis.The IR spectra were run on Shimadzu 8700 FTIR spectrophotometer using a thin film supported on KBr pellets or utilizing chloroform. 1 H NMR and 13 C NMR spectra were recorded on Bruker AC NMR spectrometer using DMSO-d6 as solvent.The mass spectra of the cyclopeptide was recorded on JMS-DX 303 Mass spectrometer (Jeol, Tokyo, Japan) operating at 70 eV by ESI-MS/MS.Optical rotation of synthesized peptide derivatives was measured on automatic polarimeter in a 2 dm tube at 25°C.Elemental analyses of all peptides were performed on Vario EL III elemental analyzer.Purity of all synthesized compounds was checked by TLC on precoated silica gel G plates utilizing chloroform / methanol as developing solvent system in different ratios (7:3 / 9:1 v/v).

General method for preparation of di/tripeptide intermediates
L-Amino acid methyl ester hydrochloride/dipeptide methyl ester (10 mmol) was dissolved in dichloromethane (DCM, 20 ml).To this, NMM (2.21 ml, 20 mmol) was added at 0°C and the reaction mixture was stirred for 15 min.Boc-L-amino acid (10 mmol) in DCM (20 ml) and DIPC/DCC (1.26 g/2.1 g, 10 mmol) were added with stirring.After 24 h, the reaction mixture was filtered and the residue was washed with DCM (30 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 recrystallized from a mixture of chloroform and petroleum ether (b.p. 40 to 60°C) followed by cooling at 0°C.

Antibacterial screening
The newly synthesized cyclopeptide 7 was evaluated for its antibacterial potential against two Gram-positive bacteria Bacillus subtilis, Staphylococcus epidermidis and two Gram-negative bacteria Pseudomonas aeruginosa and Klebsiella pneumoniae at 25-6.25 g/ml concentration by using modified Kirby-Bauer disc diffusion method (Bauer et al., 1966).MIC values of test compound was determined by Tube Dilution Technique.Synthesized cyclopolypeptide 7 was dissolved to prepare a stock solution of 1 mg/ml using DMF.Stock solution was aseptically transferred and suitably diluted with sterile broth medium to contain seven different concentrations of test compound in different test tubes.All the tubes were inoculated with one loopful of one of the test bacterium.The process was repeated with different test bacteria.Tubes inoculated with bacterial cultures were incubated at 37°C for 18 h and the presence/absence of growth of the bacteria was observed.From these results, minimum inhibitory concentration (MIC) of each test compound was determined against each test bacterium.A spore suspension in sterile distilled water was prepared from 5 days old culture of the test bacteria growing on nutrient broth media.About 20 ml of the growth medium was transferred into sterilized petri plates and inoculated with 1.5 ml of the spore suspension (spore concentration -6  10 4 spores/ml).Filter paper disks of 6 mm diameter and 2 mm thickness were sterilized by autoclaving at 121°C (15 psig) for 15 min.Each petri plate was divided into five equal portions along the diameter to place one disc.Three discs of test sample were placed on three portions together with one disc with reference drug -gatifloxacin and a disk impregnated with the solvent (DMF) as negative control.The petri plates inoculated with bacterial cultures were incubated at 37 °C for 18 h.Diameters of the zones of inhibition (in mm) were measured and the average diameters for test sample were calculated for triplicate sets.The diameters obtained for the test sample were compared with that produced by the standard drug.The results of antibacterial studies are presented in Table 1.

Antifungal screening
Serial plate dilution method was employed for the evaluation of antifungal activity against diamorphic fungal strain C. albicans and three other fungal strains, including Aspergillus niger and two cutaneous fungal strains M. audouinii and T. mentagrophytes at 25-.25 g/ml concentration (Khan, 1997).MIC values of synthesized 6 cyclopeptide 7 was determined by employing the same technique as used for antibacterial studies using DMSO instead of DMF and  tubes inoculated with fungal cultures were incubated at 37 °C for 48 h.After incubation, the presence/absence of growth of the fungi was observed and MIC of test compound was determined against each test fungus.A spore suspension in normal saline was prepared from culture of the test fungi on sabouraud's broth media.
After transferring growth medium, petri plates were inoculated with spore suspension.After drying, wells were made using an agar punch and test sample, reference drug -griseofulvin and negative control (DMSO) were placed in labeled wells in each petri plate.
The petri plates inoculated with fungal cultures were incubated at 37°C for 48 h.Antifungal activity was determined by measuring the diameter of the inhibition zone for triplicate sets.Activity of test compound was compared with reference standard.The results of antifungal studies are given in the Table 2.

Antihelmintic screening
Antihelmintic activity studies were carried out against three different species of earthworms Megascoplex konkanensis, Pontoscotex corethruses and Eudrilus species at 2 mg/ml concentration (Garg and Atal, 1963).Suspension was prepared by triturating synthesized cyclopeptide 7 (100 mg) with tween 80 (0.5%) and distilled water and the resulting mixture was stirred using a mechanical stirrer for 30 min.The suspension was diluted to contain 0.2% w/v of the test sample.Suspension of reference drugmebendazole was prepared with the same concentration in a similar way.Three sets of five earthworms of almost similar sizes (2 inch in length) were placed in petri plates of 4 inch diameter containing 50 ml of suspension of test sample and reference drug at RT. Another set of five earthworms was kept as control in 50 ml suspension of distilled water and tween 80 (0.5%).The paralyzing and death times were noted and their mean was calculated for triplicate sets.The death time was ascertained by placing the earthworms in warm water (50°C) which stimulated the movement, if the worm was alive.The results of antihelmintic studies are tabulated in the Table 3.

