Ciopińska, Agnieszka

Kieć-Kononowicz, Katarzyna

Latacz, Gniewomir

Pękala, Elżbieta

Acta Poloniae Pharmaceutica - Drug Research

English

Jagiellonian Univeristy Repository

Compared to native peptides peptidomimeticsoffers many advantages as newly designed drugs. Thebenefits include enhanced stability to proteolyticenzymes, better transport through cellular membranes,increased bio-availability, changing agonist to antago-nists potency and higher affinity in ligand-receptorsinteractions. D-amino acids are used to design andobtain new peptidomimetics by their incorporation inplace of the natural L-amino acids, either at a specificposition, or throughout the whole peptide. D-phenylala-nine is one of the preferred amino acids. Addition ofhydrophobic phenyl ring results in better transport prop-erties of peptidomimetics through cellular membranesand better enzymatic stability (1, 2).Up to now D-phenylanine and derivatives havebeen successfully used in design of new efficient pep-tidomimetics. 4-fluoro-D-phenylalanine derivativeimplicate in new agonist of melanocortin receptors(MCRs). A common structural feature present in allendogenous ligands (MSH, ACTH) of the melanocortinreceptors (MCRs) is His-Phe-Arg-Trp sequence, whichhas been identified as the minimal peptide fragmentnecessary for activating the receptors. Further structuralmodifications toward α-MSH showed that inverting Pheto the D-configuration resulted in an analog withenhanced potency and enzymatic stability. Replacementof the phenyl ring of D-Phe side chain in one of His-D-Phe-Arg-Trp analogue with 4-fluorophenyl group led topotent MCRs agonist with enhanced selectivity (3).Synthetic decapeptides, Cetrorelix and Abarelixbelong to third-generation of modified gonadotropinreleasing hormone (GnRH) antagonists and contain intheir structures D-4-chlorophenylalanine. GnRH, a lineardecapeptide synthesized by gonadotropic cells of hypo-thalamus, is a central mediator of the human reproductiveaxis. Cetrorelix and Abarelix reveal high antagonisticactivity against naturally occurring GnRH and are usedfor treatment of sex hormone-dependent disorders (4, 5).Another important example of peptidomimeticwith D-phenylalanine presence is Octreotide ñ a syn-thetic somatostatin analogue with similar but more pro-longed pharmacological effects. Octreotide is used totreat acromegaly and also to reduce flushing episodesand watery diarrhoea caused by cancerous tumors (car-cinoid syndrome) (6).Furthermore, D-amino acidñcontaining peptidesare especially important as pharmaceuticals due to theirstrong antibacterial activity. Many of them (BacitracinA, Fungisporin, Gramicidin S, Tyrocidine, Polymyxin,)contain D-phenylalanine (2).In the present work we report the application of hydan-toinase method to obtain D-phenylalanine derivatives.Hydantoinase method consist of three steps: 430UNNATURAL D-AMINO ACIDS AS BUILDING BLOCKSOF NEW PEPTIDOMIMETICSGNIEWOMIR LATACZ, ELØBIETA P KALA, AGNIESZKA CIOPI—SKAand KATARZYNA KIE∆-KONONOWICZMedical College of Jagiellonian University, Department of Technology and Biotechnology of Drugs,ul. Medyczna 9, 30-688 KrakÛw, PolandFigure 1. Figure 2.1. chemical and/or enzymatic racemization ofD,L-5-substituted hydantoin2. opening of the hydantoin ring to the N-car-bamoyl-D-amino acid by D-hydantoinase (E.C.3.5.2.2)(6).3. converting of the N-carbamoyl-D-amino acid tothe D-amino acid by diazotization or by the next enzy-matic reaction catalyzed by N-carbamylase (E.C.3.5.1)(7).In our research phenyl ring