A new family of highly potent inhibitors of microbes: Synthesis and conjugation of elastin based peptides to piperazine derivative

Article abstract of DOI:10.1007/s10989-011-9282-8

Elastin protein-based polymers have their origin in repeating sequences of the mammalian elastic protein, elastin. The sequences of elastin peptides chosen are tetrapeptides, pentapeptides and tricosamers (30 amino acids) and also aromatic amino acids. Th

Full text of DOI:10.1007/s10989-011-9282-8

Int J Pept Res Ther (2012) 18:89–98
DOI 10.1007/s10989-011-9282-8
A New Family of Highly Potent Inhibitors of Microbes: Synthesis
and Conjugation of Elastin Based Peptides to Piperazine
Derivative
•
R. Suhas S. Chandrashekar D. Channe Gowda
•
Accepted: 10 November 2011 / Published online: 22 November 2011
Ó Springer Science+Business Media, LLC 2011
Introduction
Abstract Elastin protein-based polymers have their origin
in repeating sequences of the mammalian elastic protein,
elastin. The sequences of elastin peptides chosen are tetra-
peptides, pentapeptides and tricosamers (30 amino acids)
and also aromatic amino acids. These have been conjugated
to 1-(2,3-dichlorophenyl)piperazine to study the effect of
conjugation on the activity. The conjugates so obtained were
characterized by physical and analytical techniques followed
by the antimicrobial evaluation. The study revealed that all
the conjugates have exhibited enhanced activity than the
conventional drugs. Further, the conjugates of tricosamers
have shown extraordinary activity against the fungal species
with MIC value of 3–5 lg/ml which is five fold more potent
than the antibiotic used.
The increasing incidence of infection caused by the rapid
development of bacterial resistance to most of the known
antibiotics is a serious health problem (Chu et al. 1996).
While many factors may be responsible for mutations in
microbial genomes, it has been widely demonstrated that
the incorrect use of antibiotics can greatly increase the
development of resistant genotypes (Clark 1996). As
multidrug-resistant bacterial strains proliferate, the neces-
sity for effective therapy has stimulated research into the
design and synthesis of novel antimicrobial molecules.
Thus, the design and development of new agents that
provide effective therapy for infections caused by organ-
isms resistant to older agents is an urgent need (Emami
et al. 2006).
Keywords Peptides of elastin Á Substituted piperazine Á
Conjugates Á Effective antimicrobials Á Hydrophobicity Á
Biocompatibility
The growing interest in heterocyclic compounds is
basically because of their raised biological activity and also
they make possible development of novel materials with
unique properties. One very interesting and promising class
of heterocycle is the series of piperazine and its derivatives.
Piperazines have been widely used in biological screening
resulting in numerous applications and constitute an
attractive pharmacological scaffold present in several drugs
(Foye et al. 1995). This small and rigid heterocyclic
backbone could act on various pharmacological targets.
Especially, piperazine nucleus could be found in a broad
range of biologically active compounds displaying anti-
cancer (Haga et al. 1985; Guo et al. 2003; Guo et al. 2004),
calcium channel blockers (Shanklin et al. 1991; Nomura
et al. 1995) and histamine antagonists (Hiristo et al. 1989;
Gillard et al. 2002).
Abbreviations
Boc
t-Butoxycarbonyl
EDCI 1-(3-Dimethylaminopropyl)-
3-ethyl-carbodiimide.HCl
HOBt 1-Hydroxybenzotriazole
IBCF Isobutyl chloroformate
NMM N-methyl morpholine
TFA
Trifluoroacetic acid
Electronic supplementary material The online version of this
article (doi:10.1007/s10989-011-9282-8) contains supplementary
material, which is available to authorized users.
R. Suhas Á S. Chandrashekar Á D. C. Gowda (&)
Department of Studies in Chemistry, University of Mysore,
Manasagangotri, Mysore 570 006, India
Protein-based polymers are polypeptides comprised of
repeating sequences of amino acids, having their origin in a
protein, elastin (Sandberg et al. 1985; Yeh et al. 1987). The
e-mail: dchannegowda@yahoo.co.in
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Int J Pept Res Ther (2012) 18:89–98
most striking repeating sequence (Val1-Pro2-Gly3-Val4-
Gly5)n or (VPGVG)n is apparent in the bovine and porcine
elastins and another repeat (Val1-Pro2-Gly3-Gly4)n, was
first found in porcine elastin. The monomers, oligomers,
and high polymers of these repeats have been synthesized
and conformationally characterized (Gowda et al. 1994).
Such protein-based polymers exhibit the same hierarchical
structure and mimic the parent protein. These polymers
have a number of medical and non-medical applications
(Urry et al. 1993, 1996).
pump with RP18.250-4 mm column and UV detector-UV-
VISL 7400 using a linear gradient of 0–100% acetonitrile/
1
0.1% TFA (20 min). H NMR spectra were obtained on
VARIAN 400 MHz instrument using CDCl3 and the
chemical shifts are reported as parts per million (d ppm)
using TMS as an internal standard. Mass spectra were
obtained on LCMSD-Trap-XCT instrument.
