Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 1152–1157
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Pseudo-peptides as novel antileptospiral agents: Synthesis and spectral characterization Chandan Shivamallu a, Umesha Sharanaiah a,⇑, Shiva Prasad Kollur b, Naveen Kumar R. Mallesh c, Revanasiddappa D. Hosakere c, V. Balamurugan d a
Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India PG Department of Chemistry, JSS College, Ooty Road, Mysore 570 006, Karnataka, India Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India d Project Directorate on Animal Disease Monitoring and Surveillance (PD_ADMAS), ICAR, Hebbal, Bangalore 560 024, Karnataka, India b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Syntheses of pseudo-peptides were
done. Synthesized compounds were
characterized by spectral analysis. Bacterial strains and inoculums were
prepared. Bacterial strains were confirmed by
Polymerase Chain Reaction. The characterized pseudo-peptide
compounds have been tested in vitro for biological effects.
a r t i c l e
i n f o
Article history: Received 31 July 2013 Received in revised form 8 September 2013 Accepted 26 September 2013 Available online 8 October 2013 Keywords: Pseudo-peptide Spectroscopic technique Minimum inhibitory concentration Anti-leptospiral activity
a b s t r a c t In this paper, we describe the synthesis of novel class of pseudo-peptides derived by coupling an amino acid with a heterocyclic moiety containing free amine group using suitable coupling agents. The synthesized compounds were characterized using spectral (1H NMR, 13C NMR and MS) techniques. Preliminary pharmacological assays for Leptospirosis were studied by test tube dilution (TDT) and micro dilution technique (MDT). In particular, all the analyses led to the conclusion that the synthesized compound inhibiting the Leptospira a causal organism of Leptospirosis. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Leptospira are highly susceptible to a wide variety of antimicrobial agents in vitro. It is not yet clear what the best choice of antimicrobial agents can be given to human. Transmission occurs by ⇑ Corresponding author. Tel.: +91 0821 2419884. E-mail addresses: [email protected], (U. Sharanaiah).
[email protected]
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.09.105
contact of abraded skin, conjunctiva, or mucous membranes with water contaminated by urine of infected rodents. Infection can occur after animal bites or, very rarely between humans via infected blood or urine. Rodents consider being the reservoir of the infection [1]. Based on the best available literature, currently penicillin, azithromycin, doxycyclin, benzyl penicillin, among this penicillin considered as the best of choice. Another option for treating Leptospirosis is the fluoroquinolone antimicrobial, but when this under human trial the use of this chemical is not fully supportive [2].
C. Shivamallu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 1152–1157
O
O HN
H2N
C
N H
N
HN
H2N
S
C
N H
N
OCH3
N OCH3
4b 4a O HN
H2N
C
N H
N
OH
N OH
4c Fig. 1. Structure of synthesized pseudo peptides.
Thus, the problem of increasing microbial resistant to current antibiotics straight away demanded the development of novel antimicrobial agents [3]. Peptides are among the most versatile bioactive molecules, yet they do not make good drugs, because they are quickly degraded or modified in the body. A number of studies indicated that most of antibacterial peptides exerted their activities by enhancing the permeability of pathogenic cell membranes and this kind of antibacterial peptides may be difficult to induce resistant strains of pathogens compared to classic antibacterial agents [4]. Over the past decade or so, many defense antimicrobial peptides to counter infection by microbe were isolated and characterized from a variety of natural sources [5]. However, despite the high potentials, there are still some limitations for peptides as drugs. Major disadvantages are short half-life, rapid metabolism and poor bioavailability. Nevertheless, pharmacokinetic properties of peptides can be improved by different types of modifications [6]. Peptidomimetic modifications or cyclization of linear peptides are frequently used as attractive methods to provide more conformationally constrained and thus more stable bioactive peptides [7,8].
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Taking into consideration various peptidomimetic approaches used for the design and synthesis of peptide analogs with improved pharmacological properties pseudo-peptides or peptide bond surrogates, in which peptide bonds have been replaced with other chemical groups, are especially attractive. This is mainly because such approaches create an amide bond surrogate with defined three dimensional structures similar to those of natural peptides, yet with significant differences in polarity, hydrogen bonding capability and acid-base character. Also important, the structural and stereochemical integrities of the adjacent pair of a-carbon atoms in these pseudo-peptides are unchanged. The high synthetic accessibility and the ease with which chemical functionality can be introduced illustrate the high potential of artificial pseudopeptides. In addition, their high bio stability has enabled successful applications in both in vitro and in vivo studies. It has been recently shown that pseudo-peptides constructed from pyridine and tryptophan units, were tested against the Gram-positive, Gram-negative strains of bacteria and human pathogenic fungi and proved to be a broad-spectrum antimicrobial agent, showing a significant inhibition of the growth of microorganisms [9]. Thus, we became interested in synthesizing pseudo peptides, as for our knowledge this is the first work reporting on biological activity of pseudo-peptides against Leptospira. In this article, we have synthesized and evaluated pseudo-peptides of tryptophan. In addition, we investigated anti-leptospiral activity. Experimental Materials and methods L-trypophan, 2-aminobenzothiazole,4,6-dimethoxypyrimidin2-amine and 2-aminopyrimidin-4,6-diol was obtained from Sigma Aldrich. Solvents were purified by standard procedures and were freshly distilled prior to use. Liquid state NMR spectra were recorded in CDCl3 as a solvent. 1H and 13C NMR spectra were obtained on a Bruker Avance 400 NMR spectrometer. Chemical shifts are quoted with respect to SiMe4 as internal standard. Mass spectral studies were carried out on ESI-MS micro mass
Fig. 2. 1H NMR spectra of 4a and 4b.
