Journal of Oleo Science Copyright ©2015 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess15063 J. Oleo Sci. 64, (7) 705-712 (2015)
Synthesis and Anti-microbial Activity of Novel Phosphatidylethanolamine-N-amino Acid Derivatives Tadla Vijeetha1, Marrapu Balakrishna1, Mallampalli Sri Lakshmi Karuna1, Bhamidipati Venkata Surya Koppeswara Rao1, Rachapudi Badari Narayana Prasad1＊ , Koochana Pranay Kumar2 and Upadyaula Surya Narayana Murthy2 1 2
Centre for Lipid Research, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad - 500007, India Chemical Biology Laboratory, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad - 500007, India
Abstract: The study involved synthesis of five novel amino acid derivatives of phosphatidylethanolamine isolated from egg yolk lecithin employing a three step procedure i) N-protection of L-amino acids with BOC anhydride in alkaline medium ii) condensation of - CO2H group of N-protected amino acid with free –NH2 of PE by a peptide linkage and iii) deprotection of N-protected group of amino acids to obtain phosphatidylethanolamine-N-amino acid derivatives in 60-75% yield. The five L-amino acids used were Lglycine, L-valine, L-leucine, L-isoleucine and L-phenylalanine. The amino acid derivatives were screened for anti-baterial activity against B. subtilis, S. aureus, P. aeroginosa and E. coli taking Streptomycin as reference compound and anti-fungal activity against C. albicans, S. cervisiae, A. niger taking AmphotericinB as reference compound. All the amino acid derivatives exhibited extraordinary anti-bacterial activities about 3 folds or comparable to Streptomycin and moderate or no anti-fungal activity against AmphotericinB. Key words: phosphatidylethanolamine, phosphatidylethanolamine-N-amino acids, anti-bacterial activity, anti-fungal activity 1 Introduction Phospholipids are amphiphilic molecules with unique physico-chemical properties. They are widespread as secretory and structural components of the body and mimic or enhance natural physiological processes. Phospholipids are the most important membrane building compounds that occur in human, animal and plant cells. In addition to fatty acids, they contain phosphoric acid, glycerol, inositol and nitrogenous bases such as choline and ethanolamine. Phosphatidylethanolamine（PE）is the second most abunin animal dant phospholipid after phosphatidylcholine （PC） and plant lipids and the main lipid component of several microbial membranes. It is a key building block of membrane bilayers. The unique properties of PE come from the studies of the biochemistry of E. coli, where this lipid is a major component of the membranes. PE also plays the role of receptor in adhesion of enterohemorrhagic E.coli and enteropathogenic E.coli to the host cells1, 2）. PE is required both for proper functioning and to ensure the correct folding of the enzyme lactose permease （from E. coli） in membranes3, 4）. One of the PE derivatives, N-Acyl phosphatidylethanol-
amine with an amide linkage, a bioactive lipid, containing three fatty acid chains was isolated in 1965 by Bomstein et al.5）from wheat flour. Later, it was shown to be present in all grains（pea seeds, oats and soya beans）. Its presence was also reported in microorganisms, fish and in mammalian tissues6）. In oat lipids, the major N-acylated fatty acids （heart, are palmitic, linoleic and oleic acids7）. In animal cells brain, liver and skeletal muscle）, the N-acyl chain contains frequently palmitic and stearic acids. The N-acyl-PE is widely distributed in nature and the isolation of N-fatty acyl ethanolamines is mostly reported from the hydrolysates of phospholipid fractions of arachis oil （peanut）, egg yolk and vegetable lecithins. They are produced by the reaction of PE with fatty acid anhydride8）. According to Kei-ichi et al. 9）, the immunogenicity of lipids of many naturally occurring glycosphingolipids and acidic phospholipids can be extended to some synthetic Nsubstituted phosphatidylethanolamine derivatives. Neyroz 10） synthesized N-（1-2-napthol）-PE（NAPH-PE） based on the Schiff base formation between the NH2 of PE and aldehyde moiety of 2-hydroxy-1-napthaldehyde, followed by selective reduction of imine to obtain stable sec-
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Correspondence to: Rachapudi Badari Narayana Prasad, Centre for Lipid Research, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad - 500007, India E-mail: [email protected] Accepted April 7, 2015 (received for review March 19, 2015)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
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T. Vijeetha, M. Balakrishna and M. S. L. Karuna et al.
ondary amine, NAPH-PE. Similarly, there exist many reports in literature on preparation of acetylated lecithin’ s11）. Acetylation of PE using an enzymatic approach was carried out by Karuna et al.12）, where vinyl acetate was used as acyl donor in the presence of Lipozyme from Mucor miehi as catalyst. The enzyme selectively acetylated the –NH2 of PE in lecithin. Acetylated PE helps in improving the emulsification properties of lecithin for baking applications. On the other hand, the continuous increasing of resistance seen in bacteria has promoted a pressing need for alternative anti-microbial molecules like lipopeptides to be used for clinical applications as well as in food presentation and dairy products13）. Demand for novel lipopeptides from natural sources is increasing due to their utility in human welfare too. All forms of life, both plants and animals produce anti-microbial peptides and eliminate infection14）. Most of these peptides exhibit a great structural diversity and they are typically cationic amphiphilic molecules with excess of basic amino acid residues15−17）. Peptides are excellent candidates for the development of novel therapeutic agents and complements to conventional antibiotic therapy because they generally have a broad range of activities18）. A number of naturally occurring peptides and their derivatives have been developed as novel anti-infective therapies for conditions such as oral mucositis, lung infections associated with cystic fibrosis, cancer and skin and wound infections19）. A major limitation to the therapeutic potential is the possibility of bacteria developing resistance to the peptides. Hence, providing more of the peptides as therapeutic agents increases the body resistance towards the diseases. Anti-microbial peptides have been successively incorporated into topical therapeutics as the peptides are quickly broken down when introduced into the bloodstream. With this background, an attempt was made to synthesize novel N-acylated amino acid derivatives of PE, which is an essential component of membrane bilayers. PE in the present study was isolated from egg yolk lecithin. Structurally phosphatidylethanolamine-N-amino acids contain a peptide linkage with free amino terminal. A simple and a novel 3-step procedure was adopted to synthesize the title compounds and were tested for anti-bacterial and anti-fungal activities over a wide range of bacterial and fungal strains.
2 EXPERIMENTAL 2.1 Materials Glycine, valine, leucine, isoleucine, phenylalanine and ditertiarybutyldicarbonate were purchased from M/s Fluka Chemical Company（USA）. Dicyclohexylcarbodiimide, trifluoroacetic acid, ninhydrin, sulfuric acid and n-butanol were purchased from M/s S.D. fine Chem Pvt. Ltd.,