Synthesis
Extensive work has been reported on the conjugation of
different amino acids/peptides to various biologically
active moieties (Shivakumara et al. 2007; Suresha et al.
2009; Suhas et al. 2011; Suresha et al. 2011; Banoczi et al.
2008; Dutta et al. 2009) which reveals that conjugation
plays a paramount role in exerting the activity. Also,
involving amino acids/peptides in drugs makes them low
toxic, ample bioavailability and permeability, modest
potency and good metabolic and pharmacokinetic proper-
ties (Gadek and Nicholas 2003).
The 1-(2,3-dichlorophenyl)piperazineÁHCl 1 was prepared
as previously reported method (Oshira et al. 1991). The
peptides were synthesized by classical solution phase
method using Boc chemistry. The Boc group used for
temporary Na protection was removed with 4N HCl in
dioxane or TFA. The C terminal carboxyl group was pro-
tected by the benzyl ester and its removal was effected by
hydrogenolysis using HCOONH4 as hydrogen donor and
10% Pd on carbon as catalyst (Anwer and Spatola 1980) or
hydrolysed using 1N NaOH/MeOH. The hydroxyl group of
Tyr was protected by 2,6-Cl2-Bzl and c-carboxyl group of
Glu was protected by cyclohexyl ester and removed by
In a continuous effort to develop supplementary anti-
microbial agents with improved results, the present work
involves the conjugation of highly biocompatible elastin
based peptides of varying hydrophobicity with 1-(2,3-
dichlorophenyl)piperazine.
?
treatment with polymer supported HCOO-NH3 and 10%
Pd–C (Abiraj et al. 2005). All the coupling reactions for
peptide synthesis were achieved with IBCF. The procedure
followed for the synthesis of tetrapeptides, pentapeptides
and tricosamers are outlined in the Scheme 1, 2 and 3
respectively. The protected peptides were purified by col-
umn chromatography over silica gel and characterized by
physical and analytical techniques. The purity of free
peptides was checked by HPLC and found to have purity
[95% in each case.
Materials and Methods
General
All the amino acids used except glycine were of L-con-
figuration unless otherwise mentioned. All Boc-amino
acids, EDCI, HOBt and TFA were purchased from
Advanced Chem. Tech. (Louisville, Kentucky, USA).
IBCF and NMM were purchased from Sigma Chemical Co.
(St. Louis, MO). All solvents and reagents used for the
synthesis were of analytical grade. All the chemicals and
reagents used for antimicrobial studies were of bacterio-
logical grade unless otherwise indicated. Nutrient broth and
nutrient agar were purchased from Hi-media chemicals
(Mumbai, India). Silica gel (60–120 mesh) for column
chromatography was purchased from Sisco Research
Laboratories Pvt. Ltd., (Bombay, India). The pathogens
used for the microbial studies were obtained from a local
hospital. The progress of the reaction was monitored by
TLC using silica gel coated on glass plates with the solvent
system comprising chloroform/methanol/acetic acid in the
ratio 95:5:3 throughout the study and the compounds on
TLC plates were detected by iodine vapors. Melting points
were determined on a Thomas Hoover melting point
apparatus and are uncorrected. All the HPLC analyses were
performed with Lachrom-2000 Merck-Hitachi L7100
Synthesis of Tetrapeptides, Pentapeptides
and Tricosamers
The peptides were synthesized as described in our earlier
article (Suhas et al. 2011).
General Procedure for the Hydrogenolysis of Benzyl
Esters of Tetra and Pentapeptides
Each peptide 2–6 (0.006 mol) was hydrogenolysed in
methanol (10 ml/g of peptide) using ammonium formate
(2.0 equi.) and 10% Pd–C (0.1 g/g of peptide) for 30 min at
room temperature. The completion of the reaction was
monitored by TLC. The catalyst was filtered and washed
with methanol. The combined washings and filtrate were
evaporated and the residue taken into CHCl3, washed with
water and dried over anhydrous Na2SO4. The solvent was
removed under pressure and triturated with ether, filtered,
washed with ether and dried to obtain debenzylated pep-
tides (9–13).
