Pseudo-peptides as novel antileptospiral agents: Synthesis and spectral characterization

Article abstract of DOI:10.1016/j.saa.2013.09.105

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.

Full text of DOI:10.1016/j.saa.2013.09.105

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
a
a,
⇑
b
c
Chandan Shivamallu , Umesha Sharanaiah , Shiva Prasad Kollur , Naveen Kumar R. Mallesh ,
c
d
Revanasiddappa D. Hosakere , V. Balamurugan
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
Project Directorate on Animal Disease Monitoring and Surveillance (PD_ADMAS), ICAR, Hebbal, Bangalore 560 024, Karnataka, India
b
c
d
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
a b s t r a c t
Article history:
Received 31 July 2013
Received in revised form 8 September 2013
Accepted 26 September 2013
Available online 8 October 2013
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 synthe-
sized compounds were characterized using spectral ( H NMR, C 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.
1
13
Keywords:
Pseudo-peptide
Ó 2013 Elsevier B.V. All rights reserved.
Spectroscopic technique
Minimum inhibitory concentration
Anti-leptospiral activity
Introduction
contact of abraded skin, conjunctiva, or mucous membranes with
water contaminated by urine of infected rodents. Infection can oc-
Leptospira are highly susceptible to a wide variety of antimicro-
bial agents in vitro. It is not yet clear what the best choice of anti-
microbial agents can be given to human. Transmission occurs by
cur after animal bites or, very rarely between humans via infected
blood or urine. Rodents consider being the reservoir of the infec-
tion [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 Lepto-
spirosis is the fluoroquinolone antimicrobial, but when this under
human trial the use of this chemical is not fully supportive [2].
⇑
Corresponding author. Tel.: +91 0821 2419884.
E-mail addresses: umeshgroup@yahoo.co.in, su@appbot.uni-mysore.ac.in
(
U. Sharanaiah).
1
386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.09.105

C. Shivamallu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 1152–1157
1153
OCH3
OCH3
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
O
C
N
N
O
C
H N
2
N
S
N
H
H N
HN
2
N
H
HN
4
b
4
a
OH
OH
O
C
N
H N
and stereochemical integrities of the adjacent pair of a-carbon
2
N
H
HN
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 pseudo-
peptides. In addition, their high bio stability has enabled successful
applications in both in vitro and in vivo studies.
N
4c
Fig. 1. Structure of synthesized pseudo peptides.
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 path-
ogenic fungi and proved to be a broad-spectrum antimicrobial
agent, showing a significant inhibition of the growth of microor-
ganisms [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.
Thus, the problem of increasing microbial resistant to current anti-
biotics straight away demanded the development of novel antimi-
crobial 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 anti-
bacterial peptides may be difficult to induce resistant strains of
pathogens compared to classic antibacterial agents [4].
Experimental
Over the past decade or so, many defense antimicrobial pep-
tides to counter infection by microbe were isolated and character-
ized 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 conforma-
tionally constrained and thus more stable bioactive peptides [7,8].
Materials and methods
L
-trypophan, 2-aminobenzothiazole,4,6-dimethoxypyrimidin-
2
-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
1
13
recorded in CDCl
obtained on a Bruker Avance 400 NMR spectrometer. Chemical
shifts are quoted with respect to SiMe as internal standard.
Mass spectral studies were carried out on ESI-MS micro mass
3
as a solvent. H and C NMR spectra were
4
Fig. 2. 1H NMR spectra of 4a and 4b.