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spectrometer (Waters, USA). Thin-layer chromatography analyses were carried out on pre-coated silica gel plates (Merck, USA) and spots were visualized by UV irradiation. Column chromatography was performed on glass columns loaded with silica gel or on automated flash chromatography system (Biotage, Germany) by using pre-loaded silica cartridges.
Chemistry General procedure Synthesis of compound 1 To a solution of L-tryptophan (1.0 g, 4.89 mM) in 9:1 water and 1,4-dioxane was added an aqueous solution of sodium hydroxide (1 N) dropwise at 0 °C with continuous stirring. To the above cooled solution, Boc anhydride (1.60, 7.33 mM) was added slowly
as a solution in 1,4-dioxane. The resulting mixture was stirred at 0 °C for 1 h and then at room temperature overnight. The product was extracted with ethyl acetate, and the organic layer was concentrated and dried under vacuum to give the expected Boc-tryptophan as colorless solid.
Synthesis of compound 3 To a solution of compound 1 (0.85 g, 2.79 mM) in chloroform was added triethyl amine (2 equivalents) and stirred for 10 min. The above solution was cooled (0 °C) and then EDC.HCl (0.80 g, 4.19 mM) was added. The mixture was stirred at this temperature for 20 min and then added HOBt (0.56 g, 4.19 mM). After stirring for 20 min, solution of compound 2 in chloroform was added dropwise via syringe. The resulting mixture was stirred at room temperature for an additional 10 h. The mixture was quenched with cold water and neutralized with 1 N HCl. The aqueous mixture
(a)
(b)
Fig. 3. Mass spectra of 4a and 4b.
C. Shivamallu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 1152–1157
was extracted with chloroform, and dried over Na2SO4. Removal of solvent afforded a yellow solid. Synthesis of compound 4 To a solution of compound 3 (0.75 g, 1.71 mM) in dichloromethane was added trifluoro acetic acid (0.52 mL, 6.84 mM). The mixture was stirred at room temperature for 4 h. The progress of reaction was monitored by TLC. After the completion of reaction, trifluoro acetic acid was removed under vacuum. The structures of the synthesized pseudo-peptides are shown in Fig. 1. Biology Bacterial strain and inoculum preparation The antimicrobial activity was carried against Leptospira. The following species of Leptospira were used: L. icterohaemorrhagiae, L. canicola, L. pomona, L. autumnalis, L. javanica, L. pyrogens, L. australis and L. hardjo. All the strains were procured from the repository of Project Directorate on Animal Disease Monitoring and Surveillance (PD_ADMAS), ICAR, Hebbal, Bangalore, India. All the above mentioned Leptospiral strains were grown at 30 °C in EMJH medium, their cultural characteristics and morphological features were confirmed and also subjected to standard molecular techniques before experimentation. DNA preparation The DNA was extracted from the retrieved culture after confirming the culture is positive for Leptospira using QIAgen DNA isolation kit (QIAgen, Germany). The DNA extraction was carried out according to the manufacturer’s instructions. The DNA was quantified using Nanodrop DNA quantifier and stores at 80 °C until further processed. Primers and PCR assay PCR was performed on the obtained DNA template using reported primer pair (rpoB-F-CCTCATGGGTTCCAACATGCA and rpoB-R-CGCATCCTCRAAGTTGTAWCCTT) [10]. Briefly, PCR was performed by initial denaturation at 94 °C for 3 min. Subsequent PCR amplification conducted with 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min, extension at 72 °C for 1 min followed by final extension for 20 min at 72 °C. 5 mL aliquot of each PCR product was mixed with 3 ll of 6 loading dye (Fermentas, USA) and loaded onto the wells of 2% agarose gel in TAE buffer. It was run at 80 V for about 45 min. DNA bands were visualized under UV Transilluminator and documented using gel documentation system with quantity one software (Biorad, USA).