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Int J Pept Res Ther (2012) 18:89–98
91
Gly
General Procedure for the Hydrolysis of Benzyl Esters
of Tricosapeptides
Gly
X
Pro
Boc
Boc
OH
H
OBzl
OBzl
OBzl
OBzl
Each tricosamer 7, 8 (0.006 mol) was hydrolysed in
methanol (10 ml/g of peptide) using cold solution of 1 N
NaOH (30 ml) for 2 h. The completion of the reaction was
monitored by TLC, solvent was evaporated, cooled, neu-
tralized with cold 1N HCl, extracted with CHCl3, washed
with 1N HCl followed by water and dried over anhydrous
Na2SO4. The solvent was removed under pressure and
triturated with ether, filtered, washed with ether and dried
to obtain debenzylated peptides (14, 15).
i
ii
iii
ii
Boc
Boc
H
OH
Boc
Boc
Boc
OBzl
OH
H
iii
OBzl
OH
The C terminal free peptides so obtained were charac-
terized by Rf values and M.P. (°C) and the data are as
follows: (9) Rf 0.47; M.P. 67–69 (Lit. 66) (Gowda et al.
2002) (10) Rf 0.54; M.P. 99 (Lit. 99) (Gowda et al. 2002)
(11) Rf 0.44; M.P. 106–109 (Lit. 105) (Gowda et al. 2002)
(12) Rf 0.45; M.P. 125 (Lit. 127) (Gowda et al. 2001) (13)
Rf 0.46; M.P. 117 (Lit. 118) (Gowda et al. 2001) (14) Rf
0.48; M.P. 200–203 (15) Rf 0.46; M.P. 166–169.
iv
Scheme 1 Schematic representation of the synthesis of tetrapeptides
Boc-GGXP-OH where X = Ala for GGAP; Ile for GGIP and Phe for
GGFP by stepwise approach (i) IBCF/HOBt, NMM; (ii) HCl/dioxane;
(iii) IBCF, NMM; (iv) HCOONH4/10% Pd–C
Gly
Pro
Gly
Z
Z
Boc
Boc
OH
H
OBzl
iii
General Procedure for the Coupling of Piperazine
Derivative 1 with Boc-Xaa-OH where Xaa=Phe, Trp,
Tyr(2,6-Cl2-Bzl) and Peptides 9–15
OBzl
ii
Boc
Boc
OBzl Boc
OBzl Boc
H
OH
OH
H
OBzl
OBzl
iii
iv
i
To Boc-Xaa-OH (0.005 mol) and HOBt (0.765 g, 0.005 mol)
dissolved in DMF (10 ml/g of peptide) and cooled to 0°C
was added NMM (0.55 ml, 0.005 mol). EDCI (0.956 g,
0.005 mol) was added under stirring while maintaining the
temperature at 0°C. The reaction mixture was stirred for an
additional 10 min and pre-cooled solution of 1-(2,3-dichlo-
rophenyl)piperazineÁHCl (1.340 g, 0.005 mol) and NMM
(0.55 ml, 0.005 mol) in DMF (13 ml) was added slowly.
After 20 min, pH of the solution was adjusted to 8 by the
addition of NMM and the reaction mixture was stirred over
night at room temperature. DMF was removed under reduced
pressure and the residue was poured into about 200 ml
ii
Boc
OH
H
OBzl
iii
iv
Boc
Boc
OBzl
OH
Scheme 2 Schematic representation of the synthesis of pentapeptides
Boc-GZGZ|P-OH where Z = Z| = Val for GVGVP; Z = Z| = Phe
for GFGFP; Z = Val and Z| = Phe for GVGFP; Z = Glu(OcHx) and
Z| = Phe for GE(OcHx)GFP by (3 ? 2) fragment coupling method
(i) IBCF/HOBt, NMM; (ii) HCl/dioxane; (iii) IBCF, NMM; (iv)
HCOONH4/10% Pd–C or 1N NaOH/MeOH
Scheme 3 Schematic
representation of the synthesis
of tricosapeptides by
[(5 ? 5 ? 5) ? (5 ? 5 ? 5)]
fragment coupling method
(i) EDCI/HOBt, NMM; (ii)
TFA; (iii) 1N NaOH/MeOH
GE(OcHx)GFP
GVGVP
Boc
GVGVP
Boc
GVGFP
GFGFP
GVGFP
H OBzl
OH
H
OBzl
OBzl
OBzl
OBzl
OH
i
i
Boc
Boc
H
OBzl
OBzl
OBzl
OcHx
Boc
OH
ii
ii
Boc
H
OH
OcHx
Boc
i
i
Boc
H
OcHx
Boc
ii
iii
OH
OBzl
OBzl
OcHx
Boc
i
OcHx
Boc
iii
OH
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Int J Pept Res Ther (2012) 18:89–98
Biology
ice-cold 90% saturated KHCO3 solution and stirred for
30 min. The precipitated product was taken into CHCl3 and
washed with 5% NaHCO3 solution (2 9 20 ml), water
(2 9 20 ml), 0.1N cold HCl solution (2 9 20 ml) and finally
brine solution (2 9 20 ml). The CHCl3 layer was dried over
anhydrous Na2SO4 and the solvent was removed under
reduced pressure. The products so obtained were recrystal-
lized from ether/petroleum ether to get white colored desired
conjugates (16–25). The schematic representation of the
coupling is shown in Scheme 4.