1
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C. Shivamallu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 1152–1157
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.
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
Chemistry
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,
General procedure
4
.19 mM) was added. The mixture was stirred at this temperature
Synthesis of compound 1
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 drop-
wise via syringe. The resulting mixture was stirred at room tem-
perature for an additional 10 h. The mixture was quenched with
cold water and neutralized with 1 N HCl. The aqueous mixture
To a solution of
,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
L-tryptophan (1.0 g, 4.89 mM) in 9:1 water and
1
(
(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
1155
was extracted with chloroform, and dried over Na
solvent afforded a yellow solid.
2
SO
4
. Removal of
under dark field microscope by placing a loopful of suspension
on the clear glass slide and observed under different magnifica-
tions of the dark field microscope (Zeiss, USA) [11,12]. The total
viable counts per microscopic field were compared with the posi-
tive 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
Synthesis of compound 4
To a solution of compound 3 (0.75 g, 1.71 mM) in dichlorometh-
ane was added trifluoro acetic acid (0.52 mL, 6.84 mM). The mix-
ture 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.
(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 condi-
tion for 30 min at room temperature. After incubation period, the
samples were spread on a slide by micro diluter and observed un-
der dark field microscope (Zeiss, USA) to study the inhibitory activ-
ity 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.
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.
Results and discussion
The general route for the synthesis of pseudo peptide is shown
in Scheme 1. To validate the chemically synthesized pseudo pep-
1
13
tides, H, C 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
DNA preparation
(4a)
The DNA was extracted from the retrieved culture after con-
firming the culture is positive for Leptospira using QIAgen DNA iso-
lation kit (QIAgen, Germany). The DNA extraction was carried out
according to the manufacturer’s instructions. The DNA was quanti-
fied using Nanodrop DNA quantifier and stores at À80 °C until fur-
ther processed.
1
Yield-58%, H NMR: (CDCl
3
, 400 MHz): dH 2.8–3.0 (2H, dd, CH
2
),
4
.1 (1H, t, CH), 5.7 ((2H, s, NH
2
), 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),
.8 (1H, d, ArAH), 8.0 (1H, d, ArAH), 10.9 (1H, s, NH). 13C NMR:
CDCl , 400 MHz): dC 173, 170, 158, 138, 128, 125, 122, 119, 118,
7
(
1
3
3
+
11, 108, 68, 59,53, 51, 28, 19. ESI-MS: m/z 337 (M+H) and m/z
+
75 (M+K) .
Primers and PCR assay
NMR studies
1H NMR spectra of compounds were recorded in CDCl
Bruker-400 MHz instrument. The representative spectra of com-
pounds 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
PCR was performed on the obtained DNA template using
reported primer pair (rpoB-F-CCTCATGGGTTCCAACATGCA and
rpoB-R-CGCATCCTCRAAGTTGTAWCCTT) [10]. Briefly, PCR was per-
formed by initial denaturation at 94 °C for 3 min. Subsequent PCR
amplification conducted with 40 cycles of denaturation at 94 °C for
3
using
3
0 s, annealing at 55 °C for 1 min, extension at 72 °C for 1 min fol-
lowed by final extension for 20 min at 72 °C. 5 mL aliquot of each
PCR product was mixed with 3
ll of 6Â loading dye (Fermentas,
3
3
.8 ppm in compound 4b is due to AOCH moiety. A singlet due
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 un-
der UV Transilluminator and documented using gel documentation
system with quantity one software (Biorad, USA).
to ANH group in compounds 4a and 4c appeared at
2
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 med-
ium [10]. The EMJH (Difco, USA) modified, semi-solid medium
was prepared with the addition of rabbit serum (15%) (Sigma,
6
00 bp
USA) and enriched with L-asparagin (3%), calcium chloride (1%),
5
00 bp
magnesium chloride (1%), pyruvate sodium (1%) and 0.2% agar
with the addition of 5-fluoruracil (300 mg/L) named as the selec-
tive medium. After sterility checking of the medium by placing in
the room temperature for 48 h, the Leptospira species were inocu-
lated with syringe filter. The tubes were incubated at room tem-
perature for 7 days. The inhibition patterns for each species in
different concentrations of synthetic compounds were observed
1
00 bp
Fig. 4. Agarose gel electrophoretogram showing amplification for rpoB gene of
Leptospira species. Analysis products amplified Lanes
Leptospira samples, Lane M-100 bp gene ruler.
1 to 9-PCR product of

1
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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 representa-
1
3
tive spectra of C NMR spectra of 4a and 4b shows peaks at 173,
72, 170 and 169 indicates the presence of carbonyl group (C@O)
1
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
3
3
75 and 341, respectively. A peak at m/z 337 in compound 4a and
+
41 in compound 4b represents (M+H) . A peak at m/z 375 in com-
+
pounds 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 un-
der dark field microscope. The compound 4c has shown best activ-
ity 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
inhibitory effect on all species of Leptospira, whereas compounds
a and 4b has shown best activity at 75 g/mL and also did not
lg/mL level showed significant
4
l
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 Lepto-
spira, characterized them by spectroscopic studies and the exploi-
tation 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, fur-
ther 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
1157
is no conflict of interest whatsoever among the author C.S. respon-
sible for the bench work. B.V. and S.U supervised the whole exper-
iment 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.
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