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under dark field microscope by placing a loopful of suspension on the clear glass slide and observed under different magnifications of the dark field microscope (Zeiss, USA) [11,12]. The total viable counts per microscopic field were compared with the positive control samples and images were captured. Experiments were carried out in triplicate and repeated for three times. In micro dilution technique, the viable leptospiral species were taken in all the wells of micro titer plate (Nalgene, USA) and the different concentrations of synthesized compounds 4a, 4b and 4c (5 lL in each well) were mixed in all the wells. This culture and compound mixture was well mixed and the microtitre plate was covered with fresh aluminum foil and incubated under dark condition for 30 min at room temperature. After incubation period, the samples were spread on a slide by micro diluter and observed under dark field microscope (Zeiss, USA) to study the inhibitory activity on Leptospira species. Experiments were carried out in triplicate repeated for three times. In both of these techniques, percentage of leptospiral inhibition was calculated and tabulated. Results and discussion The general route for the synthesis of pseudo peptide is shown in Scheme 1. To validate the chemically synthesized pseudo peptides, 1H, 13C NMR and mass of the compounds were recorded and as follows, 2-amino-N-(benzo[d]thiazol-2-yl)-3-(1H-indol-3-yl) propanamide (4a) Yield-58%, 1H NMR: (CDCl3, 400 MHz): dH 2.8–3.0 (2H, dd, CH2), 4.1 (1H, t, CH), 5.7 ((2H, s, NH2), 6.9 (1H, d, ArAH), 7.0 (1H, d, ArAH), 7.4 (3H, m, ArAH), 7.6 (1H, d, ArAH), 7.7 (1H, d, ArAH), 7.8 (1H, d, ArAH), 8.0 (1H, d, ArAH), 10.9 (1H, s, NH). 13C NMR: (CDCl3, 400 MHz): dC 173, 170, 158, 138, 128, 125, 122, 119, 118, 111, 108, 68, 59,53, 51, 28, 19. ESI-MS: m/z 337 (M+H)+ and m/z 375 (M+K)+. NMR studies 1
H NMR spectra of compounds were recorded in CDCl3 using Bruker-400 MHz instrument. The representative spectra of compounds 4a and 4b are given in Fig. 2. The methylene protons of C-8 appeared at d2.8–3.9 ppm as doublet. The methynic proton of C-7 at d 3.8–4.1 ppm appeared as triplet due to vicinal coupling with two protons of methylene group. A singlet appeared at d 3.8 ppm in compound 4b is due to AOCH3 moiety. A singlet due to ANH2 group in compounds 4a and 4c appeared at d
In vitro assay: Tube dilution technique and micro dilution technique The tube dilution technique was carried out by adding various concentrations of synthesized compounds 4a, 4b and 4c in the Ellinghausen, McCullough, Jensen and Harris (EMJH) liquid medium [10]. The EMJH (Difco, USA) modified, semi-solid medium was prepared with the addition of rabbit serum (15%) (Sigma, USA) and enriched with L-asparagin (3%), calcium chloride (1%), magnesium chloride (1%), pyruvate sodium (1%) and 0.2% agar with the addition of 5-fluoruracil (300 mg/L) named as the selective medium. After sterility checking of the medium by placing in the room temperature for 48 h, the Leptospira species were inoculated with syringe filter. The tubes were incubated at room temperature for 7 days. The inhibition patterns for each species in different concentrations of synthetic compounds were observed
600 bp 500 bp
100 bp
Fig. 4. Agarose gel electrophoretogram showing amplification for rpoB gene of Leptospira species. Analysis products amplified Lanes 1 to 9-PCR product of Leptospira samples, Lane M-100 bp gene ruler.
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Note: Inhibition rate of MDT and TDT of various extracts of biologically active compounds against selective species of Leptospira species MDT is micro dilution technique and TDT tube dilution technique.
100 100 80 100 – – 100 100 L. hardjo
MDT TDT
100 100
100 80
100 90
100 60
100 100
100 80
90 70
70 70
20 –
20 20
100 100
100 80
90 70
70 70
50 50
100 100 – 20 – – – – L. australis
MDT TDT
20 –
60 100
80 100
80 100
20 20
40 80
– –
– –
– 20
– –
20 20
40 80
60 60
40 40
– –
100 100 – 40 – – – 80 L. pyrogens
MDT TDT
– –
– –
– –
20 40
– –
– –
– 20
– 20
– 40
– 50
– –
– –
60 800
40 20
– 50
100 100 60 40 40 20 100 100 100 100 L. javanica
MDT TDT
80 80
100 100
100 100
100 100
80 80
100 100
20 20
40 20
60 40
60 40
80 80
100 100
80 80
60 40
100 100 80 80 100 80 70 – – – L. autumnalis
MDT TDT
10 –
20 –
60 –
80 20
10 –
20 –
– 60
– 80
20 80
– –
10 80
20 80
80 60
40 80
100 100 100 100 100 100 100 100 100 80 L. pomona
MDT TDT
100 80
100 100
100 100
100 100
100 80
100 80
100 100
100 100
100 100
70 80
100 80
100 80
100 80
70 80
100 100 100 100 100 100 100 100 100 100 L. canicola
MDT TDT
100 80
100 100
100 100
100 100
100 80
100 100
100 100
100 100
100 100
100 100
100 80
100 100
100 100
100 100
30
100 100 100 100 100 100 100 100 100 100
150 125 100 75 50
100 100 100 80
25 150
100 100 100 100
125 100
100 100 100 100
75 50
100 100 100 80 70 80 70 80
25 150 125 100
70 80 80 100 100 100 100 80
75 50 25
MDT TDT
4c 4b 4a
Inhibition rate percentage in various concentration Technique Name of the serogroup
Table 1 Effect of various concentrations of biologically active compounds against selective species of Leptospira by MDT and TDT technique.