Antibacterial Activity
In vitro antibacterial activity was evaluated against human
pathogens of both gram positive organisms namely Coag-
ulase positive staphylococcus and Klebsiella pneumoniae
and gram negative organisms namely Xanthomonas oryzae
and Escherichia coli by agar well diffusion method (Fig. 1)
as well as microdilution method (Fig. 2).
Agar Well Diffusion Method
Deprotection of 2,6-Cl2-Bzl of Tyr and OcHx of Glu
of the Conjugates 18, 24 and 25
The microorganisms were inoculated into the sterilized
nutrient broth and maintained at 37°C for 24 h. On the day of
testing, bacteria were subcultured separately into 25 ml of
sterilized nutrient broth. Inoculated subcultured broths were
kept at room temperature for the growth of inoculums. Each
test compounds (26–35) and standard drug (amoxicillin)
of 10 mg was dissolved in 10 ml of DMSO to get a con-
centration of 1 mg/ml and further diluted to get a final con-
centration of 50 lg/ml. About 15–20 ml of molten nutrient
agar was poured into each of the sterile plates. With the help
of cork borer of 6 mm diameter, the cups were punched and
scooped out of the set agar and the plates were inoculated
with the suspension of particular organism by spread plate
technique. The cups of inoculated plates were then filled with
0.1 ml of the test solution, amoxicillin solution and DMSO
(negative control). The plates were allowed to stay for 24 h at
37°C and zone of inhibition (mm) was then measured.
To a solution of the conjugates 18, 24 and 25 (0.001 mol) in
methanol (10 ml/g of compound), 10% Pd–C (100 mg) and
polymer supported formate (1 g) were added and the mixture
was stirred at room temperature for 8 h. After completion of
the reaction monitored by TLC, catalyst and the polymer
were filtered, washed with methanol. The solvent was
evaporated under reduced pressure and the product was
taken into CHCl3, washed with saturated NaCl and the sol-
vent was dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure and triturated with ether
and dried to get side chain deprotected conjugates.
General Procedure for the Deblocking of Boc-Ybb-
Heterocycle
Each conjugate (16–25) (0.001 mol) was deblocked with
TFA (10 ml/g of compound) for 40 min (Scheme 4). The
excess solvent was removed under reduced pressure, tritu-
rated with ether, filtered, washed with ether and dried under
vacuum (yield 100%) to obtain TFA.NH2-Ybb-Heterocycle
(26–35) and these were used for the antibacterial and anti-
fungal studies.
Microdilution Method
All the microorganisms were grown in Muller-Hinton
broth. After cultivation for 16–18 h at 37°C, the bacteria
were harvested and their density was determined by
measuring OD at A600. MIC of the compounds was
Cl
Cl
Cl
Cl
a
+
HCl
N
Boc
HN
Xaa
N
Boc Xaa
N
COOH
CO
16-25
1
b
Cl
Cl
Cl
Cl
c
TFA .
Ybb
NH2
N
N
CO
26-35
Boc
Ybb
N
N
CO
Scheme 4 Schematic representation of the coupling of amino acids/
peptides to heterocycle 1, Reagents and conditions: (a) EDCI/HOBt,
GVGVP GVGVP GFGFP GFGFP, GE(OcHx)GFP GVGVP GVGFP
GFGFP GVGVP GVGFP Ybb = Phe, Trp, Tyr, GGAP, GGIP,
GGFP, GVGVP, GFGFP, GEGFP GVGVP GVGVP GVGVP GFGFP
GFGFP, GEGFP GVGVP GVGFP GFGFP GVGVP GVGFP
NMM, 0°C, overnight at rt; (b) Polymer supported HCOO-NH3
10% Pd–C; (c) TFA, 40 min, rt Xaa = Phe, Trp, Tyr(2,6-Cl2-Bzl),
/
?
GGAP, GGIP, GGFP, GVGVP, GFGFP, GE(OcHx)GFP GVGVP
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Int J Pept Res Ther (2012) 18:89–98
93
Fig. 1 Diagrammatic
representation of antibacterial
activity of the synthesized
conjugates by agar well
diffusion method
E.coli
X. oryzae
K. pneumoniae
C. positive staphyloccous
25
20
15
10
5
0
26
27
28
29
30
31
32
33
34
35
Std.
Conjugated Compounds
Std. = Amoxicillin.
Fig. 2 Diagrammatic
representation of antibacterial
activity of the synthesized
conjugates by microdilution
method
E.coli
X. oryzae
K. pneumoniae
C. positive staphyloccous
40
30
20
10
0
26
27
28
29
30
31
32
33
34
35
Std.
Conjugated Compounds
Std. = Amoxicillin.