substituted 5-benzyl-hydantoins were obtained in racemic form from cheapstarting materials such as benzylaldehyde derivativesand hydantoin using first Knoevenagel condensationand next the reduction of unsaturated bond in 5-positionof the hydantoin (Figure 1). Obtained ring substituted 5-benzylhydantoin derivatives were used as substrates forimmobilized, recombinant from Escherichia coli D-hydantoinase. The enzyme catalyzed the enantioselec-tive hydrolytic ring-opening of racemic hydantoin toform N-carbamoyl-D-amino acid, which was next con-verted by diazotization to the corresponding enan-tiomerically pure D-amino acid (Figure 2).431Table 1. 5-Arylidenehydantoin (1-10) and 5-benzylhydantoin (1a-10a) derivatives.Yield m. p. Molecular massComp. R [%] [OC] formula Rf A 1 2-F 65 222-224 206,17 0,46C10H7FN2O2 1a 2-F 90 191-192 208,19 0,28C10H9FN2O22 3-F 90 210-212 206,17 0,51C10H7FN2O22a 3-F 92 192 208,19 0,21C10H9FN2O23 4-F 86 243-250 206,17 0,49C10H7FN2O23a 4-F 94 200-201 208,19 0,19C10H9FN2O24 2-Br 33,8 300-302 267,08 0,46C10H7BrN2O24a 2-Br 80 252-253 269,09 0,30C10H9BrN2O25 3-Br 65 235-237 267,08 0,60C10H7BrN2O25a 3-Br 91 193-194 269,09 0,29C10H9BrN2O26 4-Br 77 307-309 267,08 0,55C10H7BrN2O26a 4-Br 88,5 199-200 269,09 0,23C10H9BrN2O27 2-CH3 69,3 199-201 202,21 0,69C11H10N2O27a 2-CH3 63,7 175-177 204,23 0,37C11H10N2O28 3-CH3 44,5 226-228 202,21 0,70C11H10N2O28a 3-CH3 49,5 184-186 204,23 0,38C11H10N2O29 4-CH3 61,9 263-266 202,21 0,69C11H10N2O29a 4-CH3 58,8 208-210 204,23 0,38C11H10N2O210 2,4-CH3 60,2 183-185 216,24 0,68C12H12N2O210a 2,4-CH3 18,4 215-217 218,25 0,41C12H14N2O2A = chloroform/ethyl acetate (1:1, v/v)EXPERIMENTALChemicals and reagentsHydantoin, benzylaldehyde derivatives, red phos-phorus, sodium acetate, ammonium acetate and solventswhich were used in chemical synthesis of 5-benzylhy-dantoin derivatives were obtained from commercialsuppliers and used without further purification.Biotransfomations were carried out in borate buffer pH= 9 (Merck) with commercially available D-hydan-toinase (E.C 3.5.2.2), immobilized, cloned andexpressed in Escherichia coli, (Fluka, 53,1 U/g activity).General methodsAll melting points were measured in glass capil-lary tubes using Mel-TempÆ Laboratory Devices Inc.USA apparatus and are uncorrected. 1H-NMR spectrawere recorded on Varian Mercury-VX instrument at 300MHz using TMS as an internal standard in DMSO-d6.IR spectra were recorded in KBr pellets using a JascoFT/IR-410 spectrophotometer. Reactions undermicrowave irradiation were performed using SamsungM1618 domestic microwave oven.432Table 2. IR and 1H NMR spectral data of compounds 1a-10a.I RNo. (KBr)[cm-1] 1H NMR δ (ppm)NH C=O (2) C=O (4)1a 3184 1733 1781 2,85-2,90 (dd, J=6,4 Hz,1H, H -C-H); 3,00-3,05 (dd, J=4,9 Hz, 1H, H-C-H);4,29 (t, J=5,6 Hz, 1H, CH); 7,12 (q, J=7,6Hz, 2H, Ph-3-H, Ph-6-H ); 7,26 (t, J=6,9 Hz, 2H, Ph-4-H, Ph-5-H); 7,92 (s, 1H, N1H); 10,52 (s, 1H, N3H);2a 3179 1734 1780 2,93 (qu, J=7,1 Hz, 2H, CH2); 4,34 (t, J=5,2 Hz, 1H, CH); 6,99-7,07 (m, 3H, Ph-2-H, Ph-4-H, Ph-6-H); 7,31(q, J=7,4 Hz, 1H, Ph-5-H); 7,91(s, 1H, N1H); 10,47 (s, 1H, N3H);3a 3245 1708 1757 2,96 (d, J=4,9 Hz, 2H, CH2); 4,30 (t, J=4,9 Hz, 1H, CH); 7,06-7,21 (m, 4H,Ph-2-H, Ph-3-H, Ph-5-H, Ph-6-H); 7,89 (s, 1H, N1H); 10,42 (s, 1H, N3H);4a 3187 1731 1783 2,88-2,96 (dd, J=9,0 Hz, 1H, H -C-H); 3,16-3,22 (dd, J=5,0 Hz, 1H, H-C-H); 4,32 (t, J=5,0 Hz, 1H, CH); 7,17 ñ 7,24 (m, 1H, Ph-6-H); 7, 30 ñ 7, 35 (m, 2H,Ph-4-H, Ph-5-H); 7, 59 (d, J=7,0Hz, 1H, Ph-3-H); 7,91 (s, 1H, N1H); 10,67 (s,1H, N3H);5a 3239 1700 