L. icterohaemorrhagiae
Standard benzyl penicillin
5.5–5.7 ppm. The aromatic protons of C-16 in 4b appeared as singlet at d 6.2 ppm. In all the compounds the aromatic protons appeared as multiplet in the region d 6.9–8.0 ppm. The representative spectra of 13C NMR spectra of 4a and 4b shows peaks at 173, 172, 170 and 169 indicates the presence of carbonyl group (C@O) and imine group (C@N). Mass spectral studies The mass spectra of compounds 4a and 4b are shown in Fig. 3. Molecular ion peak of compounds 4a and 4b were observed at m/z 375 and 341, respectively. A peak at m/z 337 in compound 4a and 341 in compound 4b represents (M+H)+. A peak at m/z 375 in compounds 4a is due to (M+K)+ and a peak at m/z 364 in 4b is due to (M+Na)+. Biology DNA extraction by QIAamp blood extraction kit (Qiagen, Germany) yielded a substantial amount of DNA without any protein contamination. The primers had sufficient sensitivity to detect DNA concentration of 1 fg and it is specifically amplified Leptospiral DNA (Fig. 4). Good amplifications were evident for PCR reaction carried out at the annealing temperature of 55 °C for 1 min. The 600 bp amplicon was obtained and observed under UV transilluminator light using BIORAD gel documentation unit (Fig. 4). The in vitro evaluation by tube and micro dilution techniques, the compound 4c showed efficient inhibitory activity. As observed by the percentage of dead cells of Leptospires when observed under dark field microscope. The compound 4c has shown best activity at 25, 50 and 75 lg/mL, whereas other two compounds also shown activity but, less when compared to 4c and the standard drug benzyl penicillin. The compound 4c at 50 and 75 lg/mL level showed significant inhibitory effect on all species of Leptospira, whereas compounds 4a and 4b has shown best activity at 75 lg/mL and also did not shown any inhibitory effect for L. autumnalis and L. pyrogen species. Therefore, among three biologically active pseudo-peptides the compound 4c showed significant and maximum inhibition for anti-leptospiral activity. The inhibitory percentage is as shown in Table 1. Conclusions We have established a series of pseudo-peptides against Leptospira, characterized them by spectroscopic studies and the exploitation of biologically active compound against Leptospira is investigated. From the NMR and the mass studies the secondary structure of the compound is studied. The compound 4c was the most effective inhibitor compound tested for MIC. However, further investigations should be carried out through modifications in its chemical structure in order to improve further to find out more biologically active molecule. Funding This work was supported by Indian Council of Medical Research (ICMR), New Delhi, India. (Sanction order No. 80/673/2010-ECD-I, Dated 03.05.2011). Acknowledgment The senior author (Chandan, S.) is greatly acknowledges the financial assistance from the Indian Council of Medical Research, New Delhi, India, in the form of Senior Research Fellowship. There
C. Shivamallu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 1152–1157
is no conflict of interest whatsoever among the author C.S. responsible for the bench work. B.V. and S.U supervised the whole experiment and S. U planned the whole experiment, data interpretation and writing of the manuscript. The authors would like to thank Project Director, PD_ADMAS, ICAR, Hebbal, Bangalore, Karnataka, India for providing reference Leptospira isolates. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2013.09.105. Reference [1] D. Luchini, F. Meacci, M.R. Oggioni, G. Morabito, V.D. Amato, M. Gabbrielli, G. Pozzi, Int. J. Legal Med. 122 (2008) 229–233.
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[2] M.E. Griffith, D.R. Hospenthal, C.K. Morray, Curr. Opin. Infect. Dis. 19 (6) (2006) 533–537. [3] J. Travis, Science 264 (1994) 360–362. [4] K. Park, S.Y. Shin, K. Hahm, Y. Kim, Bull. Korean Chem. Soc. 24 (2003) 1478– 1484. [5] M. Zasloff, Proc. Natl. Acad. Sci. 84 (1987) 5449–5453. [6] C. Adessi, C. Soto, Curr. Med. Chem. 9 (2002) 963–978. [7] J.S. Davies, J. Pept. Sci. 9 (2003) 471–501. [8] J.M. Ahn, N.A. Boyle, M.T. MacDonald, K.D. Janda, Mini Rev. Med. Chem. 2 (2002) 463–473. [9] L. Jian, Y. Liang, L. Tingting, W. Yongmei, Bioorg. Med. Chem. Lett. 17 (2007) 1601–1607. [10] L.S. Bernard, T.B. Lan, B. Guy, A. Khamis, R. Didier, FEMS Microbiol. Lett. 263 (2006) 142–147. [11] L.M. Mathew, Comparative analysis of acetone, in: Water and Saponified Extracts of Phyllanthus niruri Against Leptospira interrogans Serogroups In Vitro, Periyar University, Salem, Tamilnadu, India, 2001. [12] N. Prabhu, P.I. Joseph, P. Chinnaswamy, K. Natarajaseenivasa, S. Lakshmi, J. Pharm. Sci. 70 (6) (2008) 788–791.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Pseudo-peptides as novel antileptospiral agents: Synthesis and spectral characterization Chandan Shivamallu a, Umesha Sharanaiah a,⇑, Shiva Prasad Kollur b, Naveen Kumar R. Mallesh c, Revanasiddappa D. Hosakere c, V. Balamurugan d a
Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India PG Department of Chemistry, JSS College, Ooty Road, Mysore 570 006, Karnataka, India Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India d Project Directorate on Animal Disease Monitoring and Surveillance (PD_ADMAS), ICAR, Hebbal, Bangalore 560 024, Karnataka, India b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Syntheses of pseudo-peptides were
done. Synthesized compounds were
characterized by spectral analysis. Bacterial strains and inoculums were
prepared. Bacterial strains were confirmed by
Polymerase Chain Reaction. The characterized pseudo-peptide
compounds have been tested in vitro for biological effects.