Agar Well Diffusion Method
determined by agar dilution method. Suspension of each
microorganism was prepared to contain approximately
(1 9 104–2 9 104 CFU/ml) and applied to the plates with
serially diluted compounds (dissolved in DMSO) to be
tested and also reference drug and incubated at 37°C
overnight. The minimum inhibitory concentration was
considered to be the lowest concentration that completely
inhibited the growth of microorganisms on the plates. Zone
of inhibition (mm) was measured after 24 h and MIC
values were determined.
The fungal strains were subcultured separately into 25 ml
of sterilized nutrient broth and incubated for 1 day to
obtain the inoculums. Each test compounds (26–35) and
standard drug (bavistin) of 10 mg was dissolved in 10 ml
of DMSO to get a concentration of 1 mg/ml and further
diluted to get a final concentration of 50 lg/ml. Molten
media of Sabouraud agar of 10–15 ml was poured into the
petriplates and allowed to solidify. Fungal subculture was
inoculated on the solidified media. With the help of 6 mm
cork borer, the cups were punched and scooped out of the
set agar. The cups of inoculated plates were then filled with
0.1 ml of the test solution, bavistin solution and DMSO
(negative control). The plates were allowed to stay for
3 days at room temperature and zone of inhibition (mm)
was then measured.
Antifungal Activity
In vitro antifungal activity was evaluated against three
fungal species namely Aspergillus niger, A. flavus and
Fusarium oxysporum by agar well diffusion method
(Fig. 3) as well as microdilution method (Fig. 4).
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Int J Pept Res Ther (2012) 18:89–98
Fig. 3 Diagrammatic
representation of antifungal
activity of the synthesized
conjugates by agar well
diffusion method
40
30
20
10
0
A. niger
A. flavus
F. oxysporum
26
27
28
29
30
31
32
33
34
35
Std.
Conjugated Compounds
Std. = Bavistin
Fig. 4 Diagrammatic
representation of antifungal
activity of the synthesized
conjugates by microdilution
method
50
40
30
20
10
0
A. niger
A. flavus
F. oxysporum
26
27
28
29
30
31
32
33
34
35
Std.
Conjugated Compounds
Std. = Bavistin
were found to be good and characterized by M.P., 1H NMR
1
and mass spectroscopic techniques. H NMR data of the
Microdilution Method
Sabouraud agar was used for the preparation of plates.
Suspension of each microorganism was prepared to contain
105 CFU/ml. The agar plates were inoculated with fungal
strains and serially diluted test compounds and reference
drug dissolved in DMSO. The plates were incubated at
25°C for 48–72 h. The minimum inhibitory concentration
was considered to be the lowest concentration that com-
pletely inhibited the growth of microorganisms on the
plates. Zone of inhibition (mm) was measured after 72 h
and MIC values were determined.
final protected peptides (2–8) are given in Table S1. The
physical and analytical data of the conjugated compounds
are provided in Table 1. The 1H NMR and mass data were
found to be in good agreement with the structures assigned.
Biological Evaluation
The efficacy of the synthesized compounds as antimicro-
bials were evaluated for their antibacterial studies against
different strains of human pathogens of both gram positive
bacteria namely K. pneumoniae and C. positive staphylo-
coccus and gram negative organisms like E. coli and
X. oryzae and antifungal studies against A. niger, A. flavus
and F. oxysporum. The results obtained as zone of inhibi-
tion (mm) and minimum inhibitory concentration (lg/ml)
are presented in Tables 2 and 3 respectively. Amoxicillin
and bavistin were used as standard drugs for antibacterial
and antifungal assays respectively.
Results and Discussion
Peptide Synthesis and Characterization
Peptides were synthesized by classical solution phase
method using Boc chemistry. These peptides were conju-
gated to piperazine derivative 1 using EDCI/HOBt as cou-
pling agent and NMM as base. The yields of the compounds
Our earlier investigation (Suhas et al. 2011) revealed
promising activity by the conjugation of benzisoxazole
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Int J Pept Res Ther (2012) 18:89–98
95
Table 1 Physical and analytical data of the conjugated compounds (16–25)
Entry Rf
Yield M.P. °C Theoretical Actual MS
1H NMR data (CDCl3, d ppm)
value (%)
Mol. Wt.
values (M?)