Chemistry
In order to carry out the first total synthesis of gypsin B 7, disconnection strategy was employed.The cyclic hexapeptide molecule was split into two dipeptide units Boc-L-Tyr-L-Phe-OMe (1), Boc-Gly-L-Leu-OMe (3) and a single amino acid unit L-Pro-OMe.HCl (2).The required dipeptide units 1 and 3 were prepared by coupling of Boc-amino acids viz.Boc-L-Tyr-OH and Boc-Gly-OH with corresponding amino acid methyl ester hydrochlorides such as L-Phe-OMe.HCl and L-Leu-OMe.HCl employing DCC as coupling agent (Bodanszky and Bodanszky, 1984).Ester group of dipeptide 1 was removed by alkaline hydrolysis with LiOH and deprotected peptide was coupled with amino acid methyl ester hydrochloride 2 using DCC/DIPC and TEA/NMM, to get the first tripeptide unit Boc-L-Tyr-L-Phe-L-Pro-OMe (4).Similarly, dipeptide 3 after deprotection at carboxyl end, was coupled with 2 to get the another tripeptide unit Boc-Gly-L-Leu-L-Pro-OMe (5).After removal of ester group of tripeptide 4 and Boc group of tripeptide 5, deprotected units were coupled to get linear hexapeptide unit Boc-L-Tyr-L-Phe-L-Pro-Gly-L-Leu-L-Pro-OMe (6).The methyl ester group of linear peptide fragment was replaced by p-nitrophenyl/pentafluorophenyl (pnp/pfp) ester group.The Boc-group of resulting compound was removed using TFA and deprotected linear fragment was now cyclized by keeping the whole contents at 0 C for 7 days in presence of catalytic amount of TEA/NMM/pyridine to get cyclic product 7 (Scheme 1).Synthesis of gypsin B 7 was carried out successfully with good yield.Cyclization of linear peptide was indicated by disappearance of absorption bands at 1749, 1268 cm -1 and 1392, 1368 cm -1 (CO stretching of ester and CH deformation of tert-Butyl group) in IR spectra of cyclopeptide 7. Formation of cyclopeptide was further confirmed by disappearance of singlet at 1.56 ppm corresponding to nine protons of tert-Butyl group of Boc and singlet at 3.66 ppm corresponding to three protons of methyl ester, in 1 H NMR spectrum of 7. Furthermore, 1 H NMR and 13 C NMR spectra of synthesized cyclic hexapeptide showed characteristic peaks confirming presence all the 46 protons and 36 carbon atoms.Presence of (M + 1) + ion peak at m/z 675.7 corresponding to the molecular formula C 36 H 46 N 6 O 7 in mass spectra of 7, along with other fragment ion peaks resulting from cleavage at 'Leu-Gly', 'Pro-Phe' and 'Pro-Leu' amide bond levels, showed exact sequence of attachment of all the six amino acid moieties in a chain.In addition, elemental analysis of 7 afforded values ( 0.03) strictly in accordance to the molecular composition.

Pharmacology
Results of antimicrobial and anthelmintic activity studies are summarized in Tables 1 to 3. Comparison of antimicrobial activity data suggested that synthesized cyclopeptide 7 exhibited high level of antidermatophyte activity against M. audouinii and T. mentagrophytes with MIC value of 6.25 μg/ml, in comparison to standard druggriseofulvin.Antibacterial activity data further indicated that 7 possessed good level of bioactivity against pathogenic fungus C. albicans and bacteria P. aeruginosa and K. pneumoniae with MIC value of 6.25 μg/ml, in comparison to standard drug -gatifloxacin.Antihelmintic activity data revealed that 7 showed moderate antihelmintic activity against M. konkanensis, P. corethruses and Eudrilus sp. at 2 mg/ml concentration, in comparison to standard drug -mebendazole.However, 7 displayed no significant activity against Gram-positive bacteria and A. niger.

Conclusion
First total synthesis of natural peptide, gypsin B 7 was accomplished with > 85% yield via coupling reactions utilizing different carbodiimides.DIPC was found to be a better coupling agent in comparison to DCC and Dahiya and Gautam 1965 pentafluorophenyl ester was proved to be better for the activation of acid functionality of linear hexapeptide unit when compared 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 cyclohexapeptide displayed significant antimicrobial activity against pathogenic T. mentagrophytes, M. audouinii, C. albicans, K. pneumoniae and P. aeruginosa.
In comparison, gram-negative bacteria were found to be more sensitive than Gram-positive bacteria towards the newly synthesized peptide.On passing toxicity tests, newly synthesized cyclopeptide 7 may prove good candidate for clinical studies and can be a novel antimicrobial drug of the future.