Himaja M et al / IJRAP 2010, 1 (1)

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1 imj M et l / IJRAP 2010, 1 (1) Reserch Article Avilble online through SYTESIS AD ATIMICRBIAL ACTIVITY F DICTMI A AALGS imj Mlipeddi *, Mlipeddi Venktrmn b, Shoo Atish Kumr c, Annd Rnjith, Krigr Asif d * VIT University, Vellore , Indi. b Aljbr Alghrbi University, Zwi, Liby c GSM Institute of Phrmceuticl Sciences, Pneer, Derlktte, Mnglore, Krntk, Indi d Mrth Mndl college of Phrmcy, Belgum, Krntk, Indi Received: ; Revised: ; Accepted: ABSTRACT A solution phse peptide synthesis ws employed to synthesize Dichotomin A nlogs. The, - dimethyltion on Tyrosine nd configurtionl chnge of L- to D- on Vline ws mde on Dichotomin A to derive the compounds 1) Cyclo-L-[Gly-Thr-Phe-Leu-(-C 3, -C 3 ) Tyr-Vl] nd 2) Cyclo-L-[Gly-Thr- Phe-Leu-(-C 3,-C 3 )Tyr-D-Vl]. The structure of these compounds ws confirmed by IR, 1 MR, nd FABMASS. The synthesized compounds were tested for their biologicl ctivities ginst bcteril nd fungl orgnisms nd were found to be ctive. Compound (2) crrying D-vline unit hve shown more ntimicrobil ctivity thn the compound crrying L-vline unit. KEYWRDS: Dichotomin A, Cyclic hexpeptide, Solution Phse Synthesis, Antimicrobil ctivity *Author for correspondence Dr (Mrs).M.imj Professor, Phrmceuticl Chemistry Division, VIT University, Vellore Tmil du, Indi. Mobile: Phone: e-mil:[email protected] Interntionl Journl of Reserch in Ayurved & Phrmcy, Volume 1, Issue 1, Sep-ct

2 imj M et l / IJRAP 2010, 1 (1) ITRDUCTI A cyclic hexpeptide dichotomin-a, Cyclo-L-[Gly-Thr-Phe-Leu-Tyr-Vl] isolted from the roots of Stellri dichotom, showed cell growth inhibitory ctivity 1 nd used s folk medicine for ntifebrile. Severl peptide ntibiotics such s geodimolides, rylomycin, clvriosporin were found to contin -methylted mino cids in their ring structures 2-4. A review of the structures of cyclic peptides exhibiting ntimicrobil ctivity showed presence of D- mino cid nd / or - methylted mino cid units in the molecule. ence two cyclic hexpeptides -methylted nlogs of dichotomin-a hve been designed. Both the cyclic hexpeptides comprise of, - dimethylted Tyr units but the second one in ddition to tht contins one D-Vl insted of L-Vl. The two molecule (1) Cyclo-L-[Gly-Thr-Phe-Leu-(-C 3,-C 3 ) Tyr-Vl] (2) Cyclo-L-[Gly-Thr- Phe-Leu-(-C 3, -C 3 ) Tyr-D-Vl] were synthesized by solution phse technique of peptide synthesis using dicyclohexylcrbodimide (DCC) s the coupling gent nd triethylmine (TEA) s bse. The enhnced biologicl ctivity by the -methyltion nd configurtionl chnges 5,6 on the cyclic peptides directed us to synthesize these compounds with intention to increse the ntimicrobil ctivity. EXPERIMETAL MATERIALS AD METDS All the rections requiring nhydrous conditions were conducted in flme dried pprtus. The mino cids used re L- nd D-mino cid, purchsed from Spectrochem Privte Limited, Mumbi, Indi. Solvents nd regents were purified by stndrd methods. Boc-mino cids, mino cid methyl ester hydrochlorides were prepred by stndrd procedures 7. -methylted mino cids were prepred using /C 3 I by Benoiton method 8. rgnic extrcts were dried over nhydrous sodium sulphte. Melting points were determined by n open cpillry method nd re uncorrected. The completion of the rection nd purity of the compounds were checked by thin lyer chromtogrphy. IR spectr were recorded on icolet impct 400 FT/IR spectrometer using KBr pressed pellet technique. 1 MR spectr were recorded on GEL-JMS D 300 (Mz) MR spectrometer. MASS spectr were recorded on Shimndzu GC-MS (t 70 ev) Mss Spectrometer using xenon s the crrier gs. Preprtion of Dipeptides: Amino cid methyl ester hydrochloride (10mmol) ws dissolved in chloroform (20ml). To this, triethylmine (4ml, 28.7mmol) ws dded t 0 0 C nd the rection mixture ws stirred for 15 mins. Boc-mino cid (10mmol) in CCl 3 (20ml) nd DCC (10mmol) were dded with stirring. After 12hrs, the rection mixture ws filtered nd the residue ws wshed with CCl 3 (30ml) nd dded to the filtrte. The filtrte ws wshed with 5% C 3 (20ml) nd sturted Cl (20ml) solutions. The orgnic lyer ws filtered nd evported in vcuum. To remove the trces of the dicyclohexylure (DCU), the product ws dissolved in minimum mount of chloroform nd cooled to 0 0 C. The crystllized DCU ws removed by filtrtion. Petroleum ether ws dded to the filtrte t 0 0 C to recrystllize the pure product. Boc-Gly-Thr- Me, Boc-Phe-Leu-Me nd Boc-L-(-C 3, -C 3 )-Tyr-L-Vl-Me, Boc-L-(-C 3, -C 3 )-Tyr-D- Vl-Me were prepred in this mnner. Preprtion of the Tetrpeptide Boc-Gly-Thr-Phe-Leu-Me: The tetrpeptide ws prepred from the dipeptide The tetrpeptide were prepred from the dipeptide Boc-Gly-Thr-Me (1) nd Boc-Phe-Leu-Me (2) units fter pproprite deprotection t the required functionl groups using DCC/Et 3 to get the protected tripeptide. Preprtion of liner hexpeptide: The Boc-group of the dipeptide Boc-L-(-C 3,-C 3 )-Tyr-L-Vl- Me nd Boc-L-(-C 3,-C 3 )-Tyr-D-Vl-Me ws removed nd the ester group of the tetrpeptide (3) ws deprotected. Both the deprotected units were coupled to get the two liner hexpeptide. Interntionl Journl of Reserch in Ayurved & Phrmcy, Volume 1, Issue 1, Sep-ct