a r t i c l e
i n f o
Article history: Received 31 July 2013 Received in revised form 8 September 2013 Accepted 26 September 2013 Available online 8 October 2013 Keywords: Pseudo-peptide Spectroscopic technique Minimum inhibitory concentration Anti-leptospiral activity
a b s t r a c t In this paper, we describe the synthesis of novel class of pseudo-peptides derived by coupling an amino acid with a heterocyclic moiety containing free amine group using suitable coupling agents. The synthesized compounds were characterized using spectral (1H NMR, 13C NMR and MS) techniques. Preliminary pharmacological assays for Leptospirosis were studied by test tube dilution (TDT) and micro dilution technique (MDT). In particular, all the analyses led to the conclusion that the synthesized compound inhibiting the Leptospira a causal organism of Leptospirosis. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Leptospira are highly susceptible to a wide variety of antimicrobial agents in vitro. It is not yet clear what the best choice of antimicrobial agents can be given to human. Transmission occurs by ⇑ Corresponding author. Tel.: +91 0821 2419884. E-mail addresses: [email protected], (U. Sharanaiah).
[email protected]
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.09.105
contact of abraded skin, conjunctiva, or mucous membranes with water contaminated by urine of infected rodents. Infection can occur after animal bites or, very rarely between humans via infected blood or urine. Rodents consider being the reservoir of the infection [1]. Based on the best available literature, currently penicillin, azithromycin, doxycyclin, benzyl penicillin, among this penicillin considered as the best of choice. Another option for treating Leptospirosis is the fluoroquinolone antimicrobial, but when this under human trial the use of this chemical is not fully supportive [2].
C. Shivamallu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 1152–1157
O
O HN
H2N
C
N H
N
HN
H2N
S
C
N H
N
OCH3
N OCH3
4b 4a O HN
H2N
C
N H
N
OH
N OH
4c Fig. 1. Structure of synthesized pseudo peptides.
Thus, the problem of increasing microbial resistant to current antibiotics straight away demanded the development of novel antimicrobial agents [3]. Peptides are among the most versatile bioactive molecules, yet they do not make good drugs, because they are quickly degraded or modified in the body. A number of studies indicated that most of antibacterial peptides exerted their activities by enhancing the permeability of pathogenic cell membranes and this kind of antibacterial peptides may be difficult to induce resistant strains of pathogens compared to classic antibacterial agents [4]. Over the past decade or so, many defense antimicrobial peptides to counter infection by microbe were isolated and characterized from a variety of natural sources [5]. However, despite the high potentials, there are still some limitations for peptides as drugs. Major disadvantages are short half-life, rapid metabolism and poor bioavailability. Nevertheless, pharmacokinetic properties of peptides can be improved by different types of modifications [6]. Peptidomimetic modifications or cyclization of linear peptides are frequently used as attractive methods to provide more conformationally constrained and thus more stable bioactive peptides [7,8].
1153
Taking into consideration various peptidomimetic approaches used for the design and synthesis of peptide analogs with improved pharmacological properties pseudo-peptides or peptide bond surrogates, in which peptide bonds have been replaced with other chemical groups, are especially attractive. This is mainly because such approaches create an amide bond surrogate with defined three dimensional structures similar to those of natural peptides, yet with significant differences in polarity, hydrogen bonding capability and acid-base character. Also important, the structural and stereochemical integrities of the adjacent pair of a-carbon atoms in these pseudo-peptides are unchanged. The high synthetic accessibility and the ease with which chemical functionality can be introduced illustrate the high potential of artificial pseudopeptides. In addition, their high bio stability has enabled successful applications in both in vitro and in vivo studies. It has been recently shown that pseudo-peptides constructed from pyridine and tryptophan units, were tested against the Gram-positive, Gram-negative strains of bacteria and human pathogenic fungi and proved to be a broad-spectrum antimicrobial agent, showing a significant inhibition of the growth of microorganisms [9]. Thus, we became interested in synthesizing pseudo peptides, as for our knowledge this is the first work reporting on biological activity of pseudo-peptides against Leptospira. In this article, we have synthesized and evaluated pseudo-peptides of tryptophan. In addition, we investigated anti-leptospiral activity. Experimental Materials and methods L-trypophan, 2-aminobenzothiazole,4,6-dimethoxypyrimidin2-amine and 2-aminopyrimidin-4,6-diol was obtained from Sigma Aldrich. Solvents were purified by standard procedures and were freshly distilled prior to use. Liquid state NMR spectra were recorded in CDCl3 as a solvent. 1H and 13C NMR spectra were obtained on a Bruker Avance 400 NMR spectrometer. Chemical shifts are quoted with respect to SiMe4 as internal standard. Mass spectral studies were carried out on ESI-MS micro mass
Fig. 2. 1H NMR spectra of 4a and 4b.