（Mumbai, India）. Egg lecithin was isolated from fresh egg yolk. Silica gel（60-120 mesh）was purchased from M/s Acmes laboratory, Acme Synthetic Chemicals（Mumbai, India）. Nutrient agar and potato dextrose agar（PDA）were purchased from M/s Himedia Laboratories （Mumbai, India）. Mass spectra were recorded using electron spray ionization-mass spectrometry（ ESI-MS）. IR（Infrared）spectra were recorded on a Perkin-Elmer FT-IR Spectrum BX. 1H NMR spectra was recorded on Brucker UXNMR（operating at 300 MHz and 500 MHz）spectrometer using CDCl 3. Chemical shifts δ are reported relative to TMS（δ＝0.0）as an internal standard. All spectra were recorded at 25℃. 2.2 Microorganisms Bacterial strains: Two gram-positive organisms, viz., Bacillus subtilis（MTCC-441）and Staphylococcus aureus （MTCC-96）and two gram-negative organisms Pseudomoand Escherichia coli （MTCCnas aeruginosa （MTCC-741） 443） ; Fungal strains: Three test organisms Saccharomyces cerevisiae（MTCC-36）, Aspergillus niger （MTCC-282）and Candida albicans （MTCC-227）were obtained from the Institute of Microbial Technology, Chandigarh, India. 2.3 Methods 2.3.1 Isolation of phosphatidylethanolamine（PE）from egg lecithin The yellow yolk（432 g）of fresh hen eggs（25 no’ s）was separated out from white portion carefully and extracted with acetone（1:5, w/v）. Acetone insoluble matter（234 g） was separated out from solubles by centrifugation. Acetone insoluble matter was stirred magnetically with ethanol （1:5, w/v）for 1 h and the ethanol insolubles obtained were separated by centrifugation. The ethanol insolubles（ 25 g） contain PE and PI as major components. Pure PE（99％ purity）was isolated from egg ethanol insolubles by silica gel column chromatography eluted with chloroform: methanol（85:15, v/v）as eluent20, 21）. 2.3.2 Typical procedure for the preparation of N-protected L-amino acids（2a-e） 2.3.2.1 N-protected glycine（2a） Ditertiarybutyldicarbonate（1.53 mL, 7 mmol）was added dropwise to a stirred solution of L-glycine（1a, 0.390 g, 5 mmol）and 1.0 N NaOH（6 mL） at 0℃ for 30 min. After the addition, the temperature of the contents was raised to 35℃ over a period of 10 min and stirring was continued at this temperature for an additional 2.5 h. The course of the reaction was monitored by TLC using solvent system chloroform: methanol: water（65:24:4, v/v/v）. The formation of the product was confirmed by negative ninhydrin test（disappearance of pink colour）. After the reaction, the product was taken in hexane and extracted with water（2×25 mL） followed by aqueous saturated sodium carbonate（2×25 mL）. The combined aqueous layer was adjusted to pH 1 using potassium hydrogen sulphate, followed by extraction
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J. Oleo Sci. 64, (7) 705-712 (2015)
Synthesis and Anti-microbial Activity of Novel Phosphatidylethanolamine-N-amino Acid Derivatives
of the aqueous layer with ethyl acetate and then washed with distilled water（2×25 mL）. The ethyl acetate layer was passed through a sodium sulphate to remove the residual moisture and the organic layer was concentrated on a rotary evaporator to obtain the product 2a （0.890 g, 98％ yield）. 1 H NMR（CDCl3, 300 MHz）: δ 7.71（bs, 1H, COOH）, 6.82 （m, 1H, NH）, 4.12（m, 2H, -NH-CH2-COOH）, 1.49（s, 9H, O-C （CH3）3）. ＋ ＋ , 198.52 ［M＋Na］ . ESI-MS: 175.34 ［M＋1］ 2.3.2.2 N-protected valine （2b） N-Protected valine（2b, 1.0 g, 99％ yield）was prepared from L-valine （1b, 0.580 g, 5 mmol） following the procedure as described in 2.3.2.1. 1 H-NMR （CDCl3, 300 MHz）: δ 10.19 （bs, 1H, COOH）, 6.54 （m, 1H, NH） , 5.16 （m,1H, NH-CH-COOH） , 2.23 （m,1H, CH（s, 9H, O-C （CH3） ） , 0.98 （ d, 6H, CH（CH3）2） . （CH3）2）, 1.51 3 ＋ ＋ , 240.28 ［M＋Na］ . ESI-MS: 217.51 ［M＋1］ 2.3.2.3 N-Protected leucine （2c） N-Protected leucine（2c, 1.0 g, 99％ yield）was prepared from L-leucine（1c, 0.650g, 5 mmol）following the procedure as described in 2.3.2.1. 1 H-NMR （CDCl3, 300 MHz）: δ 11.04 （bs, 1H, COOH）. 6.64 （m, 1H, NH-CO-O（CH3）3）, 4.09（m, 1H, NH-CH-COOH）, 1.78（ m, 2H, CH 2-CH（CH 3）2）, 1.57（ m, 1H, CH-（CH 3）2）, , 0.93 （d, 6H, CH（CH3） . 1.45 （s, 9H, O-C（CH3）3） 2） ＋ ＋ , 254.75 ［M＋Na］ . ESI-MS: 231.43 ［M＋1］ 2.3.2.4 N-Protected isoleucine （2d） N-Protected isoleucine（2d, 1.0 g, 99％ yield）was prepared from L-isoleucine（1d, 0.650 g, 5 mmol）following the procedure as described in 2.3.2.1. 1 H-NMR （CDCl3, 300 MHz）: δ 10.07 （bs, 1H, -COOH） , 6.64 （m, 1H, NH-CO-OC（CH3）3）, 5.09（m, 1H, NH-CH-COOH）, 1.87（m, 1H, -CH-CH2-CH3）, 1.48（m, 11H, O-C（CH3）3, CH（d, 3H, CH-CH3） , 0.96 （t, 3H, CH2-CH3） . CH2-CH3）, 1.21 ＋ ［M＋Na］ . ESI-MS: 231.19 ［M＋1］＋, 254.43 2.3.2.5 N-Protected phenylalanine （2e） 2e, N-Protected phenylalanine（ 1.27 g, 98％ yield）was prepared from L-phenylalanine （1e, 0.82 g, 5 mmol）following the procedure as described in 2.3.2.1. 1 H-NMR （CDCl3, 300 MHz）: δ 8.48 （bs, 1H, -COOH）, 7.157.31（m, 5H, Aromatic protons）, 6.56（m, 1H, NH-CO-OC （CH3）3）, 5.09（m, 1H, NH-CH-COOH）, 3.17（m, 2H, CH2. Ar）, 1.41 （s, 9H, -O-C （CH3）3） ＋ ＋ , 288.46 ［M＋Na］ . ESI-MS: 265.72 ［M＋1］ 2.3.3 Typial procedure for the preparation of phosphatidylethanolamine-N-protected amino acids （4a-e） 2.3.3.1 P hosphatidylethanolamine-N-protected glycine （4a） Phosphatidylethanolamine（ 3, 0.364 g, 0.5 mmol）and BOC protected glycine（2a, 0.125 g, 0.7 mmol）were taken in R B flask containing DCC（0.185 g, 0.9 mmol）in chloroform（10 mL）. The contents were stirred at 30℃ for 12 h. The course of the reaction was monitored by TLC using
solvent system of chloroform: methanol: water（65:25:4, v/ v/v）. The formation of the product was confirmed by disappearance of pink colour after spraying with ninhydrin. After 12 h, the solvent was removed by rotary evaporator from reaction mixture and washed with hexane untill free from dicyclohexylurea（ DCU）to obtain the product 4a （0.430 g, 95％ yield）. 1 H-NMR（CDCl3, 500 MHz）: δ 7.64（m, 1H, CH2-NH-CO, 7.12（m, 1H, CH2-NH-COO） , 5.32（m, CH＝CH）, 4.38 CH2） , 4.28（m, 2H, CH-CH2-OCO） , 4.13（m, （m, 1H, CH2-CH-CH2） 2H, CH-CH2-OPO）, 3.99（m, 2H, CH2-CH2-OPO）, 3.78（m, 2H, NH-CO-CH2-NH）, 3.41（m, 2H, CH2-CH2-NH）, 2.26（m, CH2-COO）, 2.01（m, CH2-CH＝CH-CH2）, 1.59（m, CH2-CH2COO）, 1.39（s, 9H, -O-C（CH3）3）, 1.25（m, CH2-CH2-CH2）, . 0.89（m, -CH2-CH3） IR（neat, cm−1）: 1735（O-C＝O-）, 1454（CH2-P-）, 1244 （C-N） , 1053（P-O-C） , 2361（2°amide）. 2.3.3.2 Phosphatidylethanolamine-N-protected valine（4b） Phosphatidylethanolamine-N-protected valine（4b, 0.453 g, 96％ yield）was prepared from phosphatidylethanolfollowing the procedure as deamine（3, 0.364 g, 0.5 mmol） scribed in 2.3.3.1. 1 H-NMR（CDCl3, 500 MHz）: δ 7.73（m, 1H, CH2-NH-COCH）, 6.98（m, 1H, CH-NH-COO）, 5.31（m, CH＝CH）, 4.98 （m, 1H, NH-CO-CH-NH）, 4.40（m, 1H, CH2-CH-CH2）, 4.32 （m, 2H, CH-CH2-OCO）, 4.09（m, 2H, CH-CH2-OPO）, 3.92 （m, 2H, CH2-CH2-OPO）, 3.51（m, 2H, CH2-CH2-NH）, 2.81 （m, 1H, CH（CH3）2）, 2.29（m, CH2-COO）, 1.98（m, CH2-CH ＝CH-CH 2）, 1.58（ m, CH 2-CH 2-COO）, 1.40（ s, 9H, -O-C （CH 3）3）, 1.25（ m, CH 2-CH 2-CH 2）, 0.88（ m, CH 2-CH 3, CH . （CH3） 2） : 2365（2°amide）. IR（neat, cm−1） 2.3.3.3 Phosphatidylethanolamine-N-protected leucine （4c） Phosphatidylethanolamine-N-protected leucine（ 4c, 0.445 g, 93％ yield）was prepared from phosphatidylethafollowing the procedure as nolamine（3, 0.364 g, 0.5 mmol） described in 2.3.3.1. 1 H-NMR（CDCl3, 500 MHz）: δ 8.32（m, 1H, CH2-NH-COCH）, 6.89（m, 1H, CH-NH-COO）, 5.30（m, CH＝CH）, 4.87 （m, 1H, NH-CO-CH-NH）, 4.38（m, 1H, CH2-CH-CH2）, 4.29 （m, 2H, CH-CH2-OCO）, 4.09（m, 2H, CH-CH2-OPO）, 3.95 （m, 2H, CH2-CH2-OPO）, 3.67（m, 2H, CH2-CH2-NH）, 2.25 （m, CH2-COO）, 1.99（m, CH2-CH＝CH-CH2）, 1.78（m, 2H, CH2-CH（CH3）2）, 1.61（m, CH2-CH2-COO）, 1.46（m, 1H, CH （CH3）2）, 1.38（s, 9H, -O-C（CH3）3）, 1.26（m, CH2-CH2-CH2）, （CH3）2） . 0.89（m, CH2-CH3, -CH : 2363（2°amide）. IR （neat, cm−1） 2.3.3.4 Phosphatidylethanolamine-N-protected isoleucine （4d） Phosphatidylethanolamine-N-protected leucine（ 4d, 0.445 g, 93％ yield）was prepared from phosphatidylethafollowing the procedure as nolamine（3, 0.364 g, 0.5 mmol） described in 2.3.3.1. 707
J. Oleo Sci. 64, (7) 705-712 (2015)
