Synthesis , characterization and biological evaluation of cyclomontanin D

In present study, it was of interest to synthesize a natural proline-rich cyclooligopeptide cyclomontanin D 8 by coupling of tripeptide Boc-L-Asn(bzh)-L-Pro-Gly-OH and tetrapeptide L-Leu-LPro-L-Tyr-L-Ala-OMe followed by cyclization of linear polypeptide fragment. Structure of synthesized cyclopeptide was confirmed by detailed spectral analysis including FTIR, H NMR, C NMR, ESIMS/MS and elemental analysis. From the results of biological evaluation, it was concluded that newly synthesized cyclooligopeptide possessed good bioactivity against Gram-negative bacteria Klebsiella pneumonia, Pseudomonas aeruginosa as well as potent antidermatophyte activity against Microsporum audouinii and Trichophyton mentagrophytes, in comparison to reference drugs ciprofloxacin and griseofulvin. Moreover, 8 exhibited moderate antifungal activity against pathogenic Candida albicans with minimum inhibitory concentration (MIC) value of 6 μg/ml.

A natural cycloheptapeptide, cyclomontanin D has been isolated from the seeds of annonaceous plant Annona montana Macf.(mountain soursop) and its structure was elucidated on basis of ESIMS/MS fragment evidence, 2D NMR analysis and chemical means (Chuang et al., 2008).
Keeping in view the wide range of pharmacological activities exhibited by natural plant-originated cyclopeptides (Tan and Zhou, 2006;Morita and Takeya, 2010) and in continuation of our previous investigations on peptides (Dahiya and Pathak, 2006;Dahiya and Gautam, 2011), present investigation was directed toward the synthesis of a natural cyclic heptapeptide 8 employing solution-phase technique.Furthermore, synthesized product was also subjected to antibacterial and antifungal activity studies.

General experimental part
Melting point of the synthesized product was determined in open glass capillary on melting point apparatus and was uncorrected.Laboratory chemicals were supplied by SpectroChem Ltd.The purity of the compounds was checked by thin layer chromatography (TLC) on precoated silica gel G plates utilizing CHCl3/MeOH as developing solvent.IR spectra were recorded on Shimadzu 8700 FTIR spectrophotometer using KBr pellets for cyclopeptide and CHCl3 as solvent for intermediate semisolids. 1 H NMR and 13 C NMR spectra were recorded on Bruker AC NMR spectrometer (300 MHz / 100 MHz).Chemical shifts are expressed in δ (ppm) relative to TMS as an internal standard using C5D5N as solvent.Mass spectra was recorded on JMS-DX 303 Mass spectrometer operating at 70 eV using ESIMS/MS technique.Elemental analyses of newly synthesized compounds were performed on Vario EL III elemental analyzer.

Antimicrobial activity
Antibacterial activity of synthesized cyclooligopeptide 8 were carried out against four bacterial strains Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, according to modified disc diffusion method at 12.5-6 g/ml (Bauer et al., 1966).Antifungal screening was performed against four fungal strains Candida albicans, Microsporum audouinii, Trichophyton mentagrophytes, Aspergillus niger) according to serial plate dilution method at 12.5 to 6 g/ml (Khan, 1997).Ciprofloxacin and griseofulvin were used as standard drugs against bacterial and fungal strains.Minimum inhibitory concentration (MIC) values of test compounds were determined by tube dilution technique using sterile DMF.The Petri plates inoculated with bacterial and fungal cultures were incubated at 37°C for 18 and 48 h, respectively.The average diameters of the zones of inhibition (in mm) of test compounds were calculated for triplicate sets and compared with that produced by the standard drugs (Tables 1 and 2).Experimental details of antibacterial and antifungal activity procedures are given in previously published reports by Dahiya and Pathak (2007).6) 20 ( 6) 20 ( 6) 18 (12.5)† in mm; ‡ DMSO.