16
17
0.83 88.0
0.81 90.0
Gummy 478
478.4
Boc = 1.41 (9H, s); Phe = 3.20–3.51 (2H, d, –bCH2), 4.94 (1H, t, -aCH),
6.89–7.23 (5H, m, ArH), 8.10 (1H, s, –NH); Heterocycle = 3.51–3.74 (8H,
m, –CH2), 6.89–7.23 (3H, m, ArH)
Boc = 1.43 (9H, s); Trp = 3.14–3.34 (2H, d, –bCH2), 4.95 (1H, t, –aCH),
7.08 (1H, s, –CH of indole), 6.64–7.39 (4H, m, ArH), 10.21 (1H, s, -NH of
indole), 8.08 (1H, s, –NH); Heterocycle = 3.54–3.71 (8H, m, –CH2),
6.64–7.39 (3H, m, ArH)
Boc = 1.43 (9H, s); Tyr(2,6-Cl2-Bzl) = 3.21-3.54 (2H, d, –bCH2), 4.93 (1H,
t, –aCH), 5.27 (2H, s, –CH2 of side chain protecting group), 6.72–7.49 (7H,
m, ArH), 8.12 (1H, s, –NH); Heterocycle = 3.47–3.74 (8H, m, –CH2),
6.72–7.49 (3H, m, ArH)
Boc = 1.40 (9H, s); Gly1 = 3.87 (2H, s, –aCH); Gly2 = 4.14 (2H, s, –aCH);
Ala3 = 1.30 (3H, d, –bCH3), 4.62 (1H, m, –aCH); Pro4 = 1.97–2.10 (2H, m,
–cCH2), 2.11–2.18 (2H, m, –bCH2), 3.64, 3.79 (2H, m, –dCH2), 4.60 (1H, m,
–aCH); Heterocycle = 3.47–3.74 (8H, m, –CH2), 6.72–7.49 (3H, m, ArH)
Boc = 1.42 (9H, s); Gly1 = 3.81 (2H, s, –aCH); Gly2 = 3.97 (2H, s, –aCH);
Ile3 = 1.04–1.06 (6H, d, –(CH3)2), 1.43 (2H, m, –cCH2), 2.11 (1H, m,
–bCH), 4.60 (1H, m, –aCH); Pro4 = 2.12–3.65 (6H, m, –CH2), 4.57 (1H, m,
–aCH); Heterocycle = 3.66–3.74 (8H, m, –CH2), 6.90–7.26 (3H, m, ArH)
Boc = 1.44 (9H, s); Gly1 = 3.83 (2H, s, –aCH); Gly2 = 3.93 (2H, s, –aCH);
Phe3 = 3.18, 3.57 (2H, d, –bCH2), 4.93 (1H, t, –aCH); 6.99–7.26 (5H, m,
ArH); Pro4 = 1.99–2.00 (2H, m, –cCH2), 2.05, 2.23 (2H, m, –bCH2),
3.57–3.62 (2H, m, –dCH2), 4.95 (1H, m, –aCH); Heterocycle = 2.23–2.94
(8H, m, –CH2), 6.99–7.26 (3H, m, ArH)
129
517
653
614
656
690
517.4
653.4
613.5
655.6
18
19
20
21
0.84 96.0
0.72 90.0
0.72 90.0
0.64 91.0
79
84–86
78–81
60–62
711.2
[M??Na]
22
23
24
0.62 94.0
0.66 89.0
0.73 88.0
116–118 741
104–106 837
167–170 3136
740.7
Boc = 1.41 (9H, s); Gly1 = 3.82 (2H, s, –aCH); Val2 = 0.96 (6H, m,
(–CH3)2), 2.58 (1H, m, –bCH), 4.50 (1H, m, –aCH); Gly3 = 4.13 (2H, s,
–aCH); Val4 = 0.96 (6H, m, (–CH3)2), 2.63 (1H, m, –bCH), 4.49 (1H, m,
–aCH); Pro5 = 2.11–3.64 (6H, m, –CH2), 4.52 (1H, m, –aCH);
Heterocycle = 2.23–2.94 (8H, m, –CH2), 6.99–7.26 (3H, m, ArH)
Boc = 1.42 (9H, s); Gly1 = 3.82 (2H, s, –aCH); Phe2 = 3.52, 3.58 (2H, m,
–bCH2), 4.64 (1H, m, –aCH), 6.91–7.28 (5H, m, ArH); Gly3 = 4.12 (2H, s,
–aCH); Phe4 = 3.47, 3.60 (2H, m, –bCH2), 4.66 (1H, m, –aCH), 6.91–7.28
(5H, m, ArH); Pro5 = 2.11–3.64 (6H, m, –CH2), 4.51 (1H, m, –aCH);
Heterocycle = 3.40–3.73 (8H, m, –CH2), 6.91–7.28 (3H, m, ArH)
858.3
[M??Na]
3142.2
Boc = 1.67 (9H, s); Gly = 4.09, 4.92 (24H, m, –aCH); Val = 0.98, 1.01
(36H, m, (–CH3)2), 2.45, 2.65 (6H, m, –bCH), 4.41 (6H, m, –aCH);
Glu = 1.15–1.25 (10H, m, –CH2 of cyclohexyl ring), 2.07, 2.19 (4H, m,
–
b,cCH2), 3.71 (1H, m, –CH of cyclohexyl ring), 4.74 (1H, m, –aCH);
Phe = 3.52, 3.72 (10H, m, –bCH2), 4.21, 4.67 (5H, m, –aCH), 7.19–7.62
(25H, m, ArH); Pro = 2.02, 3.55, 3.69 (36H, m, –CH2), 4.75 (6H, m, a–CH);
Heterocycle = 3.40, 3.64 (8H, m, –CH2), 7.19–7.62 (3H, m, ArH)
25
0.74 89.0
185–188 3136
3142.2
Boc = 1.62 (9H, s); Gly = 4.08, 4.90 (24H, m, –aCH); Val = 0.98, 1.00
(36H, m, (–CH3)2), 2.44, 2.62 (6H, m, –bCH), 4.41 (6H, m, –aCH);
Glu = 1.09–1.25 (10H, m, –CH2 of cyclohexyl ring), 2.07, 2.17 (4H, m,
–
b,cCH2), 3.68 (1H, m, –CH of cyclohexyl ring), 4.70 (1H, m, –aCH);