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spectrometer (Waters, USA). Thin-layer chromatography analyses were carried out on pre-coated silica gel plates (Merck, USA) and spots were visualized by UV irradiation. Column chromatography was performed on glass columns loaded with silica gel or on automated flash chromatography system (Biotage, Germany) by using pre-loaded silica cartridges.
Chemistry General procedure Synthesis of compound 1 To a solution of L-tryptophan (1.0 g, 4.89 mM) in 9:1 water and 1,4-dioxane was added an aqueous solution of sodium hydroxide (1 N) dropwise at 0 °C with continuous stirring. To the above cooled solution, Boc anhydride (1.60, 7.33 mM) was added slowly
as a solution in 1,4-dioxane. The resulting mixture was stirred at 0 °C for 1 h and then at room temperature overnight. The product was extracted with ethyl acetate, and the organic layer was concentrated and dried under vacuum to give the expected Boc-tryptophan as colorless solid.
Synthesis of compound 3 To a solution of compound 1 (0.85 g, 2.79 mM) in chloroform was added triethyl amine (2 equivalents) and stirred for 10 min. The above solution was cooled (0 °C) and then EDC.HCl (0.80 g, 4.19 mM) was added. The mixture was stirred at this temperature for 20 min and then added HOBt (0.56 g, 4.19 mM). After stirring for 20 min, solution of compound 2 in chloroform was added dropwise via syringe. The resulting mixture was stirred at room temperature for an additional 10 h. The mixture was quenched with cold water and neutralized with 1 N HCl. The aqueous mixture
(a)
(b)
Fig. 3. Mass spectra of 4a and 4b.
C. Shivamallu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 1152–1157
was extracted with chloroform, and dried over Na2SO4. Removal of solvent afforded a yellow solid. Synthesis of compound 4 To a solution of compound 3 (0.75 g, 1.71 mM) in dichloromethane was added trifluoro acetic acid (0.52 mL, 6.84 mM). The mixture was stirred at room temperature for 4 h. The progress of reaction was monitored by TLC. After the completion of reaction, trifluoro acetic acid was removed under vacuum. The structures of the synthesized pseudo-peptides are shown in Fig. 1. Biology Bacterial strain and inoculum preparation The antimicrobial activity was carried against Leptospira. The following species of Leptospira were used: L. icterohaemorrhagiae, L. canicola, L. pomona, L. autumnalis, L. javanica, L. pyrogens, L. australis and L. hardjo. All the strains were procured from the repository of Project Directorate on Animal Disease Monitoring and Surveillance (PD_ADMAS), ICAR, Hebbal, Bangalore, India. All the above mentioned Leptospiral strains were grown at 30 °C in EMJH medium, their cultural characteristics and morphological features were confirmed and also subjected to standard molecular techniques before experimentation. DNA preparation The DNA was extracted from the retrieved culture after confirming the culture is positive for Leptospira using QIAgen DNA isolation kit (QIAgen, Germany). The DNA extraction was carried out according to the manufacturer’s instructions. The DNA was quantified using Nanodrop DNA quantifier and stores at 80 °C until further processed. Primers and PCR assay PCR was performed on the obtained DNA template using reported primer pair (rpoB-F-CCTCATGGGTTCCAACATGCA and rpoB-R-CGCATCCTCRAAGTTGTAWCCTT) [10]. Briefly, PCR was performed by initial denaturation at 94 °C for 3 min. Subsequent PCR amplification conducted with 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min, extension at 72 °C for 1 min followed by final extension for 20 min at 72 °C. 5 mL aliquot of each PCR product was mixed with 3 ll of 6 loading dye (Fermentas, USA) and loaded onto the wells of 2% agarose gel in TAE buffer. It was run at 80 V for about 45 min. DNA bands were visualized under UV Transilluminator and documented using gel documentation system with quantity one software (Biorad, USA).