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H-NMR（CDCl3, 500 MHz）: δ 7.60（m, 1H, CH2-NH-COCH）, 6.89（m, 1H, CH-NH-COO）, 5.29（m, CH＝CH）, 4.92 （m, 1H, NH-CO-CH-NH）, 4.40（m, 1H, CH2-CH-CH2）, 4.32 （m, 2H, CH-CH2-OCO）, 4.10（m, 2H, CH-CH2-OPO）, 3.89 （m, 2H, CH2-CH2-OPO）, 3.53（m, 2H, CH2-CH2-NH）, 2.25 （m, CH2-COO）, 1.98（m, CH2-CH＝CH-CH2）, 1.59（m, CH2（CH2CH3））, 1.37（s, 9H, CH2-COO）, 1.49（m, 2H, CH（CH3） -O-C（CH 3）3）, 1.25（ m, CH 2-CH 2-CH 2, CH-CH 3）, 0.88（ m, . CH2-CH3, CH（CH3）2） （2°amide） . IR （neat, cm−1）: 2369 2.3.3.5 Phosphatidylethanolamine-N-protected phenylalanine （4e） Phosphatidylethanolamine-N-protected leucine（ 4e, 0.461 g, 93％ yield）was prepared from phosphatidylethanolamine（3, 0.364 g, 0.5 mmol） following the procedure as described in 2.3.3.1. 1 H-NMR（CDCl3, 500 MHz）: δ 8.16（m, 1H, CH2-NH-COCH）, 7.24-7.31 （m, 5H, Aromatic protons） , 6.90 （m, 1H, CHNH-COO）, 5.31（ m, CH＝CH）, 4.98（ m, 1H, NH-CO-CHNH）, 4.39（ m, 1H, CH 2-CH-CH 2）, 4.31（ m, 2H, CH-CH 2OCO）, 4.12（m, 2H, CH-CH2-OPO）, 3.99（m, 2H, CH2-CH2OPO）, 3.51（m, 2H, CH2-CH2-NH）, 2.68（m, 2H, CH2-Ar）, 2.26（m, CH2-COO）, 1.98（m, CH2-CH＝CH-CH2）, 1.58（m, -CH2-CH2-COO）, 1.40（s, 9H, -O-C（CH3）3）, 1.27（m, CH2. CH2-CH2）, 0.89（m, CH2-CH3） （2°amide） . IR （neat, cm−1）: 2367 2.3.4 Typical procedure for the preparation of phosphatidylethanolamine-N-amino acids （5a-e） 2.3.4.1 Phosphatidylethanolamine-N-glycine （5a） Trifluoroacetic acid（1％ of wt of substrate）was added drop wise to a stirred solution of phosphatidylethanolamine-N-protected glycine（4a, 0.272 g, 0.3 mmol）in difor 5 min at 0℃. The contents were chloromethane（10 mL） stirred for 3 h at 0℃. The course of the reaction was monitored by TLC using solvent system chloroform: methanol: water （65:25:4, v/v/v）till the appearance of pink colour with ninhydrin spray. At the end of reaction, the product was taken in dichloromethane and washed with saturated aqueous sodium bicarbonate solution and passed through sodium sulphate. The solvent was evaporated under rotary evaporator. The crude product was purified by silica gel column chromatography using chloroform: methanol （85:15 v/v）as eluent to obtain the title product 5b（0.177 g, 75％ yield）. 1 H-NMR（CDCl3, 500 MHz）: δ 8.09（m, 1H, CH2-NH-CO）, 5.39（ m, CH＝CH）, 5.08（ m, 2H, NH2-CH 2）, 4.41（ m, 1H, CH2-CH-CH2）, 4.30（m, CH-CH2-OCO）, 4.12（m, 2H, CHCH2-OPO）, 3.92（m, 2H, CH2-CH2-OPO）, 3.78（m, 2H, CH2NH2）, 3.45（m, 2H, CH2-NH-CO）, 2.35（m, CH2-COO）, 2.01 （m, CH2-CH＝CH-CH2）, 1.58（m, CH2-CH2-COO）, 1.24（m, （m, CH2-CH3） . CH2-CH2-CH2）, 0.89 （-NH2） , 2361 （2°amine） . IR （neat, cm−1）: 3328 ESI-MS: 774（M＋1） , 796 （M＋Na） , 802 （M＋1） , 824（M＋ Na）. 1
2.3.4.2 Phosphatidylethanolamine-N-valine（5b） Phosphatidylethanolamine-N-valine（5b, 0.161 g, 65％ yield）was prepared from 4b by the following procedure as described in 2.3.4.1. 1 H-NMR（CDCl3, 500 MHz）: δ 8.69（m, 2H, NH2-CH）, 7.98 , 5.39（m, CH＝CH） , 4.62（m, 1H, CH2（m, 1H, CH2-NH-CO） , 4.38 （m, 2H, CH-CH2-OCO） , 4.19 （m, 2H, CH-CH2CH-CH2） OPO）, 4.01（m, 2H, CH2-CH2-OPO）, 3.51（m, 2H, CH2-NH, 2.39（m, CH2-COO, CH （CH3） , CO） , 3.40 （m, 1H, CH-NH2） 2） 2.09（m, CH2-CH＝CH-CH2）, 1.70（m, CH2-CH2-COO）, 1.38 , 0.82（m, CH2-CH3, CH-CH3） . （m, CH2-CH2-CH2） : 3331（-NH2） , 2362（2°amine）. IR（neat, cm−1） ESI-MS: 816（M＋1） , 838（M＋Na）, 844（M＋1） , 866（M＋ Na）. 2.3.4.3 Phosphatidylethanolamine-N-leucine（5c） Phosphatidylethanolamine-N-leucine（5c, 0.163 g, 65％ yield）was prepared from 4c by the following procedure as described in 2.3.4.1. 1 H-NMR（CDCl3, 500 MHz）: δ 8.70（m, 2H, NH2-CH）, 8.05 , 5.22（m, CH＝CH） , 4.56（m, 1H, CH2（m, 1H, CH2-NH-CO） , 4.41 （m, 2H, CH-CH2-OCO） , 4.27 （m, 2H, CH-CH2CH-CH2） OPO）, 4.08（m, 2H, CH2-CH2-OPO）, 3.60（m, 2H, CH2-NHCO）, 3.48（m, 1H, CH-NH2）, 2.28（m, CH2-COO,）, 2.09（m, CH2-CH＝CH-CH2）, 1.71（m, 1H, CH（CH3）2）, 1.60（m, CH2, 0.88（m, CH2-COO）, 1.23（m, CH2-CH2-CH2, CH2-CH（CH3） 2） . CH2-CH3, CH-CH3） : 3328（-NH2） , 2363（2°amine）. IR （neat, cm−1） ESI-MS: 830（M＋1） , 852（M＋Na）, 858（M＋1） , 880（M＋ Na） . 2.3.4.4 Phosphatidylethanolamine-N-isoleucine（5d） Phosphatidylethanolamine-N- isoleucine（5d, 0.156 g, 60％ yield）was prepared from 4d by the following procedure as described in 2.3.4.1. 1 H-NMR（CDCl3, 500 MHz）: δ 8.70（m, 2H, NH2-CH）, 8.08 , 5.19（m, CH＝CH） , 4.40（m, 1H, CH2（m, 1H, CH2-NH-CO） , 4.29 （m, 2H, CH-CH2-OCO） , 4.13 （m, 2H, CH-CH2CH-CH2） OPO）, 3.91（m, 2H, CH2-CH2-OPO）, 3.41（m, 3H, CH-NH2, CH2-NH-CO）, 2.30（m, CH2-COO,）, 2.01（m, CH2-CH＝CH（C2H5））, 1.30 CH2-）, 1.59（m, CH2-CH2-COO, CH-CH（CH3） （m, CH2-CH2-CH2）, 1.18（d, 3H, CH-CH3）, 0.89（ m, CH2. CH3） : 3330（-NH2） , 2366（2°amine）. IR （neat, cm−1） ESI-MS: 830（M＋1） , 852（M＋Na）, 858（M＋1） , 880（M＋ Na）. 2.3.4.5 Phosphatidylethanolamine-N-phenylalanine（5e） Phosphatidylethanolamine-N-isoleucine（ 5e, 0.162 g, 62％ yield）was prepared from 4e by the following procedure as described in 2.3.4.1. 1 H-NMR（CDCl3, 500 MHz）: δ 8.71（m, 2H, NH2-CH）, 8.09 , （m, 1H, CH2-NH-CO）, 7.27-41（m, 5H, Aromatic protons） , 4.41（m, 2H, 5.39（m, CH＝CH） , 4.60（m, 1H, CH2-CH-CH2） CH-CH2-OCO）, 4.28（m, 2H, CH-CH2-OPO）, 3.99（m, 2H, , 3.50 （m, 2H, CH2-NH-CO） , 3.31 （d, CH2-CH2-OPO, CH-NH2） 2H, CH2-Ar）, 2.30（m, CH2-COO,）, 2.01（m, CH2-CH＝CH-
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Synthesis and Anti-microbial Activity of Novel Phosphatidylethanolamine-N-amino Acid Derivatives
CH2）, 1.60 （m, CH2-CH2-COO） , 1.25 （m, CH2-CH2-CH2） , 0.89 （m, CH2-CH3）. （-NH2） , 2361 （2°amine） . IR（neat, cm−1）: 3330 ESI-MS: 864（M＋1） , 886 （M＋Na） , 892 （M＋1） , 914（M＋ Na）. 2.3.5 Biological activity 2.3.5.1 Anti-bacterial activity The minimum inhibitory concentration（MIC）of the various evaluated compounds were tested against two representative gram-positive organisms, viz. Bacillus subtilis （MTCC-441）, Staphylococcus aureus（ MTCC-96）, and gram-negative organisms, viz. Escherichia coli（MTCC443）, Pseudomonas aeruginosa（MTCC- 741）by the well diffusion method recommended by the National Committee for Clinical Laboratory（NCCL）standards22）. Three to five identical colonies from each agar plate were lifted with a sterile wire loop and transferred into a tube containing 5 ml of nutrient agar. The turbidity of each bacterial suspension was adjusted to reach an optical comparison to that of a 0.5 McFarland standard; resulting in a suspension containing approximately 1 to 2×108 CFU/mL. Nutrient agar plates were inoculated by streaking the swab over the entire sterile agar surface. This procedure was repeated by streaking 2 more times, rotating the plate approximately 60°each time to ensure even distribution of the inoculums. As a final step, the rim of the agar was also swabbed. After allowing the inoculums to dry at room temperature, 6-mmdiameter wells were bored in the agar. Each extract was checked for antibacterial activity by introducing 50 µL of a 100 mg/mL concentration into duplicate wells. The plates were allowed to stand at room temperature for 1 h for extract to diffuse into the agar and then they were incubated at 37℃ for 24 h. Subsequently, the plates were examined for bacterial growth inhibition and the inhibition zone diameter（IZD）measured to the nearest millimeter. Streptomycin was used as positive controls and dimethylsulfoxide （DMSO） as a negative control for antibacterial activity. 2.3.5.2 Anti-fungal Activity The anti-fungal activities were assayed against four fungal strains, namely Candida albicans（MTCC-227）, Saccharomyces cerevisiae（MTCC-36）and Aspergillus niger（MTCC-282）, by the agar-well diffusion method23）. Cultures of test organisms were maintained on potato dextrose agar（PDA）slants and subcultured on petri dishes before examining for antifungal activity employing the agar cup bioassay method. A commercially prepared PDA medium（composition: potato infusion, 200 g; dextrose, 20 g; agar, 15 g）was used in this evaluation study. The PDA medium（39.0 g）was suspended in distilled water（ 1000 mL）and heated to boiling until it dissolved completely （pH 5.6）. The medium and the Petri dishes were autoclaved at 120℃ at a pressure of 15 psi, for 20 min. The medium was poured into sterile petri dishes under aseptic conditions in
a laminar flow chamber. When the medium in the plates had solidified, 0.5 mL of the test culture was inoculated and uniformly spread over the agar surface. Solutions were prepared at 150 µg/mL concentration by dissolving the test compounds in chloroform. After inoculation, cup was scooped out with a 6-mm sterile cork borer and the lids of the dishes were replaced. The test solution in 150 µg/mL concentration was added to cup. The controlled cup was treated at 27℃ for 48 h. The diameters（mm）of the inhibition zones were then measured. Amphotericin B was taken as positive controls and DMSO as negative control. Three replicates were maintained for each treatment.