3 imj M et l / IJRAP 2010, 1 (1) Preprtion of Cyclic hexpeptide (1) nd (2): The cyclistion of the liner hexpeptide unit ws crried out by the p-nitrophenyl ester method of Bodnszky 9 with certin modifictions. The ester group of the liner frgment ws removed nd the p-nitrophenyl ester group ws introduced by stirring it for 12 hrs in CCl 3 with p-nitrophenol t 0 0 C. The rection mixture ws wshed severl times with sturted C 3 until the unrected p-nitrophenol ws removed completely nd wshed with 5% Cl to get Boc-peptide-pnp ester. The Boc-group lso ws removed, dded CCl 3 nd pyridine nd the rection mixture ws kept t 0 0 C for 10 dys. The mixture ws finlly wshed with 5% Cl, dried nd evported in vcuum to get the cyclised product. Cyclic hexpeptide (1): Cyclo-L-[Gly-Thr-Phe-Leu-(-C 3,-C 3 ) Tyr-Vl]: IR (KBr): 3676, 3328, , , , 1575; 1 MR (CDCl 3 ): δ (m, 9, Ar-), 7.6 (s, 1, -), (s, 3, -), 5.5 (s, 1, -), 4.6 (m, 2,) (m, 3,) 3.7 (s, 3, C 3 ), (m, 2, -C 2 ), (m, 2, - C 2 ), 2.2 ( s, 3, -C 3 ), 2.0 (m, 2, -C 2 ), 1.6 (m, 3, -C), (m, 15, 5(-C 3 ). FABMASS: m/z: 708 (M+1). Cyclic hexpeptide 2: Cyclo-L-[Gly-Thr-Phe-Leu-(-C 3, -C 3 ) Tyr-D-Vl]: IR (KBr): 3640, , , , , ; 1 MR (CDCl 3 ): 7.6 (s, 1, -), (m, 9, Ar-), 7.1 (s, 3, -), 6.85 (s, 1, -), 4.7(s, 1, -), (m, 2), (m, 3), 3.75 (s, 3, -C 3 ), (m, 2, -C 2 ), (m, 2, -C 2 ), (s, 3, -C 3 ), (m, 2, -C 2 ). FABMASS: m/z 708 (M+1) EVALUATI F ATIMICRBIAL ACTIVITY Both the compounds were screened for ntibcteril nd ntifungl ctivities using the cup-plte gr diffusion method 10 by mesuring the zone of inhibiton. A 24 h culture of bcteril strins of S. ureus, B. subtilis, p. eruginos nd E. coli were cultivted in nutrient broth medium nd the fungl strins of A. flvus, A. fumigtes nd C. lbicns were cultivted in Fluid Sbrud s medium respectively. Both the compounds were tested t concentrtion level of 25 μg/ml. Dimethyl formmide ws used s solvent nd s control. Ciprofloxcin nd Fluconzole were used s stndrd for comprison of the results. The dimeter of zone of inhibition ws mesured fter 24h incubtion t 37 0 C. RESULTS AD DISCUSSI The cyclic hexpeptides were obtined in moderte yield (Tble 1) nd their structure ws confirmed by IR, 1 MR nd FABMASS spectroscopy. The compounds were screened for ntibcteril nd ntifungl ctivities using the bcteril strins of Stphylococcus ureus, Bcillus subtilis, Escherichi coli, Pseudomons eruginos nd the fungl strins of Asperigillus flvus, Asperigillus fumigtes nd Cndid lbicns respectively. Both the cyclic hexpeptides hve shown good ntimicrobil ctivities compred to their stndrd drugs but the ntifungl ctivity is more prominent. Compound (2) showed enhnced ctivity thn Compound (1) in both the cses. The incresed ctivity of the compounds my be ssumed due to the chnge in the hydrogen bond formtion nd incresed liphophilic chrcter of the molecule which enhnces the permebility of the molecule into the bcteri nd fungi. In ddition to, -dimethyltion the configurtionl chnges lso ply n importnt role to increse the ntimicrobil ctivity. Further studies on other mino cid units re necessry to know the effect of -methyltion nd configurtionl chnges to conclude some structure ctivity reltionship of Dichotomin A. Interntionl Journl of Reserch in Ayurved & Phrmcy, Volume 1, Issue 1, Sep-ct