Chemistry
In present investigation, disconnection strategy was utilized to carry out the first total synthesis of cyclomontanin D 8. The amino group of L-amino acids was protected by stirring with Boc 2 O (di-tertbutylpyrocarbonate) and isopropanol.Carboxamide side chain of Boc-L-asparagine was protected using benzhydrol whereas Boc/benzhydrol groups were removed by treatment with trifluoroacetic acid (TFA).The carboxyl group of L-amino acids was protected by esterification with MeOH/SOCl 2 whereas methyl ester group was removed by alkaline hydrolysis with LiOH.All the above protections and deprotections were done according to the previously reported procedures (Dahiya and Kumar, 2007).All the coupling reactions were perfomed utilizing dicyclohexylcarbodiimide/diisopropylcarbodiimide (DCC/DIPC) as coupling agents and pyridine as base (Bodanzsky and Bodanzsky, 1984).
The cyclopeptide molecule was split into a single amino acid unit Boc-Asn-OH 1 and three dipeptide units Boc-L-Pro-Gly-OMe 2, Boc-L-Leu-L-Pro-OMe 3, Boc-L-Tyr-L-Ala-OMe 4. The carboxamide side chain of 1 was protected using benzhydrol to get Boc-L-Asn(bzh)-OH 1a.Dipeptide units 2-4 were prepared by coupling of Bocamino acids viz.Boc-L-Pro-OH, Boc-L-Leu-OH and Boc-L-Tyr-OH with corresponding amino acid methyl ester hydrochlorides such as Gly-OMe.HCl, L-Pro-OMe.HCl and L-Ala-OMe.HCl employing DCC as coupling agent.Boc-groups of dipeptides 2 and 4 were removed using TFA to get deprotected units 2a and 4a, and dipeptide 3 was deprotected at carboxyl end by alkaline hydrolysis to obtain 3a.Deprotected dipeptide 2a was coupled with 1a to get the tripeptide unit Boc-L-Asn(bzh)-L-Pro-Gly-OMe 5 whereas deprotected dipeptde units 3a and 4a were coupled together to obtain tetrapeptide unit Boc-L-Leu-L-Pro-L-Tyr-L-Ala-OMe 6.Now, 5 was deprotected at carboxyl end and coupled with 6 deprotected at amino terminal using DCC/DIPC and pyridine as base, to get the linear heptapeptide unit Boc-L-Asn(bzh)-L-Pro-Gly-L-Leu-L-Pro-L-Tyr-L-Ala-OMe 7. The methyl ester group of linear peptide fragment was replaced by pentafluorophenyl (pfp) ester group.Boc and bzh groups of resulting unit were 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 triethylamine (TEA) / Nmethylmorpholine (NMM) to get final cyclized product 8 (Scheme 1).Structure of the newly synthesized cyclic heptapeptide as well as intermediates linear di/tri/tetra/heptapeptides were confirmed by spectral as well as elemental analysis.
Synthesis of cyclopeptide 8 was accomplished with 89% yield and NMM was proved to be a yield effective base for cyclization of linear heptapeptide fragment.Cyclization of linear peptide fragment was indicated by disappearance of absorption bands at 1756, 1271 and 1390, 1376 cm −1 (C=O str / C−O str of ester and C−H bend of tert-Butyl group).Deprotection of asparagine was confirmed by presence of Amide I and II bands (1655, 1623 cm -1 ) and bands at 3349, 3175 and 1409 cm −1 due to N−H and C−N str of the -CONH 2 moiety and disapppearance of strong out-of-plane deformation bands at 725-721 cm −1 and 688-683 cm −1 due to aromatic rings of bzh, in IR spectra and disapppearance of multiplet at 7.28 to 7.17 and 7.12 to 7.05 ppm due to 10 protons of phenyl rings of bzh group, in 1 H NMR spectra of 8. Formation of cyclopeptide was further confirmed by disappearance of singlets at 3.61 and 1.51 ppm corresponding to three protons of methyl ester group and nine protons of tert-Butyl group of Boc, in and 'Ala-Asn' amide bond levels, showed exact sequence of attachment of all the seven amino acid moieties in a chain.In addition, presence of immonium ion peaks at m/z 136, 70, 87, 86, 44 and 30 further confirmed all seven amino acid moieties in cyclopeptide structure.Furthermore, elemental analysis of 8 afforded values with tolerance of ± 0.03.

Pharmacology
The results of antibacterial and antifungal activity studies of cyclopeptide 8 against eight pathogenic microbes are summarized in Tables 1 and 2. Overall observation from the results of antimicrobial data revealed that 8 possesses potent antibacterial activity against P. displayed less bioactivity against pathogenic bacteria and fungi when compared to corresponding cyclic form 8. This is because, cyclization of peptides reduces the degree of freedom for each constituent within the ring and thus substantially leads to reduced flexibility, increased potency and selectivity of cyclic peptides.Further, inherent flexibility of linear peptides lead to different conformations which can bind to more than one receptor molecules, resulting in undesirable adverse effects.Antibacterial and antifungal data was subjected to one way ANOVA with post test using GraphPad InStat software.The P value for antibacterial data was 0.0016 which was considered very significant and the variations among column means were significantly greater than expected by chance (Tables 3 and 4).The F calc was found to be 13.649 and 3.238 whereas F tab was 4.07 and 4.35 for antibacterial and antifungal data respectively.As the value of F calc is greater than F tab in case of bacterial species, the significant difference exists between the values of zone of inhibition of species tested.In case of fungal species, value of F calc is less than F tab , indicating that significant difference does not exist between the values of zone of inhibition of species tested.

Conclusion
First total synthesis of natural proline-rich cyclooligopeptide 8 was accomplished with 89% yield utilizing different carbodiimides and bases.Pentafluorophenyl ester was found to be better for the activation of acid functionality of linear heptapeptide unit.NMM was found to be a good base for intramolecular cyclization of linear peptide fragment, in comparison to TEA.In all coupling reactions, DIPC was proved to be a yield-effective coupling agent, in comparison to DCC.Significant level of biopotential was observed for synthesized cycloheptapeptide against Gram-negative bacteria and dermatophytes.Gram-positive bacteria were found to be less sensitive toward newly synthesized peptide, in comparison to Gram-negative bacteria.On passing toxicity tests, proline-rich cyclopeptide 8 may prove good candidate for clinical studies and can be a novel antibacterial and antidermatophyte drug of the future.

Table 2 .
Antifungal profile of newly synthesized linear/cyclic heptapeptide

Table 3 .
ANOVA table for bacterial species.

Table 4 .
ANOVA table for fungal species.
S. aureus nor pathogenic fungus A. niger.Analysis of antimicrobial activity data revealed that linear peptide 7