3 Results and discussion Several studies on modified phosphatidylethanolamine （PE）particularly at –NH2 group（N-acyl, N-methyl amino, N,N-dimethylamino etc）revealed that these compounds exhibited significant biological activities24−28）. Hence, there is a large potential to exploit this area by derivatizing –NH2 group of PE with biologically important L-amino acids. In the present study, five L-amino acids namely L-glycine, Lvaline, L-leucine, L-isoleucine and L-phenylalnine（1a-e） were reacted with PE to obtain PE-N-amino acids employing a novel three step procedure. The method involves i） Nprotection of L-amino acid with BOC anhydride in alkaline medium ii）condensation of –CO2H group of N-protected amino acid with free –NH2 of PE by a peptide linkage and iii）deprotection of N- protected group of amino acids to obtain PE-N-amino acids. The L-amino acids were protected using ditertiarybutyldicarbonate（BOC anhydride）in the presence of 1 N NaOH solution to get the corresponding N-protected amino acids （2a-e）with excellent yields（98-99％）. N-Protected amino acids were characterized by 1H NMR（2a）which showed disappearance of primary amine proton and a sharp peak at 1.49 δ accounting for 9 protons of the 3 methyl groups of , an amide bond formed during the proBOC （-O-C（CH3）3） tection of free –NH2 of amino acids. A multiplet at 4.12 δ accounted for two protons of methylene group attached to -NH-BOC, a multiplet at 6.82 δ accounting for one proton attached to nitrogen（-NH-BOC）and an acidic proton as a broad singlet at 7.71 δ was observed. PE isolated from egg yolk lecithin with 99％ purity was coupled with N-protected amino acids in presence of DCC in chloroform to obtain PE-N-protected amino acids （4a-e） . The disappearance of the pink color spot of PE with the ninhydrin spray indicated that the –NH2 group of PE is linked to BOC-protected amino acid. The by-product（Dicyclohexylurea）formed during the reaction was separated by filtration after dissolving the product in hexane. The crude product was subjected to silica gel column chromatography using chloroform:methanol （90:10, v/v）as eluent to obtain 709
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Scheme Synthesis of phosphatidylethanolamine-N-amino acid derivatives. the pure product in 93-96％ yield. PE-N-protected amino acids （4a-e）were characterized by 1H NMR and IR. The 1H NMR data of 4a-e showed a singlet peak at 1.39 δ account（CH3）3） , ing for 9 protons of 3 methyl groups of BOC （-O-Ca multiplet at 3.78 δ accounting for 2 protons of methylene , a multiplet at 4.13 δ acgroup of glycine （NH-CO-CH2-NH） counting for two protons of methylene group attached to phosphate moiety（ P-O）and other characteristic peaks related to fatty acids and glycerol back bone of PE were observed. Deprotection of BOC protected amino acids of PE was carried out using trifluoroacetic acid to obtain PE-N-amino acids （5a-e）with good yields in 60-75％. 5a-e were characterized by 1H NMR and IR. The 1H NMR of 5a-e showed a multiplet at 3.78 δ accounting for 2 protons of methylene adjacent to NH2 and a multiplet at 3.45 δ accounting for 2 protons of methylene adjacent to amide nitrogen was found. A multiplet at 5.08 δ accounting for 2 proton of primary amine group and a broad singlet at 8.09 δ accounting for one proton of amide nitrogen group and other characteristic signals related to PE were observed. 3.1 Biological evaluation PE-N-amino acids（5a-e）prepared in the present study were evaluated for their anti-microbial activity, i.e. antibacterial and anti-fungal activities. The anti-bacterial activity was evaluated against two gram-positive bacteria namely Bacillus subtilis, Staphylococcus aureus and two gram-negative bacteria namely Escherichia coli, Pseudomonas aeruginosa. Anti-fungal activity was tested against three fungal stains namely Saccharomyces cerevisiae, Aspergillus niger and Candida albicans. Controls were maintained with DMSO and streptomycin（150 μg/mL）and
. amphotericin-B （150 μg/mL） 3.1.1 Anti-bacterial activity Anti-bacterial activity was assayed by well diffusion method. The zone of inhibition was observed after 24 h of incubation and the diameter of the zone was measured. The MIC values of PE-N-amino acid analogues are tabulated in Table 1. It was interesting to find that amino acid derivatives（5a-e）exhibited about three folds much superior activities compared to streptomycin as reference compound against the tested Gram-positive and Gram-negative bacteria. This further revealed that lipopeptide functionalities play a very important role in the activity compared to the amido glucoside functionality present in streptomycin. Further the series 5a and 5d showed extraordinary activities against Gram-positive bacterial strain, B. subtilis compared to other analogues of PE and Gram-negative bacterial strains. Also it was observed that shift from non-polar aliphatic functionalities as seen in 5a-d to a non-polar aromatic functionality（5e）increased the MIC values. This can be explained that decrease in basicity of amino group decreased the activity. The observation was similar to that reported by Lee et al.29）on ethambutol derivatives. Hence, it can be concluded that the presence of ethanolamine as in PE and non-polar aliphatic chain as seen in 5a-d were crucial for anti-bacterial activity against Gram-positive bacteria. The mode of action of anti-microbial activity of these PE-N-amino acid derivatives could be explained similar to the model described by Straus et al.30）for hydrophilic peptides. According to their model, the molecules upon interaction into the bacterial membrane adopt a three-dimensional structure. They fold into amphiphilic molecules with the induction of positive membrane curvature interacting directly with the lipid head groups. The bacterial cells are
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Table 1 Anti-bacterial activity of synthesized compounds 5a-e. Test Compounds 5a 5b 5c 5d 5e Streptomycin
*Gram +ve (μg/mL) B. subtilis S. aureus 8 12 12 14 10 20 7 18 22 27 24 30
*Gram –ve (μg/mL) P. aeruginosa E. coli 14 11 10 10 11 12 16 12 23 24 31 33
Inhibitory zone diameters are in mm, Concentration, μg/mL, Negative Control (DMSO). DMSO did not show any activity. *MIC values reported are an average of three triplicate analysis.
Table 2 Anti-fungal activities of synthesized compounds 5a-e. Test Compounds 5a 5b 5c 5d 5e Amphotericin-B
Yeast (μg/mL) C. albicans 99 115 96 110 88 23.5
Filamentous Fungi (μg/mL) S. cervisiae A. niger − − − 88 − 66 − − 40 110 22 25
Inhibitory zone diameters are in mm; Concentration, μg/mL. Negative Control (DMSO). DMSO did not show any activity. finally killed either by membrane perforation or some other membrane-associated event. 3.1.2 Anti-fungal activity 5a showed good activity against yeast, C. albicans while no activity against other two fungal species. 5b showed moderate activity against yeast, C. albicans and good activity against filamentous fungi, Aspergillus niger. 5c showed good activity against C. albicans and, Aspergillus niger while 5d showed moderate activity against only C. albicans. 5e showed good activity against yeast, C. albicans and filamentous fungi, S. cerevisiae and moderate activity against Aspergillus niger. It was observed that upon derivatization of free –NH2 in PE, the compounds 5a-d did not exhibit any activity against filamentous fungi, S. cervisiae. However, the aromatic amino acid PE derivative, 5e showed good activity against S. cervisiae and moderate activity against Aspergillus niger. Compounds 5a-d exhibited moderate activity against C. albicans and no activity against the filamentous fungi. Presence of aromatic group helped compound 5e to exhibit moderate ac. tivity against both the yeast and fungi （Table 2）
4 Conclusion A simple and novel method was developed for the preparation of phosphatidylethanolamine-N-amino acid deriva-
tives using L-glycine, L-leucine, L-valine, L-isoleucine and L-phenylalanine. All the amino acid derivatives 5（ a-e） when evaluated for anti-bacterial exhibited extraordinary to moderate anti-bacterial activity compared to streptomycin as reference compound. While, moderate to poor antifungal activity compared to amphotericin-B as reference compound.
Acknowledgements The author T.Vijeetha acknowledges the financial support from D B T, New Delhi, India.
References 1）Foster, B. D.; Philpo, D. H.; Mabul, M. M.; Sherman, P. M.; Lingwood, L. A. Phosphatidylethanolamine recognition promotes enteropathogenic E. coli and enterohemorrhagic E. coli host cell attachment. Microb. Pathog. 27, 289-301（1999）. 2）Foster, B. D.; Abul, M.; Huesca, M.; Lingwood, C. A. Enterohemorrhagic Escherichia coli Induces apoptosis which augments bacterial binding and phosphatidylethanolamine exposure on the plasma membrane outer leaflet. Infect. Immuno. 68, 3108-3115（2000）. 711
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3）Mandel, H.; Sharf, R.; Berant, M.; Wanders, R. J. A.; Vreken, P.; Aviram, M. Plasmalogen phospholipids are involved in HDL-mediated cholesterol efflux: Insights from investigations with plasmalogen-deficient cells. Biochem. Biophys. Res. Comm. 250, 369-373 （1998）. 4）Brites, P.; Waterham, H. R.; Wanders, R. J. A. Functions and biosynthesis of plasmalogens in health and disease. Biochem. Biophys. Acta 1636, 219-231 （2004） . 5）Bomestein, R. A. A new class of phosphatides isolated from soft wheat flour. Biochem. Biophys. Res. Comm. 21, 49-54 （1965） . 6）Schmid, H. H.; Schmid, P. C.; Natarajan, V. N-acylated glycerophospholipids and their derivatives. Prog. Lip. id Res. 29, 1-43 （1990） 7）Holmback, J.; Karlsson, A.; Arnoldsson, K. C. Characterization of N-acylphosphatidylethanolamine and acylphosphatidylglycerol in Oats. Lipids 36, 153-165 （2001） . 8）Aneja, R.; Chadha, J. S.; Knaggs, J. A. N-acylphosphatidylethanolamines: Occurrence in nature, structure and stereochemistry. Biochem. Biophys. Res. Comm. 36, 401-406 （1969） . 9）Uemura, K.; Nicolotti, R. A.; Six, H. R.; Kinsky, S. C. Antibody formation in response to liposomal model membranes sensitized with N-substituted phosphatidylethanolamine derivatives. Biochemistry 13, 1572 （1974） . 10）Neyroz, P.; Franzoni, L.; Spisni, A.; Masotti, L.; Brand, L. The chemical synthesis of N-［1-（2-naphthol）］ -phosphatidylethanolamine, a fluorescent phospholipid for excited-state proton transfer studies. Chem. （1992） . Phys. Lipids 61, 255-263 11）Karuna, M. S. L.; Vandana, V.; Vijaya Lakshmi, P.; Prasad, R. B. N. US Patent No 6, 403, 344 （2002） . 12）Karuna, M. S. L.; Vandana, V.; Vijaya Lakshmi, P.; Prasad, R. B. N. Simple enzymatic approach for selective acetylation of phosphatidylethanolamine. J. Am. Oil Chem. Soc. 90, 369-375 （2013） . 13）Meena, K. R.; Kanwar, S. S. Lipopeptides as the Antifungal and Antibacterial Agents: Applications in Food Safety and Therapeutics. Bio. Med. Res. Int. 2015, 1-9 （2015）. 14）Hancock, R. E. W.; Lehrer, R. Cationic peptides: a new source of antibiotics. Trends Biotechnol. 16, 82-88 （1998） . 15）Zasloff, M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precur. sor. Proc. Natl. Acad. Sci. 84, 5449-5453 （1987） 16）Wade, D.; Andreu, D.; Mithell, S. A.; Silveira, A. M. V.; Boman, A.; Boman, H. G.; Merrified R. B. Antibacterial peptides designed as analogs or hybrids of cecropins and melittin. Int. J. Pept. Protein Res. 40, 429-436