1756 2.94 (t, J= 5,-Hz, 2H, CH2); 4,36 (t, J=5,0 Hz, 1H, CH); 7,13-7,18 (m, 1H, Ph-2-H);7,26-7,35 (m, 4H, Ph-4-H, Ph-5-H, Ph-6-H), 7,93 (s, 1H, N1H); 10,72 (s, 1H,N3H);6a 3241 1706 1756 2,89(q, J=10,0 Hz, 2H, CH2); 3,34 (t, J=10,0 Hz, 1H, CH); 7,35 (d, J=8,0 Hz 2H, Ph-2-H, Ph-6-H); 7,48 (d, J=8,0 Hz, 2H, Ph-3-H, Ph-5-H); 7,94 (s, 1H, N1H); 10,48 (s, 1H, N3H);7a 3185 1730 1784 2,26 (s, 3H,CH3);2,80-2,87 (dd, J=14,4 Hz, 1H, H -C-H); 2,96-3.02 (dd, J=14,4 Hz, 1H, H -C-H); 4,28 (m, 1H, CH); 7,06 (m, 4H, Ph-2-H, Ph-3-H,Ph-4-H, Ph-5-H); 7,89(s, 1H, N1H); 10, 52(s, 1H, N3H);8a 3225 1728 1782 2,24(s, 3H, CH3); 2,79-2,92(m, 2H, CH2); 4,27(t, J=4,6 Hz, 1H, CH); 6,95-7,03(m, 3H, Ph-2-H, Ph-4-H, Ph-6-H); 7,14(t, J=7,4 Hz, 1H, Ph-5-H); 7,89(s,1H, N1H); 10, 41(s, 1H, N3H);9a 3246 1706 1755 2,24(s, 3H, CH3); 2,85-2,86(d, J=4,9 Hz, 2H, CH2); 4,25-4,29(m, 1H, CH); 7,05(s, 4H, Ph-2-H, Ph-3-H, Ph-5-H, Ph-6-H); 7,86(s, 1H, N1H); 10, 36(s, 1H, N3H);10a 3221 1730 1779 2,20 (s, 3H,CH3); 2,22 (s, 3H,CH3);2,76-2,83 (dd, 14,4 Hz 1H, H -C-H); 2,90-2,97 (dd, J=14,4 Hz) 1H, H -C-H); 4,23-4,37 (m, 1H, CH); 6,87-6,93 (m, 2H, Ph-4-H, Ph-5-H); 7,00 (d, J=7,7 Hz, 1H, Ph-2-H); 7,86(s, 1H, N1H); 10, 45 (s, 1H, N3H);Figure 3. The image shows chiral TLC confirmation of the presenceof D-amino acids.Analysis by TLC and Chiral TLCThe reactions were monitored by thin-layer chro-matography using commercially available TLC alu-minium sheets (silica gel 60 F254 Merck) using A: chlo-roform/ethyl acetate (1:1, v/v), B: butanol/aceticacid/water (4:1:1, v/v/v) eluents and visualized by UVlight (254 nm). The enantiomeric purities of D-aminoacids were determined using chiral thin layer chro-matography. The samples were applied to ChiralplateÆMacherey-Nagel, 0.25 mm silica gel, amino acids wereeluted with C: methanol/water/acetonitrile (50:50:200,v/v/v). Spots were visualized after spraying with 0,3%ninhydrin and heating at 100OC for 3 ñ 5 min.Synthesis of racemic phenyl ring-substituted 5-ben-zylhydantoins5-Arylidenehydantoins (1-6) were synthesizedaccording to Knoevenagel procedure. A mixture of 0.05mol (5.05 g) of hydantoin, 0,05 mol of appropriate ben-zaldehyde, 0.2 mol (16.7 g) of anhydrous sodium acetateand 100 mL of 99,5% acetic acid was stirred and refluxedfor 5 h. The mixture was allowed to stand overnight atambient temperature. The precipitated crystalline materialwas filtered off, washed with water and after dryingrecrystallized from acetic acid. The reaction was moni-tored by TLC with A: chloroform/ethyl acetate (1:1, v/v).5-Arylidenehydantoins (7-10) were synthesized bycondensation of 0.01 mol (1.01 g) of hydantoin and 0.01mol of appropriate benzaldehyde which was carried outunder microwave irradiation in the presence of 0.01 mol(0.77 g) of ammonia acetate at 450 W of power for 1min, and was continued after cooling for 0.5 min. Thecrude 5-arylidenehydantoin was suspended in 30 mL of99.5% acetic acid and refluxed for 0.5 h. The solutionwas cooled and allowed to stand at 4OC overnight. Theprecipitated crystalline material was filtered off andwashed with water.5-Benzylhydantoins (1a-10a) were synthesized byreduction of 0.005 mol of appropriate 5-arylidenehydan-toin (1-10) with 4.5 mL of 57% hydroiodic acid in thepresence of 1 g of red phosphorus. The mixture was heat-ed under reflux for 0.5 h. Afterward 30 mL of distilledwater was added and the precipitate was filtered off (8). Physicochemical propertiesThe obtained compounds (1-10) and (1a-10a) arepresented in Table 1. The IR and 1H NMR spectral dataof