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under dark field microscope by placing a loopful of suspension on the clear glass slide and observed under different magnifications of the dark field microscope (Zeiss, USA) [11,12]. The total viable counts per microscopic field were compared with the positive control samples and images were captured. Experiments were carried out in triplicate and repeated for three times. In micro dilution technique, the viable leptospiral species were taken in all the wells of micro titer plate (Nalgene, USA) and the different concentrations of synthesized compounds 4a, 4b and 4c (5 lL in each well) were mixed in all the wells. This culture and compound mixture was well mixed and the microtitre plate was covered with fresh aluminum foil and incubated under dark condition for 30 min at room temperature. After incubation period, the samples were spread on a slide by micro diluter and observed under dark field microscope (Zeiss, USA) to study the inhibitory activity on Leptospira species. Experiments were carried out in triplicate repeated for three times. In both of these techniques, percentage of leptospiral inhibition was calculated and tabulated. Results and discussion The general route for the synthesis of pseudo peptide is shown in Scheme 1. To validate the chemically synthesized pseudo peptides, 1H, 13C NMR and mass of the compounds were recorded and as follows, 2-amino-N-(benzo[d]thiazol-2-yl)-3-(1H-indol-3-yl) propanamide (4a) Yield-58%, 1H NMR: (CDCl3, 400 MHz): dH 2.8–3.0 (2H, dd, CH2), 4.1 (1H, t, CH), 5.7 ((2H, s, NH2), 6.9 (1H, d, ArAH), 7.0 (1H, d, ArAH), 7.4 (3H, m, ArAH), 7.6 (1H, d, ArAH), 7.7 (1H, d, ArAH), 7.8 (1H, d, ArAH), 8.0 (1H, d, ArAH), 10.9 (1H, s, NH). 13C NMR: (CDCl3, 400 MHz): dC 173, 170, 158, 138, 128, 125, 122, 119, 118, 111, 108, 68, 59,53, 51, 28, 19. ESI-MS: m/z 337 (M+H)+ and m/z 375 (M+K)+. NMR studies 1
H NMR spectra of compounds were recorded in CDCl3 using Bruker-400 MHz instrument. The representative spectra of compounds 4a and 4b are given in Fig. 2. The methylene protons of C-8 appeared at d2.8–3.9 ppm as doublet. The methynic proton of C-7 at d 3.8–4.1 ppm appeared as triplet due to vicinal coupling with two protons of methylene group. A singlet appeared at d 3.8 ppm in compound 4b is due to AOCH3 moiety. A singlet due to ANH2 group in compounds 4a and 4c appeared at d
In vitro assay: Tube dilution technique and micro dilution technique The tube dilution technique was carried out by adding various concentrations of synthesized compounds 4a, 4b and 4c in the Ellinghausen, McCullough, Jensen and Harris (EMJH) liquid medium [10]. The EMJH (Difco, USA) modified, semi-solid medium was prepared with the addition of rabbit serum (15%) (Sigma, USA) and enriched with L-asparagin (3%), calcium chloride (1%), magnesium chloride (1%), pyruvate sodium (1%) and 0.2% agar with the addition of 5-fluoruracil (300 mg/L) named as the selective medium. After sterility checking of the medium by placing in the room temperature for 48 h, the Leptospira species were inoculated with syringe filter. The tubes were incubated at room temperature for 7 days. The inhibition patterns for each species in different concentrations of synthetic compounds were observed
600 bp 500 bp
100 bp
Fig. 4. Agarose gel electrophoretogram showing amplification for rpoB gene of Leptospira species. Analysis products amplified Lanes 1 to 9-PCR product of Leptospira samples, Lane M-100 bp gene ruler.
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Note: Inhibition rate of MDT and TDT of various extracts of biologically active compounds against selective species of Leptospira species MDT is micro dilution technique and TDT tube dilution technique.
100 100 80 100 – – 100 100 L. hardjo
MDT TDT
100 100
100 80
100 90
100 60
100 100
100 80
90 70
70 70
20 –
20 20
100 100
100 80
90 70
70 70
50 50
100 100 – 20 – – – – L. australis
MDT TDT
20 –
60 100
80 100
80 100
20 20
40 80
– –
– –
– 20
– –
20 20
40 80
60 60
40 40
– –
100 100 – 40 – – – 80 L. pyrogens
MDT TDT
– –
– –
– –
20 40
– –
– –
– 20
– 20
– 40
– 50
– –
– –
60 800
40 20
– 50
100 100 60 40 40 20 100 100 100 100 L. javanica
MDT TDT
80 80
100 100
100 100
100 100
80 80
100 100
20 20
40 20
60 40
60 40
80 80
100 100
80 80
60 40
100 100 80 80 100 80 70 – – – L. autumnalis
MDT TDT
10 –
20 –
60 –
80 20
10 –
20 –
– 60
– 80
20 80
– –
10 80
20 80
80 60
40 80
100 100 100 100 100 100 100 100 100 80 L. pomona
MDT TDT
100 80
100 100
100 100
100 100
100 80
100 80
100 100
100 100
100 100
70 80
100 80
100 80
100 80
70 80
100 100 100 100 100 100 100 100 100 100 L. canicola
MDT TDT
100 80
100 100
100 100
100 100
100 80
100 100
100 100
100 100
100 100
100 100
100 80
100 100
100 100
100 100
30
100 100 100 100 100 100 100 100 100 100
150 125 100 75 50
100 100 100 80
25 150
100 100 100 100
125 100
100 100 100 100
75 50
100 100 100 80 70 80 70 80
25 150 125 100
70 80 80 100 100 100 100 80
75 50 25
MDT TDT
4c 4b 4a
Inhibition rate percentage in various concentration Technique Name of the serogroup
Table 1 Effect of various concentrations of biologically active compounds against selective species of Leptospira by MDT and TDT technique.