Phe = 3.52, 3.70 (10H, m, –bCH2), 4.21, 4.59 (5H, m, –aCH), 7.20–7.62
(25H, m, ArH); Pro = 2.02, 3.55, 3.69 (36H, m, –CH2), 4.75 (6H, m, a–CH);
Heterocycle = 3.41, 3.64 (8H, m, –CH2), 7.20–7.62 (3H, m, ArH)
derivative with elastin based peptides. Encouraged by this,
in the present study it was felt worthy to link piperazine
moiety to these peptides. It is evident from the results that
all the heterocycle conjugated amino acids/peptides have
exhibited enhanced activity compared to heterocycle or
free peptides tested alone which are inactive or weakly
active ([50 lg/ml).
Earlier studies report the significance of activity
revealed by aromaticity and hydrophobicity (Shivakumara
et al. 2007; Suresha et al. 2009, 2011; Suhas et al. 2011)
and hence initially more hydrophobic and aromatic amino
acids such as Phe, Trp and Tyr were selected for conju-
gation. The results revealed that the Phe coupled hetero-
cycle (26) has shown improved activity. Surprisingly, other
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Int J Pept Res Ther (2012) 18:89–98
Table 2 Inhibitory zone (diameter) mm of the synthesized conjugates (26–35) against tested bacterial and fungal strains by agar well diffusion
method
Entry
Antibacterial activity
Antifungal activity
Zone of inhibitiona (mm) SD (n = 3)
E. coli
X. oryzae
K. pneumoniae
C. positive staphylococcus
A. niger
A. flavus
F. oxysporum
26
13 0.26
06 0.21
05 0.26
15 0.47
16 0.25
18 0.28
20 0.30
24 0.15
22 0.20
20 0.30
03 0.15
10 0.20
–
11 0.20
05 0.10
05 0.05
14 0.30
16 0.05
17 0.25
18 0.37
20 0.36
21 0.36
18 0.32
04 0.25
08 0.66
–
10 0.30
05 0.10
03 0.30
12 0.36
14 0.30
15 0.10
17 0.40
19 0.23
21 0.26
20 0.40
02 0.17
08 0.11
–
09 0.20
04 0.11
04 0.26
11 0.47
15 0.43
15 0.20
17 0.17
20 0.30
22 0.30
20 0.05
02 0.32
07 0.25
–
15 0.36
07 0.20
05 0.10
18 0.40
22 0.41
24 0.36
29 0.52
31 0.40
38 0.43
37 0.47
04 0.26
–
11 0.15
05 0.05
04 0.41
14 0.25
16 0.34
17 0.25
19 0.26
21 0.32
29 0.32
26 0.26
03 0.20
–
15 0.10
05 0.15
05 0.47
17 0.45
19 0.30
20 0.55
22 0.25
23 0.41
31 0.30
29 0.10
03 0.20
–
27
28
29
30
31
32
33
34
35
Heterocycle
Amoxicillin
Bavistin
a
11 0.25
08 0.15
11 0.17
Values are mean of three determinations, the ranges of which are \5% of the mean in all cases
two amino acid conjugates 27 and 28 have shown moderate
activity in spite of being aromatic and also having indole
group in Trp and phenolic group in Tyr. Hence, it was
found that substitution of Phe by other aromatic amino
acids is not effective in exerting potent activity.
increase in the activity (*two times) which is evident from
the conjugate GGFP (31). Similarly, the remaining two
tetrapeptide conjugates GGIP and GGAP have shown
increased activity compared to Phe alone (26). Also, when
the length of the peptide chain increased as well as two Phe
residues were introduced (greater hydrophobicity) as in
GFGFP (33) has exhibited two times greater activity than
the standard drugs. This trend was followed in GVGVP
conjugate (32) also. Hence, it can be inferred that as the
length of the peptide chain as well as the hydrophobicity
increases the activity also increases (Suresha et al. 2009;
Suhas et al. 2011). Thus, the order of activity of the
heterocyclic conjugated peptides is found to be
GFGFP [ GVGVP [ GGFP [ GGIP [ GGAP [ Phe [
Trp [ Tyr.