（1992）. 17）Hancock, R. E. W. Peptide antibiotics. Lancet 349, 418-422（1997）. 18）Reddy, K. V.; Yedery, R. D.; Aranha, C. Antimicrobial peptides: premises and promises. Int. J. Antimicrob. Agents 24, 536–547（2004）. 19）O’ Driscoll, N. H.; Labovitiadi, O.; Cushnie, T. P. T.; Matthews, K. H.; Mercer, D. K.; Lamb, A. J. Production and evaluation of an antimicrobial peptide-containing wafer formulation for topical application. Curr. Microb. 66, 271-278（2013）. 20）Mangold, H. K. Thin-layer chromatography of lipids. J. Am. Oil Chem. Soc. 38, 708-727 （1961）. 21）Vashkovsky, V. E.; Kostetsky, G. Y. J. Lipid Res. 9, 936-940（1968）. 22）National committee for clinical laboratory standards approved standards（NCCLS）: M2-A6（1997）Performance standards for antimicrobial disk susceptibility testing. NCCLS （6th edn）. 23）Linday, E. M. Practical introduction to microbiology. E. & F.N. Spon, London, p. 178 （1962）. 24）Wellner, N.; Diep, T. A.; Janfelt, C.; Hansen, H. S. N-acylation of phosphatidylethanolamine and its biological functions in mammals. Biochem. Biophys. Acta 1831, 652-662（2013）. 25）Terova, B.; Petersen, G.; Hansen, H. S.; Slotte, J. P. Nacyl phosphatidylethanolamines affect then lateral distribution of cholesterol in membranes. Biochim. Biophys. Acta 1715, 49-56 （2005）. 26）Ogawa, Y.; Epand, R. M. Effect of N-acyl phosphatidylethanolamine on the membrane fusion between sendai virus and liposome. Biosci. Rep. 17, 401-408（1997）. 27）Garcia, A. M.; Rosell, F.; Sagrista, M. L.; Mora, M.; DeMadariaga, M. A. Structural organization of N-acyl derivatives of egg phosphatidylethanolamine into bilayers: polymorphic and thermotropic studies. Colloid. Surface. A: Physicochem. Eng. Asp. 115, 73-82 （1996）. 28）Basu, A.; Prence, E.; Garrett, K.; Glew, R. H.; Ellingson, J. S. Comparison of N-acyl phosphatidylethanolamines with different N-acyl groups as activators of s disglucocerebrosidase in various forms of Gaucher’ ease. Arch. Biochem. Biophys. 243, 28-34（1985）. 29）Lee, R. E.; Protopopova, M.; Crooks, E.; Slayden, R. A.; Terrot, M.; Barry, C. E. Combinatorial lead optimization of［1,2］-diamines based on ethambutol as potential anti-tuberculosis preclinical candidates. J. Comb. Chem. 5, 172-187（2003）. 30）Straus, S. K.; Hancock, R. E. W. Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: Comparison with cationic antimicrobial peptides and lipopeptides. Biochim. Biophys. Acta 1758, 1215-1223（2006）.
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Synthesis and Anti-microbial Activity of Novel Phosphatidylethanolamine-N-amino Acid Derivatives Tadla Vijeetha1, Marrapu Balakrishna1, Mallampalli Sri Lakshmi Karuna1, Bhamidipati Venkata Surya Koppeswara Rao1, Rachapudi Badari Narayana Prasad1＊ , Koochana Pranay Kumar2 and Upadyaula Surya Narayana Murthy2 1 2
Centre for Lipid Research, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad - 500007, India Chemical Biology Laboratory, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad - 500007, India
Abstract: The study involved synthesis of five novel amino acid derivatives of phosphatidylethanolamine isolated from egg yolk lecithin employing a three step procedure i) N-protection of L-amino acids with BOC anhydride in alkaline medium ii) condensation of - CO2H group of N-protected amino acid with free –NH2 of PE by a peptide linkage and iii) deprotection of N-protected group of amino acids to obtain phosphatidylethanolamine-N-amino acid derivatives in 60-75% yield. The five L-amino acids used were Lglycine, L-valine, L-leucine, L-isoleucine and L-phenylalanine. The amino acid derivatives were screened for anti-baterial activity against B. subtilis, S. aureus, P. aeroginosa and E. coli taking Streptomycin as reference compound and anti-fungal activity against C. albicans, S. cervisiae, A. niger taking AmphotericinB as reference compound. All the amino acid derivatives exhibited extraordinary anti-bacterial activities about 3 folds or comparable to Streptomycin and moderate or no anti-fungal activity against AmphotericinB. Key words: phosphatidylethanolamine, phosphatidylethanolamine-N-amino acids, anti-bacterial activity, anti-fungal activity 1 Introduction Phospholipids are amphiphilic molecules with unique physico-chemical properties. They are widespread as secretory and structural components of the body and mimic or enhance natural physiological processes. Phospholipids are the most important membrane building compounds that occur in human, animal and plant cells. In addition to fatty acids, they contain phosphoric acid, glycerol, inositol and nitrogenous bases such as choline and ethanolamine. Phosphatidylethanolamine（PE）is the second most abunin animal dant phospholipid after phosphatidylcholine （PC） and plant lipids and the main lipid component of several microbial membranes. It is a key building block of membrane bilayers. The unique properties of PE come from the studies of the biochemistry of E. coli, where this lipid is a major component of the membranes. PE also plays the role of receptor in adhesion of enterohemorrhagic E.coli and enteropathogenic E.coli to the host cells1, 2）. PE is required both for proper functioning and to ensure the correct folding of the enzyme lactose permease （from E. coli） in membranes3, 4）. One of the PE derivatives, N-Acyl phosphatidylethanol-
amine with an amide linkage, a bioactive lipid, containing three fatty acid chains was isolated in 1965 by Bomstein et al.5）from wheat flour. Later, it was shown to be present in all grains（pea seeds, oats and soya beans）. Its presence was also reported in microorganisms, fish and in mammalian tissues6）. In oat lipids, the major N-acylated fatty acids （heart, are palmitic, linoleic and oleic acids7）. In animal cells brain, liver and skeletal muscle）, the N-acyl chain contains frequently palmitic and stearic acids. The N-acyl-PE is widely distributed in nature and the isolation of N-fatty acyl ethanolamines is mostly reported from the hydrolysates of phospholipid fractions of arachis oil （peanut）, egg yolk and vegetable lecithins. They are produced by the reaction of PE with fatty acid anhydride8）. According to Kei-ichi et al. 9）, the immunogenicity of lipids of many naturally occurring glycosphingolipids and acidic phospholipids can be extended to some synthetic Nsubstituted phosphatidylethanolamine derivatives. Neyroz 10） synthesized N-（1-2-napthol）-PE（NAPH-PE） based on the Schiff base formation between the NH2 of PE and aldehyde moiety of 2-hydroxy-1-napthaldehyde, followed by selective reduction of imine to obtain stable sec-
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Correspondence to: Rachapudi Badari Narayana Prasad, Centre for Lipid Research, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad - 500007, India E-mail: [email protected] Accepted April 7, 2015 (received for review March 19, 2015)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
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T. Vijeetha, M. Balakrishna and M. S. L. Karuna et al.
ondary amine, NAPH-PE. Similarly, there exist many reports in literature on preparation of acetylated lecithin’ s11）. Acetylation of PE using an enzymatic approach was carried out by Karuna et al.12）, where vinyl acetate was used as acyl donor in the presence of Lipozyme from Mucor miehi as catalyst. The enzyme selectively acetylated the –NH2 of PE in lecithin. Acetylated PE helps in improving the emulsification properties of lecithin for baking applications. On the other hand, the continuous increasing of resistance seen in bacteria has promoted a pressing need for alternative anti-microbial molecules like lipopeptides to be used for clinical applications as well as in food presentation and dairy products13）. Demand for novel lipopeptides from natural sources is increasing due to their utility in human welfare too. All forms of life, both plants and animals produce anti-microbial peptides and eliminate infection14）. Most of these peptides exhibit a great structural diversity and they are typically cationic amphiphilic molecules with excess of basic amino acid residues15−17）. Peptides are excellent candidates for the development of novel therapeutic agents and complements to conventional antibiotic therapy because they generally have a broad range of activities18）. A number of naturally occurring peptides and their derivatives have been developed as novel anti-infective therapies for conditions such as oral mucositis, lung infections associated with cystic fibrosis, cancer and skin and wound infections19）. A major limitation to the therapeutic potential is the possibility of bacteria developing resistance to the peptides. Hence, providing more of the peptides as therapeutic agents increases the body resistance towards the diseases. Anti-microbial peptides have been successively incorporated into topical therapeutics as the peptides are quickly broken down when introduced into the bloodstream. With this background, an attempt was made to synthesize novel N-acylated amino acid derivatives of PE, which is an essential component of membrane bilayers. PE in the present study was isolated from egg yolk lecithin. Structurally phosphatidylethanolamine-N-amino acids contain a peptide linkage with free amino terminal. A simple and a novel 3-step procedure was adopted to synthesize the title compounds and were tested for anti-bacterial and anti-fungal activities over a wide range of bacterial and fungal strains.