L. icterohaemorrhagiae
Standard benzyl penicillin
5.5–5.7 ppm. The aromatic protons of C-16 in 4b appeared as singlet at d 6.2 ppm. In all the compounds the aromatic protons appeared as multiplet in the region d 6.9–8.0 ppm. The representative spectra of 13C NMR spectra of 4a and 4b shows peaks at 173, 172, 170 and 169 indicates the presence of carbonyl group (C@O) and imine group (C@N). Mass spectral studies The mass spectra of compounds 4a and 4b are shown in Fig. 3. Molecular ion peak of compounds 4a and 4b were observed at m/z 375 and 341, respectively. A peak at m/z 337 in compound 4a and 341 in compound 4b represents (M+H)+. A peak at m/z 375 in compounds 4a is due to (M+K)+ and a peak at m/z 364 in 4b is due to (M+Na)+. Biology DNA extraction by QIAamp blood extraction kit (Qiagen, Germany) yielded a substantial amount of DNA without any protein contamination. The primers had sufficient sensitivity to detect DNA concentration of 1 fg and it is specifically amplified Leptospiral DNA (Fig. 4). Good amplifications were evident for PCR reaction carried out at the annealing temperature of 55 °C for 1 min. The 600 bp amplicon was obtained and observed under UV transilluminator light using BIORAD gel documentation unit (Fig. 4). The in vitro evaluation by tube and micro dilution techniques, the compound 4c showed efficient inhibitory activity. As observed by the percentage of dead cells of Leptospires when observed under dark field microscope. The compound 4c has shown best activity at 25, 50 and 75 lg/mL, whereas other two compounds also shown activity but, less when compared to 4c and the standard drug benzyl penicillin. The compound 4c at 50 and 75 lg/mL level showed significant inhibitory effect on all species of Leptospira, whereas compounds 4a and 4b has shown best activity at 75 lg/mL and also did not shown any inhibitory effect for L. autumnalis and L. pyrogen species. Therefore, among three biologically active pseudo-peptides the compound 4c showed significant and maximum inhibition for anti-leptospiral activity. The inhibitory percentage is as shown in Table 1. Conclusions We have established a series of pseudo-peptides against Leptospira, characterized them by spectroscopic studies and the exploitation of biologically active compound against Leptospira is investigated. From the NMR and the mass studies the secondary structure of the compound is studied. The compound 4c was the most effective inhibitor compound tested for MIC. However, further investigations should be carried out through modifications in its chemical structure in order to improve further to find out more biologically active molecule. Funding This work was supported by Indian Council of Medical Research (ICMR), New Delhi, India. (Sanction order No. 80/673/2010-ECD-I, Dated 03.05.2011). Acknowledgment The senior author (Chandan, S.) is greatly acknowledges the financial assistance from the Indian Council of Medical Research, New Delhi, India, in the form of Senior Research Fellowship. There
C. Shivamallu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 1152–1157
is no conflict of interest whatsoever among the author C.S. responsible for the bench work. B.V. and S.U supervised the whole experiment and S. U planned the whole experiment, data interpretation and writing of the manuscript. The authors would like to thank Project Director, PD_ADMAS, ICAR, Hebbal, Bangalore, Karnataka, India for providing reference Leptospira isolates. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2013.09.105. Reference [1] D. Luchini, F. Meacci, M.R. Oggioni, G. Morabito, V.D. Amato, M. Gabbrielli, G. Pozzi, Int. J. Legal Med. 122 (2008) 229–233.
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[2] M.E. Griffith, D.R. Hospenthal, C.K. Morray, Curr. Opin. Infect. Dis. 19 (6) (2006) 533–537. [3] J. Travis, Science 264 (1994) 360–362. [4] K. Park, S.Y. Shin, K. Hahm, Y. Kim, Bull. Korean Chem. Soc. 24 (2003) 1478– 1484. [5] M. Zasloff, Proc. Natl. Acad. Sci. 84 (1987) 5449–5453. [6] C. Adessi, C. Soto, Curr. Med. Chem. 9 (2002) 963–978. [7] J.S. Davies, J. Pept. Sci. 9 (2003) 471–501. [8] J.M. Ahn, N.A. Boyle, M.T. MacDonald, K.D. Janda, Mini Rev. Med. Chem. 2 (2002) 463–473. [9] L. Jian, Y. Liang, L. Tingting, W. Yongmei, Bioorg. Med. Chem. Lett. 17 (2007) 1601–1607. [10] L.S. Bernard, T.B. Lan, B. Guy, A. Khamis, R. Didier, FEMS Microbiol. Lett. 263 (2006) 142–147. [11] L.M. Mathew, Comparative analysis of acetone, in: Water and Saponified Extracts of Phyllanthus niruri Against Leptospira interrogans Serogroups In Vitro, Periyar University, Salem, Tamilnadu, India, 2001. [12] N. Prabhu, P.I. Joseph, P. Chinnaswamy, K. Natarajaseenivasa, S. Lakshmi, J. Pharm. Sci. 70 (6) (2008) 788–791.