4 imj M et l / IJRAP 2010, 1 (1) REFERECES 1. Morit, Kyshit T, Shishido A, Tkey K, Itokw, Shiro M. Dichotomin A: A ew Cyclic expeptide from Stellri dichotom L vr. lnceolt Bge. Bio-org nd Med chem lett 1995;5: Morit, Kyshit T, Shishido A, Tkey K, Itokw. Shiro M, Dichotomins A - E, ew Cyclic Peptides from Stellri dichotom L vr lnceolt Bge. Tetrhedron 1996; 52: Morit, Iizuk T, Choo CY, Chn KL, Itokw, nd Tkey K. Dichotomins J nd K:Vsodiltor Cyclic Peptides from Stellri dichotom. J t Prod;2005;68: Morit, Shishido A, Kyshit, T, Tkey K, nd ideji I. J t Prod; 1997; 60: Chn WR, Tinto WF, Mnchnd PS, Todro LJ. Totl synthesis of the mixed peptide-polypropionte bsed cyclodepsipeptide (+)-geodimolide B. Tetrhedron lett; 1988; 29: Schimn J, Gebhrdt K, oltzel A, Schmid DG, Sussmuth R, Muller J et l. Journl of ntibiotics. 2002; 55: Bodnszky M, Bodnszky A. Prctice of Peptide synthesis. 1 st Ed. ew York:Springer- Verlg;1984: Cheung ST., Benoiton L. -Methylmino cids in peptide synthesis-v. The synthesis of -tertbutyloxycrbonyl--methylmino cids by -methyltion. Cn J Chem; 1977; 55: imj M, Rnjith A. Anticncer ctivity of synthetic Winunumide: A proline rich cyclic heptpeptide. Ind Drugs; 2009; 46: Suzuki, Mkoto, Youji S. J Antibiot; 2001; 54:17. Interntionl Journl of Reserch in Ayurved & Phrmcy, Volume 1, Issue 1, Sep-ct

5 imj M et l / IJRAP 2010, 1 (1) Boc-Gly- + -Thr-Me Boc-Gly-Thr-Me b Boc-Phe- + -Leu-Me Boc-Phe-Leu-Me c Boc-L-Tyr-Me C 3 I, DMF Boc-L-(-C 3, -C 3 )Tyr-Me b Boc-Gly-Thr- + -Phe-Leu- Boc-L-(-C 3, -C 3 ) Tyr- + X-Vl-Me Boc-L-(-C 3, -C 3 ) Tyr-L-X-Vl-Me Boc-Gly-Thr-Phe-Leu- + c L-(-C 3, -C 3 ) Tyr-X-Vl-Me Boc-Gly-Thr-Phe-Leu-L-(-C 3, -C 3 ) Tyr-X-Vl-Me b, d Boc-Gly-Thr-Phe-Leu-L-(-C 3, -C 3 ) Tyr-X-Vl-PP c,e C 3 C 3 C 3 C 3 Where X= L nd D PP = 2 = CCl 3, DCC, Et 3, 24h, RT, b = TF: 2 (1:1), Li, 1h, RT, c = CF 3 C/CCl 3, 1h, RT d = p-nitrophenol, e = CCl 3, pyridine, 10 dys, 0 0 C Scheme-I Interntionl Journl of Reserch in Ayurved & Phrmcy, Volume 1, Issue 1, Sep-ct

6 imj M et l / IJRAP 2010, 1 (1) Tble 1: Physicl dt of synthesized Cyclic hexpeptides Compounds Composition Physicl stte % yield CP-1 CP-2 Cyclo L-[Gly-Thr-Phe-Leu- (-C 3,-C3)Tyr-L-Vl] Cyclo L-[Gly-Thr-Phe-Leu- (-C 3,-C3)Tyr -D-Vl] brown solid brown solid Tble 2: Antibcteril nd ntifungl ctivities of the compounds Comp. no rgnism Dimeter of zone of inhibition(mm) S. ureus B. subtilis E. coli P. er C. lbicns A. flvus CP CP Ciprofloxcin Fluconzole A. fumigtus ( ) indictes no inhibition zone (no ctivity) Source of support: il, Conflict of interest: one Declred Interntionl Journl of Reserch in Ayurved & Phrmcy, Volume 1, Issue 1, Sep-ct