2 EXPERIMENTAL 2.1 Materials Glycine, valine, leucine, isoleucine, phenylalanine and ditertiarybutyldicarbonate were purchased from M/s Fluka Chemical Company（USA）. Dicyclohexylcarbodiimide, trifluoroacetic acid, ninhydrin, sulfuric acid and n-butanol were purchased from M/s S.D. fine Chem Pvt. Ltd.,
（Mumbai, India）. Egg lecithin was isolated from fresh egg yolk. Silica gel（60-120 mesh）was purchased from M/s Acmes laboratory, Acme Synthetic Chemicals（Mumbai, India）. Nutrient agar and potato dextrose agar（PDA）were purchased from M/s Himedia Laboratories （Mumbai, India）. Mass spectra were recorded using electron spray ionization-mass spectrometry（ ESI-MS）. IR（Infrared）spectra were recorded on a Perkin-Elmer FT-IR Spectrum BX. 1H NMR spectra was recorded on Brucker UXNMR（operating at 300 MHz and 500 MHz）spectrometer using CDCl 3. Chemical shifts δ are reported relative to TMS（δ＝0.0）as an internal standard. All spectra were recorded at 25℃. 2.2 Microorganisms Bacterial strains: Two gram-positive organisms, viz., Bacillus subtilis（MTCC-441）and Staphylococcus aureus （MTCC-96）and two gram-negative organisms Pseudomoand Escherichia coli （MTCCnas aeruginosa （MTCC-741） 443） ; Fungal strains: Three test organisms Saccharomyces cerevisiae（MTCC-36）, Aspergillus niger （MTCC-282）and Candida albicans （MTCC-227）were obtained from the Institute of Microbial Technology, Chandigarh, India. 2.3 Methods 2.3.1 Isolation of phosphatidylethanolamine（PE）from egg lecithin The yellow yolk（432 g）of fresh hen eggs（25 no’ s）was separated out from white portion carefully and extracted with acetone（1:5, w/v）. Acetone insoluble matter（234 g） was separated out from solubles by centrifugation. Acetone insoluble matter was stirred magnetically with ethanol （1:5, w/v）for 1 h and the ethanol insolubles obtained were separated by centrifugation. The ethanol insolubles（ 25 g） contain PE and PI as major components. Pure PE（99％ purity）was isolated from egg ethanol insolubles by silica gel column chromatography eluted with chloroform: methanol（85:15, v/v）as eluent20, 21）. 2.3.2 Typical procedure for the preparation of N-protected L-amino acids（2a-e） 2.3.2.1 N-protected glycine（2a） Ditertiarybutyldicarbonate（1.53 mL, 7 mmol）was added dropwise to a stirred solution of L-glycine（1a, 0.390 g, 5 mmol）and 1.0 N NaOH（6 mL） at 0℃ for 30 min. After the addition, the temperature of the contents was raised to 35℃ over a period of 10 min and stirring was continued at this temperature for an additional 2.5 h. The course of the reaction was monitored by TLC using solvent system chloroform: methanol: water（65:24:4, v/v/v）. The formation of the product was confirmed by negative ninhydrin test（disappearance of pink colour）. After the reaction, the product was taken in hexane and extracted with water（2×25 mL） followed by aqueous saturated sodium carbonate（2×25 mL）. The combined aqueous layer was adjusted to pH 1 using potassium hydrogen sulphate, followed by extraction
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Synthesis and Anti-microbial Activity of Novel Phosphatidylethanolamine-N-amino Acid Derivatives
of the aqueous layer with ethyl acetate and then washed with distilled water（2×25 mL）. The ethyl acetate layer was passed through a sodium sulphate to remove the residual moisture and the organic layer was concentrated on a rotary evaporator to obtain the product 2a （0.890 g, 98％ yield）. 1 H NMR（CDCl3, 300 MHz）: δ 7.71（bs, 1H, COOH）, 6.82 （m, 1H, NH）, 4.12（m, 2H, -NH-CH2-COOH）, 1.49（s, 9H, O-C （CH3）3）. ＋ ＋ , 198.52 ［M＋Na］ . ESI-MS: 175.34 ［M＋1］ 2.3.2.2 N-protected valine （2b） N-Protected valine（2b, 1.0 g, 99％ yield）was prepared from L-valine （1b, 0.580 g, 5 mmol） following the procedure as described in 2.3.2.1. 1 H-NMR （CDCl3, 300 MHz）: δ 10.19 （bs, 1H, COOH）, 6.54 （m, 1H, NH） , 5.16 （m,1H, NH-CH-COOH） , 2.23 （m,1H, CH（s, 9H, O-C （CH3） ） , 0.98 （ d, 6H, CH（CH3）2） . （CH3）2）, 1.51 3 ＋ ＋ , 240.28 ［M＋Na］ . ESI-MS: 217.51 ［M＋1］ 2.3.2.3 N-Protected leucine （2c） N-Protected leucine（2c, 1.0 g, 99％ yield）was prepared from L-leucine（1c, 0.650g, 5 mmol）following the procedure as described in 2.3.2.1. 1 H-NMR （CDCl3, 300 MHz）: δ 11.04 （bs, 1H, COOH）. 6.64 （m, 1H, NH-CO-O（CH3）3）, 4.09（m, 1H, NH-CH-COOH）, 1.78（ m, 2H, CH 2-CH（CH 3）2）, 1.57（ m, 1H, CH-（CH 3）2）, , 0.93 （d, 6H, CH（CH3） . 1.45 （s, 9H, O-C（CH3）3） 2） ＋ ＋ , 254.75 ［M＋Na］ . ESI-MS: 231.43 ［M＋1］ 2.3.2.4 N-Protected isoleucine （2d） N-Protected isoleucine（2d, 1.0 g, 99％ yield）was prepared from L-isoleucine（1d, 0.650 g, 5 mmol）following the procedure as described in 2.3.2.1. 1 H-NMR （CDCl3, 300 MHz）: δ 10.07 （bs, 1H, -COOH） , 6.64 （m, 1H, NH-CO-OC（CH3）3）, 5.09（m, 1H, NH-CH-COOH）, 1.87（m, 1H, -CH-CH2-CH3）, 1.48（m, 11H, O-C（CH3）3, CH（d, 3H, CH-CH3） , 0.96 （t, 3H, CH2-CH3） . CH2-CH3）, 1.21 ＋ ［M＋Na］ . ESI-MS: 231.19 ［M＋1］＋, 254.43 2.3.2.5 N-Protected phenylalanine （2e） 2e, N-Protected phenylalanine（ 1.27 g, 98％ yield）was prepared from L-phenylalanine （1e, 0.82 g, 5 mmol）following the procedure as described in 2.3.2.1. 1 H-NMR （CDCl3, 300 MHz）: δ 8.48 （bs, 1H, -COOH）, 7.157.31（m, 5H, Aromatic protons）, 6.56（m, 1H, NH-CO-OC （CH3）3）, 5.09（m, 1H, NH-CH-COOH）, 3.17（m, 2H, CH2. Ar）, 1.41 （s, 9H, -O-C （CH3）3） ＋ ＋ , 288.46 ［M＋Na］ . ESI-MS: 265.72 ［M＋1］ 2.3.3 Typial procedure for the preparation of phosphatidylethanolamine-N-protected amino acids （4a-e） 2.3.3.1 P hosphatidylethanolamine-N-protected glycine （4a） Phosphatidylethanolamine（ 3, 0.364 g, 0.5 mmol）and BOC protected glycine（2a, 0.125 g, 0.7 mmol）were taken in R B flask containing DCC（0.185 g, 0.9 mmol）in chloroform（10 mL）. The contents were stirred at 30℃ for 12 h. The course of the reaction was monitored by TLC using
solvent system of chloroform: methanol: water（65:25:4, v/ v/v）. The formation of the product was confirmed by disappearance of pink colour after spraying with ninhydrin. After 12 h, the solvent was removed by rotary evaporator from reaction mixture and washed with hexane untill free from dicyclohexylurea（ DCU）to obtain the product 4a （0.430 g, 95％ yield）. 1 H-NMR（CDCl3, 500 MHz）: δ 7.64（m, 1H, CH2-NH-CO, 7.12（m, 1H, CH2-NH-COO） , 5.32（m, CH＝CH）, 4.38 CH2） , 4.28（m, 2H, CH-CH2-OCO） , 4.13（m, （m, 1H, CH2-CH-CH2） 2H, CH-CH2-OPO）, 3.99（m, 2H, CH2-CH2-OPO）, 3.78（m, 2H, NH-CO-CH2-NH）, 3.41（m, 2H, CH2-CH2-NH）, 2.26（m, CH2-COO）, 2.01（m, CH2-CH＝CH-CH2）, 1.59（m, CH2-CH2COO）, 1.39（s, 9H, -O-C（CH3）3）, 1.25（m, CH2-CH2-CH2）, . 0.89（m, -CH2-CH3） IR（neat, cm−1）: 1735（O-C＝O-）, 1454（CH2-P-）, 1244 （C-N） , 1053（P-O-C） , 2361（2°amide）. 2.3.3.2 Phosphatidylethanolamine-N-protected valine（4b） Phosphatidylethanolamine-N-protected valine（4b, 0.453 g, 96％ yield）was prepared from phosphatidylethanolfollowing the procedure as deamine（3, 0.364 g, 0.5 mmol） scribed in 2.3.3.1. 1 H-NMR（CDCl3, 500 MHz）: δ 7.73（m, 1H, CH2-NH-COCH）, 6.98（m, 1H, CH-NH-COO）, 5.31（m, CH＝CH）, 4.98 （m, 1H, NH-CO-CH-NH）, 4.40（m, 1H, CH2-CH-CH2）, 4.32 （m, 2H, CH-CH2-OCO）, 4.09（m, 2H, CH-CH2-OPO）, 3.92 （m, 2H, CH2-CH2-OPO）, 3.51（m, 2H, CH2-CH2-NH）, 2.81 （m, 1H, CH（CH3）2）, 2.29（m, CH2-COO）, 1.98（m, CH2-CH ＝CH-CH 2）, 1.58（ m, CH 2-CH 2-COO）, 1.40（ s, 9H, -O-C （CH 3）3）, 1.25（ m, CH 2-CH 2-CH 2）, 0.88（ m, CH 2-CH 3, CH . （CH3） 2） : 2365（2°amide）. IR（neat, cm−1） 2.3.3.3 Phosphatidylethanolamine-N-protected leucine （4c） Phosphatidylethanolamine-N-protected leucine（ 4c, 0.445 g, 93％ yield）was prepared from phosphatidylethafollowing the procedure as nolamine（3, 0.364 g, 0.5 mmol） described in 2.3.3.1. 1 H-NMR（CDCl3, 500 MHz）: δ 8.32（m, 1H, CH2-NH-COCH）, 6.89（m, 1H, CH-NH-COO）, 5.30（m, CH＝CH）, 4.87 （m, 1H, NH-CO-CH-NH）, 4.38（m, 1H, CH2-CH-CH2）, 4.29 （m, 2H, CH-CH2-OCO）, 4.09（m, 2H, CH-CH2-OPO）, 3.95 （m, 2H, CH2-CH2-OPO）, 3.67（m, 2H, CH2-CH2-NH）, 2.25 （m, CH2-COO）, 1.99（m, CH2-CH＝CH-CH2）, 1.78（m, 2H, CH2-CH（CH3）2）, 1.61（m, CH2-CH2-COO）, 1.46（m, 1H, CH （CH3）2）, 1.38（s, 9H, -O-C（CH3）3）, 1.26（m, CH2-CH2-CH2）, （CH3）2） . 0.89（m, CH2-CH3, -CH : 2363（2°amide）. IR （neat, cm−1） 2.3.3.4 Phosphatidylethanolamine-N-protected isoleucine （4d） Phosphatidylethanolamine-N-protected leucine（ 4d, 0.445 g, 93％ yield）was prepared from phosphatidylethafollowing the procedure as nolamine（3, 0.364 g, 0.5 mmol） described in 2.3.3.1. 707