In view of this, interest was generated to include pep-
tides having improved hydrophobicity profile as well as a
polar species, Glu in the chain which is exhibited by
tricosamers 7 and 8. Tricosamers prepared elsewhere in
order to study the hydrophobicity-induced pKa shifts were
used as such. Though the antibacterial activity of the
conjugates of tricosamers 34 and 35 has shown almost two
fold more potent activity than the reference drug it is less
compared to pentapeptide conjugate 33. The reason for this
could be attributed to the long peptide chain length irre-
spective of being a more hydrophobic entity which would
have resulted in the difficult passage of the molecule across
the bacterial cell membrane.
Based on this observation we were very much projected
towards the conjugation of elastin based peptides with
varying chain length and hydrophobicity. We focused our
attention on tetrapeptide elastin sequences with varying
hydrophobicity. Among the analogs tested GGAP (29),
GGIP (30) and GGFP (31), the latter has exhibited more
activity i.e., GGFP with Phe unit has exerted nearly two
times more potent activity than the antibiotics used where
as the other two tetrapeptides conjugated heterocycle have
shown comparatively less activity than GGFP which could
be due to less hydrophobicity of amino acids present at the
second position. Thus the order of activity among tetra-
peptide conjugates is GGFP [ GGIP [ GGAP.
In the light of the above findings, our subsequent goal
was to incorporate GVGVP and GFGFP pentapeptide
elastin sequences for conjugation. The pentapeptide con-
jugates have shown more than two fold potent activity
compared to reference drugs used. Among these, GFGFP
conjugate (33) has shown slight enhancement in the
activity over GVGVP conjugate (32) which may be due to
the presence of two more hydrophobic Phe units in 33.
Inspection of the results further revealed that the con-
jugate 26 having single Phe residue has shown enhanced
activity. On the other hand, retaining of only one Phe unit
and increasing the peptide chain length has caused further
On the contrary, tricosamers conjugated compounds 34
and 35 have exerted enhanced antifungal activity which
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Int J Pept Res Ther (2012) 18:89–98
97
Table 3 Minimum inhibitory concentration (MIC) in lg/ml of the synthesized conjugates (26–35) against tested bacterial and fungal strains by
microdilution method
Entry
Antibacterial activity
Antifungal activity
Minimum inhibitory concentration (MIC) in lg/mla
E. coli
X. oryzae
K. pneumoniae
C. positive staphylococcus
A. niger
A. flavus
F. oxysporum
26
20
39
41
17
15
14
11
08
10
12
[50
[50
24
–
16
27
29
14
12
12
10
08
09
11
[50
[50
18
–
17
31
35
14
13
11
09
06
09
10
[50
[50
19
–
18
35
38
15
13
11
10
07
12
13
[50
[50
23
–
18
32
34
16
14
12
08
06
03
04
[50
[50
–
21
42
48
17
16
15
09
08
03
03
[50
[50
–
21
39
43
19
18
16
11
07
04
05
[50
[50
–
27
28
29
30
31
32
33
34
35
Heterocycle
Peptides
Amoxicillin
Bavistin
a
23
26
25
Values are mean of three determinations, the ranges of which are \5% of the mean in all cases
were able to arrest the fungal growth at a concentration of
3–5 lg/ml which is five fold more potent than bavistin, the
antibiotic used in the assay. Hence, the conjugates of
tricosamers could be identified as highly potent antifungal
agents.
exhibited potent antimicrobial activity (6–19 lg/ml) than
either heterocycle or peptides ([50 lg/ml) whereas the
conjugates of amino acids displayed moderate activity. Of
particular importance, the conjugates of tricosamers
deserves a special mention as they have revealed extraor-
dinary activity (3–5 lg/ml) in arresting the growth of all
the fungal strains tested which is found to be five fold more
potent than the antibiotic used. Hence, these could be
developed as lead antimicrobial agents.
The theme interest of the present investigation was to
shed some light on the importance of the consequences of
conjugation. Hence, we performed the antimicrobial assay
in which equal amount of peptide and heterocycle were just
mixed in required volume of DMSO to get the concentra-
tion of 50 lg/ml. The results revealed that there was only
very less or no inhibition of the growth of microorganisms
by this technique ([50 lg/ml). This observation led to the
fact that the highly potent activity of these analogs (26–35)
is because of conjugation which aids in providing addi-
tional interaction with the cell membrane there by arresting
the growth of microbes.
Acknowledgments One of the authors RS gratefully acknowledges
Lady Tata Memorial Trust, Mumbai for the award of Junior Research
Scholarship.
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