compounds (1a-10a) are presented in Table 2.Compounds (1-10) were described previously byThenmozhiyal et al. (9). Compounds 7 and 7a wereobtained and described by Hosztafi et al. (8). Compound9 was obtained by Tan et al. (10).Biotransformation of phenyl ring-substituted 5-ben-zylohydantoins 0,125 mmol of 5-benzylhydantoin (1a-10a) wasdissolved in 0.5 mL DMSO and added to 15 mL ofbuffer solution (H3BO3/HCl/NaOH, pH = 9). In thismixture 0,3 g D-hydantoinase (53.1 U/g) was suspend-ed. The incubation was kept at 40OC for 72 h. Theprocess was monitored by TLC with B: butanol/aceticacid/water (4:1:1, v/v/v). Spots were visualized by UVlight (254 nm). The obtained N-carbamoyl-D-aminoacids (1b-10b) were not isolated (Figure 2).DiazotizationThe reaction mixture was acidified with 3.5 M HClto pH = 2-3. N-carbamoyl-D-amino acids (1b-10b)were converted into the corresponding D-amino acids(1c-10c) by means of diazotization ñ 140 µL of 3.5 MNaNO2 solution was added and stored in 0OC for 6 h. IdentificationThe enantiomeric purities of D-amino acids weredetermined using the chiral thin layer chromatography inthe presence of commercial D,L-4-fluorophenylalanine(Nutritional Biochem. Corp.) and D,L-p-chlorophenylala-nine (Sigma). The samples were applied to ChiralplateÆMacherey-Nagel, 0.25 mm silica gel and were eluted withC: methanol/water/acetonitrile (50:50:200, v/v/v). Aminoacids were detected after spraying with 0.3% ninhydrinand heating at 100OC for 3-5 min (Figure 3).CONCLUSIONSUsing hydantoinase method enantiomerically pureD-phenylalanine derivatives (1c-10c) were obtained.Chiral TLC confirmed the presence of D-enantiomer inthe reaction mixture in high optical yield (Figure 3).Obtained D-amino acids were not recovered from thereaction mixture thus the reaction efficiency of hydan-toinase method was not examined. The presence of lit-tle amounts of unreacted 5-benzylhydantoins (1a-10a)and N-carbamoyl-D-amino acids (1b-10b) in reactionmixture were observed by TLC. However, all testedphenyl ring-substituted 5-benzylohydantoins (1a-10a)were tolerated as substrates by immobilized, cloned andexpressed in Escherichia coli D-hydantoinase (Fluka).Condensation of compounds (7-10) was carried outunder microwave irradiation. Reactions were performedusing domestic microwave oven and gave several advan-tages: easy work-up after the reaction, reduction of theby-products formed in usual thermal degradation andshort reaction time compared to conventional heating. REFERENCES1. Peptide Proteins Research Ltd. (2006), http://www.peptidesynthetics.co.uk2. Mamoru W., Yoshimune K., Hirose Y., MoriguchiM.: J. Mol. Cat. B 23, 71 (2003).3. Tian, X., Chen X., Gan, L., et al.: Bioorg. Med.Chem. Lett. 16, 1721 (2006).4. Sˆderh‰ll A.J., Polymeropoulos E.E., Paulini K.,G¸nther E., K¸hne R.: Biochem. Biophys. Res.Commun. 333, 568 (2005).5. Wong L.S., Lau T.-W.D., Baughman A.S.,Menchaca D., Garnick B.M.; Clin. Pharmacol.Ther. 73, 304 (2003).6. Leong L.L., Pasieka L.J.: J. Clin. Oncol. 79, 180(2002).7. Wilms B., Wiese A., Syldatk C., Mattes R.,Altenbuchner J.: J. Biotechnol. 86, 19 (2001).8. Marton J., Enisz J., Hosztafi S., Timar T.: J. Agric.Food. Chem. 41, 148 (1993).9. Thenmozhiyal J.C., Wong T-H.P., Chui W-K.: J.Med. 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Unnatural d-amino acids as building blocks of new peptidomimetics

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Unnatural d-amino acids as building blocks of new peptidomimetics

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