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H-NMR（CDCl3, 500 MHz）: δ 7.60（m, 1H, CH2-NH-COCH）, 6.89（m, 1H, CH-NH-COO）, 5.29（m, CH＝CH）, 4.92 （m, 1H, NH-CO-CH-NH）, 4.40（m, 1H, CH2-CH-CH2）, 4.32 （m, 2H, CH-CH2-OCO）, 4.10（m, 2H, CH-CH2-OPO）, 3.89 （m, 2H, CH2-CH2-OPO）, 3.53（m, 2H, CH2-CH2-NH）, 2.25 （m, CH2-COO）, 1.98（m, CH2-CH＝CH-CH2）, 1.59（m, CH2（CH2CH3））, 1.37（s, 9H, CH2-COO）, 1.49（m, 2H, CH（CH3） -O-C（CH 3）3）, 1.25（ m, CH 2-CH 2-CH 2, CH-CH 3）, 0.88（ m, . CH2-CH3, CH（CH3）2） （2°amide） . IR （neat, cm−1）: 2369 2.3.3.5 Phosphatidylethanolamine-N-protected phenylalanine （4e） Phosphatidylethanolamine-N-protected leucine（ 4e, 0.461 g, 93％ yield）was prepared from phosphatidylethanolamine（3, 0.364 g, 0.5 mmol） following the procedure as described in 2.3.3.1. 1 H-NMR（CDCl3, 500 MHz）: δ 8.16（m, 1H, CH2-NH-COCH）, 7.24-7.31 （m, 5H, Aromatic protons） , 6.90 （m, 1H, CHNH-COO）, 5.31（ m, CH＝CH）, 4.98（ m, 1H, NH-CO-CHNH）, 4.39（ m, 1H, CH 2-CH-CH 2）, 4.31（ m, 2H, CH-CH 2OCO）, 4.12（m, 2H, CH-CH2-OPO）, 3.99（m, 2H, CH2-CH2OPO）, 3.51（m, 2H, CH2-CH2-NH）, 2.68（m, 2H, CH2-Ar）, 2.26（m, CH2-COO）, 1.98（m, CH2-CH＝CH-CH2）, 1.58（m, -CH2-CH2-COO）, 1.40（s, 9H, -O-C（CH3）3）, 1.27（m, CH2. CH2-CH2）, 0.89（m, CH2-CH3） （2°amide） . IR （neat, cm−1）: 2367 2.3.4 Typical procedure for the preparation of phosphatidylethanolamine-N-amino acids （5a-e） 2.3.4.1 Phosphatidylethanolamine-N-glycine （5a） Trifluoroacetic acid（1％ of wt of substrate）was added drop wise to a stirred solution of phosphatidylethanolamine-N-protected glycine（4a, 0.272 g, 0.3 mmol）in difor 5 min at 0℃. The contents were chloromethane（10 mL） stirred for 3 h at 0℃. The course of the reaction was monitored by TLC using solvent system chloroform: methanol: water （65:25:4, v/v/v）till the appearance of pink colour with ninhydrin spray. At the end of reaction, the product was taken in dichloromethane and washed with saturated aqueous sodium bicarbonate solution and passed through sodium sulphate. The solvent was evaporated under rotary evaporator. The crude product was purified by silica gel column chromatography using chloroform: methanol （85:15 v/v）as eluent to obtain the title product 5b（0.177 g, 75％ yield）. 1 H-NMR（CDCl3, 500 MHz）: δ 8.09（m, 1H, CH2-NH-CO）, 5.39（ m, CH＝CH）, 5.08（ m, 2H, NH2-CH 2）, 4.41（ m, 1H, CH2-CH-CH2）, 4.30（m, CH-CH2-OCO）, 4.12（m, 2H, CHCH2-OPO）, 3.92（m, 2H, CH2-CH2-OPO）, 3.78（m, 2H, CH2NH2）, 3.45（m, 2H, CH2-NH-CO）, 2.35（m, CH2-COO）, 2.01 （m, CH2-CH＝CH-CH2）, 1.58（m, CH2-CH2-COO）, 1.24（m, （m, CH2-CH3） . CH2-CH2-CH2）, 0.89 （-NH2） , 2361 （2°amine） . IR （neat, cm−1）: 3328 ESI-MS: 774（M＋1） , 796 （M＋Na） , 802 （M＋1） , 824（M＋ Na）. 1
2.3.4.2 Phosphatidylethanolamine-N-valine（5b） Phosphatidylethanolamine-N-valine（5b, 0.161 g, 65％ yield）was prepared from 4b by the following procedure as described in 2.3.4.1. 1 H-NMR（CDCl3, 500 MHz）: δ 8.69（m, 2H, NH2-CH）, 7.98 , 5.39（m, CH＝CH） , 4.62（m, 1H, CH2（m, 1H, CH2-NH-CO） , 4.38 （m, 2H, CH-CH2-OCO） , 4.19 （m, 2H, CH-CH2CH-CH2） OPO）, 4.01（m, 2H, CH2-CH2-OPO）, 3.51（m, 2H, CH2-NH, 2.39（m, CH2-COO, CH （CH3） , CO） , 3.40 （m, 1H, CH-NH2） 2） 2.09（m, CH2-CH＝CH-CH2）, 1.70（m, CH2-CH2-COO）, 1.38 , 0.82（m, CH2-CH3, CH-CH3） . （m, CH2-CH2-CH2） : 3331（-NH2） , 2362（2°amine）. IR（neat, cm−1） ESI-MS: 816（M＋1） , 838（M＋Na）, 844（M＋1） , 866（M＋ Na）. 2.3.4.3 Phosphatidylethanolamine-N-leucine（5c） Phosphatidylethanolamine-N-leucine（5c, 0.163 g, 65％ yield）was prepared from 4c by the following procedure as described in 2.3.4.1. 1 H-NMR（CDCl3, 500 MHz）: δ 8.70（m, 2H, NH2-CH）, 8.05 , 5.22（m, CH＝CH） , 4.56（m, 1H, CH2（m, 1H, CH2-NH-CO） , 4.41 （m, 2H, CH-CH2-OCO） , 4.27 （m, 2H, CH-CH2CH-CH2） OPO）, 4.08（m, 2H, CH2-CH2-OPO）, 3.60（m, 2H, CH2-NHCO）, 3.48（m, 1H, CH-NH2）, 2.28（m, CH2-COO,）, 2.09（m, CH2-CH＝CH-CH2）, 1.71（m, 1H, CH（CH3）2）, 1.60（m, CH2, 0.88（m, CH2-COO）, 1.23（m, CH2-CH2-CH2, CH2-CH（CH3） 2） . CH2-CH3, CH-CH3） : 3328（-NH2） , 2363（2°amine）. IR （neat, cm−1） ESI-MS: 830（M＋1） , 852（M＋Na）, 858（M＋1） , 880（M＋ Na） . 2.3.4.4 Phosphatidylethanolamine-N-isoleucine（5d） Phosphatidylethanolamine-N- isoleucine（5d, 0.156 g, 60％ yield）was prepared from 4d by the following procedure as described in 2.3.4.1. 1 H-NMR（CDCl3, 500 MHz）: δ 8.70（m, 2H, NH2-CH）, 8.08 , 5.19（m, CH＝CH） , 4.40（m, 1H, CH2（m, 1H, CH2-NH-CO） , 4.29 （m, 2H, CH-CH2-OCO） , 4.13 （m, 2H, CH-CH2CH-CH2） OPO）, 3.91（m, 2H, CH2-CH2-OPO）, 3.41（m, 3H, CH-NH2, CH2-NH-CO）, 2.30（m, CH2-COO,）, 2.01（m, CH2-CH＝CH（C2H5））, 1.30 CH2-）, 1.59（m, CH2-CH2-COO, CH-CH（CH3） （m, CH2-CH2-CH2）, 1.18（d, 3H, CH-CH3）, 0.89（ m, CH2. CH3） : 3330（-NH2） , 2366（2°amine）. IR （neat, cm−1） ESI-MS: 830（M＋1） , 852（M＋Na）, 858（M＋1） , 880（M＋ Na）. 2.3.4.5 Phosphatidylethanolamine-N-phenylalanine（5e） Phosphatidylethanolamine-N-isoleucine（ 5e, 0.162 g, 62％ yield）was prepared from 4e by the following procedure as described in 2.3.4.1. 1 H-NMR（CDCl3, 500 MHz）: δ 8.71（m, 2H, NH2-CH）, 8.09 , （m, 1H, CH2-NH-CO）, 7.27-41（m, 5H, Aromatic protons） , 4.41（m, 2H, 5.39（m, CH＝CH） , 4.60（m, 1H, CH2-CH-CH2） CH-CH2-OCO）, 4.28（m, 2H, CH-CH2-OPO）, 3.99（m, 2H, , 3.50 （m, 2H, CH2-NH-CO） , 3.31 （d, CH2-CH2-OPO, CH-NH2） 2H, CH2-Ar）, 2.30（m, CH2-COO,）, 2.01（m, CH2-CH＝CH-
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Synthesis and Anti-microbial Activity of Novel Phosphatidylethanolamine-N-amino Acid Derivatives
CH2）, 1.60 （m, CH2-CH2-COO） , 1.25 （m, CH2-CH2-CH2） , 0.89 （m, CH2-CH3）. （-NH2） , 2361 （2°amine） . IR（neat, cm−1）: 3330 ESI-MS: 864（M＋1） , 886 （M＋Na） , 892 （M＋1） , 914（M＋ Na）. 2.3.5 Biological activity 2.3.5.1 Anti-bacterial activity The minimum inhibitory concentration（MIC）of the various evaluated compounds were tested against two representative gram-positive organisms, viz. Bacillus subtilis （MTCC-441）, Staphylococcus aureus（ MTCC-96）, and gram-negative organisms, viz. Escherichia coli（MTCC443）, Pseudomonas aeruginosa（MTCC- 741）by the well diffusion method recommended by the National Committee for Clinical Laboratory（NCCL）standards22）. Three to five identical colonies from each agar plate were lifted with a sterile wire loop and transferred into a tube containing 5 ml of nutrient agar. The turbidity of each bacterial suspension was adjusted to reach an optical comparison to that of a 0.5 McFarland standard; resulting in a suspension containing approximately 1 to 2×108 CFU/mL. Nutrient agar plates were inoculated by streaking the swab over the entire sterile agar surface. This procedure was repeated by streaking 2 more times, rotating the plate approximately 60°each time to ensure even distribution of the inoculums. As a final step, the rim of the agar was also swabbed. After allowing the inoculums to dry at room temperature, 6-mmdiameter wells were bored in the agar. Each extract was checked for antibacterial activity by introducing 50 µL of a 100 mg/mL concentration into duplicate wells. The plates were allowed to stand at room temperature for 1 h for extract to diffuse into the agar and then they were incubated at 37℃ for 24 h. Subsequently, the plates were examined for bacterial growth inhibition and the inhibition zone diameter（IZD）measured to the nearest millimeter. Streptomycin was used as positive controls and dimethylsulfoxide （DMSO） as a negative control for antibacterial activity. 2.3.5.2 Anti-fungal Activity The anti-fungal activities were assayed against four fungal strains, namely Candida albicans（MTCC-227）, Saccharomyces cerevisiae（MTCC-36）and Aspergillus niger（MTCC-282）, by the agar-well diffusion method23）. Cultures of test organisms were maintained on potato dextrose agar（PDA）slants and subcultured on petri dishes before examining for antifungal activity employing the agar cup bioassay method. A commercially prepared PDA medium（composition: potato infusion, 200 g; dextrose, 20 g; agar, 15 g）was used in this evaluation study. The PDA medium（39.0 g）was suspended in distilled water（ 1000 mL）and heated to boiling until it dissolved completely （pH 5.6）. The medium and the Petri dishes were autoclaved at 120℃ at a pressure of 15 psi, for 20 min. The medium was poured into sterile petri dishes under aseptic conditions in
a laminar flow chamber. When the medium in the plates had solidified, 0.5 mL of the test culture was inoculated and uniformly spread over the agar surface. Solutions were prepared at 150 µg/mL concentration by dissolving the test compounds in chloroform. After inoculation, cup was scooped out with a 6-mm sterile cork borer and the lids of the dishes were replaced. The test solution in 150 µg/mL concentration was added to cup. The controlled cup was treated at 27℃ for 48 h. The diameters（mm）of the inhibition zones were then measured. Amphotericin B was taken as positive controls and DMSO as negative control. Three replicates were maintained for each treatment.
3 Results and discussion Several studies on modified phosphatidylethanolamine （PE）particularly at –NH2 group（N-acyl, N-methyl amino, N,N-dimethylamino etc）revealed that these compounds exhibited significant biological activities24−28）. Hence, there is a large potential to exploit this area by derivatizing –NH2 group of PE with biologically important L-amino acids. In the present study, five L-amino acids namely L-glycine, Lvaline, L-leucine, L-isoleucine and L-phenylalnine（1a-e） were reacted with PE to obtain PE-N-amino acids employing a novel three step procedure. The method involves i） Nprotection of L-amino acid with BOC anhydride in alkaline medium ii）condensation of –CO2H group of N-protected amino acid with free –NH2 of PE by a peptide linkage and iii）deprotection of N- protected group of amino acids to obtain PE-N-amino acids. The L-amino acids were protected using ditertiarybutyldicarbonate（BOC anhydride）in the presence of 1 N NaOH solution to get the corresponding N-protected amino acids （2a-e）with excellent yields（98-99％）. N-Protected amino acids were characterized by 1H NMR（2a）which showed disappearance of primary amine proton and a sharp peak at 1.49 δ accounting for 9 protons of the 3 methyl groups of , an amide bond formed during the proBOC （-O-C（CH3）3） tection of free –NH2 of amino acids. A multiplet at 4.12 δ accounted for two protons of methylene group attached to -NH-BOC, a multiplet at 6.82 δ accounting for one proton attached to nitrogen（-NH-BOC）and an acidic proton as a broad singlet at 7.71 δ was observed. PE isolated from egg yolk lecithin with 99％ purity was coupled with N-protected amino acids in presence of DCC in chloroform to obtain PE-N-protected amino acids （4a-e） . The disappearance of the pink color spot of PE with the ninhydrin spray indicated that the –NH2 group of PE is linked to BOC-protected amino acid. The by-product（Dicyclohexylurea）formed during the reaction was separated by filtration after dissolving the product in hexane. The crude product was subjected to silica gel column chromatography using chloroform:methanol （90:10, v/v）as eluent to obtain 709
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Scheme Synthesis of phosphatidylethanolamine-N-amino acid derivatives. the pure product in 93-96％ yield. PE-N-protected amino acids （4a-e）were characterized by 1H NMR and IR. The 1H NMR data of 4a-e showed a singlet peak at 1.39 δ account（CH3）3） , ing for 9 protons of 3 methyl groups of BOC （-O-Ca multiplet at 3.78 δ accounting for 2 protons of methylene , a multiplet at 4.13 δ acgroup of glycine （NH-CO-CH2-NH） counting for two protons of methylene group attached to phosphate moiety（ P-O）and other characteristic peaks related to fatty acids and glycerol back bone of PE were observed. Deprotection of BOC protected amino acids of PE was carried out using trifluoroacetic acid to obtain PE-N-amino acids （5a-e）with good yields in 60-75％. 5a-e were characterized by 1H NMR and IR. The 1H NMR of 5a-e showed a multiplet at 3.78 δ accounting for 2 protons of methylene adjacent to NH2 and a multiplet at 3.45 δ accounting for 2 protons of methylene adjacent to amide nitrogen was found. A multiplet at 5.08 δ accounting for 2 proton of primary amine group and a broad singlet at 8.09 δ accounting for one proton of amide nitrogen group and other characteristic signals related to PE were observed. 3.1 Biological evaluation PE-N-amino acids（5a-e）prepared in the present study were evaluated for their anti-microbial activity, i.e. antibacterial and anti-fungal activities. The anti-bacterial activity was evaluated against two gram-positive bacteria namely Bacillus subtilis, Staphylococcus aureus and two gram-negative bacteria namely Escherichia coli, Pseudomonas aeruginosa. Anti-fungal activity was tested against three fungal stains namely Saccharomyces cerevisiae, Aspergillus niger and Candida albicans. Controls were maintained with DMSO and streptomycin（150 μg/mL）and
. amphotericin-B （150 μg/mL） 3.1.1 Anti-bacterial activity Anti-bacterial activity was assayed by well diffusion method. The zone of inhibition was observed after 24 h of incubation and the diameter of the zone was measured. The MIC values of PE-N-amino acid analogues are tabulated in Table 1. It was interesting to find that amino acid derivatives（5a-e）exhibited about three folds much superior activities compared to streptomycin as reference compound against the tested Gram-positive and Gram-negative bacteria. This further revealed that lipopeptide functionalities play a very important role in the activity compared to the amido glucoside functionality present in streptomycin. Further the series 5a and 5d showed extraordinary activities against Gram-positive bacterial strain, B. subtilis compared to other analogues of PE and Gram-negative bacterial strains. Also it was observed that shift from non-polar aliphatic functionalities as seen in 5a-d to a non-polar aromatic functionality（5e）increased the MIC values. This can be explained that decrease in basicity of amino group decreased the activity. The observation was similar to that reported by Lee et al.29）on ethambutol derivatives. Hence, it can be concluded that the presence of ethanolamine as in PE and non-polar aliphatic chain as seen in 5a-d were crucial for anti-bacterial activity against Gram-positive bacteria. The mode of action of anti-microbial activity of these PE-N-amino acid derivatives could be explained similar to the model described by Straus et al.30）for hydrophilic peptides. According to their model, the molecules upon interaction into the bacterial membrane adopt a three-dimensional structure. They fold into amphiphilic molecules with the induction of positive membrane curvature interacting directly with the lipid head groups. The bacterial cells are
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Table 1 Anti-bacterial activity of synthesized compounds 5a-e. Test Compounds 5a 5b 5c 5d 5e Streptomycin
*Gram +ve (μg/mL) B. subtilis S. aureus 8 12 12 14 10 20 7 18 22 27 24 30
*Gram –ve (μg/mL) P. aeruginosa E. coli 14 11 10 10 11 12 16 12 23 24 31 33
Inhibitory zone diameters are in mm, Concentration, μg/mL, Negative Control (DMSO). DMSO did not show any activity. *MIC values reported are an average of three triplicate analysis.
Table 2 Anti-fungal activities of synthesized compounds 5a-e. Test Compounds 5a 5b 5c 5d 5e Amphotericin-B
Yeast (μg/mL) C. albicans 99 115 96 110 88 23.5
Filamentous Fungi (μg/mL) S. cervisiae A. niger − − − 88 − 66 − − 40 110 22 25
Inhibitory zone diameters are in mm; Concentration, μg/mL. Negative Control (DMSO). DMSO did not show any activity. finally killed either by membrane perforation or some other membrane-associated event. 3.1.2 Anti-fungal activity 5a showed good activity against yeast, C. albicans while no activity against other two fungal species. 5b showed moderate activity against yeast, C. albicans and good activity against filamentous fungi, Aspergillus niger. 5c showed good activity against C. albicans and, Aspergillus niger while 5d showed moderate activity against only C. albicans. 5e showed good activity against yeast, C. albicans and filamentous fungi, S. cerevisiae and moderate activity against Aspergillus niger. It was observed that upon derivatization of free –NH2 in PE, the compounds 5a-d did not exhibit any activity against filamentous fungi, S. cervisiae. However, the aromatic amino acid PE derivative, 5e showed good activity against S. cervisiae and moderate activity against Aspergillus niger. Compounds 5a-d exhibited moderate activity against C. albicans and no activity against the filamentous fungi. Presence of aromatic group helped compound 5e to exhibit moderate ac. tivity against both the yeast and fungi （Table 2）
4 Conclusion A simple and novel method was developed for the preparation of phosphatidylethanolamine-N-amino acid deriva-
tives using L-glycine, L-leucine, L-valine, L-isoleucine and L-phenylalanine. All the amino acid derivatives 5（ a-e） when evaluated for anti-bacterial exhibited extraordinary to moderate anti-bacterial activity compared to streptomycin as reference compound. While, moderate to poor antifungal activity compared to amphotericin-B as reference compound.
Acknowledgements The author T.Vijeetha acknowledges the financial support from D B T, New Delhi, India.
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