Purification, structural elucidation and bioactivity of tryptophan containing diketopiperazines, from Comamonas testosteroni associated with a rhabditid entomopathogenic nematode against major human-pathogenic bacteria

Article abstract of DOI:10.1016/j.peptides.2013.09.019

The cell free culture filtrate of a Comamonas testosteroni associated with an Entomopathogenic nematode (EPN), Rhabditis (Oscheius) sp. exhibited promising antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by

Full text of DOI:10.1016/j.peptides.2013.09.019

Peptides 53 (2014) 48–58
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Peptides
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Purification, structural elucidation and bioactivity of tryptophan
containing diketopiperazines, from Comamonas testosteroni
associated with a rhabditid entomopathogenic nematode against
major human-pathogenic bacteria
S. Nishanth Kumara,b,∗, C. Mohandasa,b, Bala Nambisana,b
a Division of Crop Protection, Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram 695017, India
b Division of Crop Utilisation, Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram 695017, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The cell free culture filtrate of a Comamonas testosteroni associated with an Entomopathogenic nematode
(EPN), Rhabditis (Oscheius) sp. exhibited promising antimicrobial activity. The ethyl acetate extract of
the bacterial culture filtrate was purified by silica gel column chromatography to obtain five diketopiper-
azines or cyclic dipeptides (DKP 1–5). The structure and absolute stereochemistry of the compounds were
Received 29 August 2013
Received in revised form
28 September 2013
Accepted 30 September 2013
Available online 10 October 2013
determined based on extensive spectroscopic analyses (HR-MS, 1HNMR, 13CNMR, 1H–1H COSY, 1H–13
C
HMBC) and Marfey’s method. Based on the spectral data the compounds were identified as Cyclo-(L-Trp-L-
Pro) (1), Cyclo-(L-Trp-L-Tyr) (2), Cyclo-(L-Trp-L-Ile) (3), Cyclo-(L-Trp-L-Leu) (4) and Cyclo-(L-Trp-L-Phe)
(5), respectively. Three diketopiperazines (DKP 2, 3 and 5) were active against all the ten bacteria tested.
The highest activity of 0.5 ␮g/ml by Cyclo-(L-Trp-L-Phe) was recorded against Vibrio cholerae followed by
Salmonella typhi (1 ␮g/ml) a human pathogen responsible for life threatening diseases like profuse watery
diarrhea and typhoid or enteric fever. The activity of this compound against V. cholerae and S. typhi is
more effective than ciprofloxacin and ampicillin, the standard antibiotics. Cyclo-(L-Trp-L-Phe) recorded
significant antibacterial activity against all the test bacteria when compared to other compounds. Five
diketopiperazines were active against all the test fungi and are more effective than bavistin the standard
fungicide. Diketopiperazines recorded no cytotoxicity to FS normal fibroblast and VERO cells (African
green monkey kidney) except DKP 3 and 4. To our best knowledge this is the first report of antimicrobial
activity of the tryptophan containing diketopiperazines against the human pathogenic microbes. The
production of cyclic dipeptides by C. testosteroni is also reported here for the first time. We conclude
that the C. testosteroni is promising sources of natural bioactive secondary metabolites against human
pathogenic bacteria which may receive great benefit in the field of human medicine in near future.
© 2013 Elsevier Inc. All rights reserved.
Keywords:
Comamonas testosteroni
Tryptophan
Cyclic dipeptide
Antibacterial
Vibrio cholerae
1. Introduction
secondary metabolites having antimicrobial property [5]. How-
ever, bacterial resistance emerges when an antimicrobial agent is
Antibiotics are one of the pillars of modern medicine. Through-
out the ages, natural products have been the most consistently
successful sources of lead compounds that have found many appli-
cations in the fields of human medicine, pharmacy and agriculture
[52,54]. Microbial natural products have been the source of most of
the antibiotics in current use for the treatment of various infectious
diseases. Many terrestrial born bacteria are reported to produce
introduced into the market or just after its introduction due to
misuse [4]. So there is a continuous need for new chemothera-
peutants, especially novel antibiotics, to combat new diseases and
drug-resistant pathogens that are becoming a significant threat to
public health [56]. The discovery and development of new drugs
from natural products (NPs) has played a significant role over the
last few decades. Over 28% of the new chemical entities and 42%
of the anticancer drugs introduced into the market can be traced
back to NPs [42]. The majority of these have been isolated from
terrestrial-borne microbes. Meanwhile, the emergence of severe
resistance to antibiotics in microbial pathogens, such as methi-
cillin and vancomycin-resistant Staphylococcus aureus (MRSA &
VRSA) and vancomycin-resistant Enterococci (VRE), and the current
∗
Corresponding author at: Division of Crop Protection, Central Tuber Crops
Research Institute, Sreekariyam, Thiruvananthapuram 695017, India.
Tel.: +91 471 2598551x214; fax: +91 471 2590063.
E-mail address: nishanthctcri@gmail.com (S. Nishanth Kumar).
0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.peptides.2013.09.019

S. Nishanth Kumar et al. / Peptides 53 (2014) 48–58
49
increase in the number of new diseases/pathogens, e.g., Gram-
2.2. Pathogen microbial targets
negative New Delhi metallo-beta-lactmase (NMD-1) bacteria have
caused a revival of interest in the discovery of Microbial based
Natural Products (MNPs) with unique scaffolds to meet the urgent
demand for new drugs [57]. Recent trends in drug discovery under-
line that terrestrial microorganisms are a potentially productive
source of novel secondary metabolites and have great potential to
increase the number of NPs in clinical trials.
Gram positive bacteria: Bacillus subtilis MTCC 2756, S. aureus
MTCC 902, Staphylococcus epidermis MTCC 435 and Staphylococ-
cus simulans MTCC 3610; Gram negative bacteria: Escherichia coli
MTCC 2622, Klebsiella pneumoniae MTCC 109, Proteus mirabilis
MTCC 425, Vibrio cholerae MTCC 3905, Pseudomonas aeruginosa
MTCC 2642, and Salmonella typhi MTCC 3216; medically important
fungi: Aspergillus flavus MTCC 183, Candida tropicalis MTCC 184,
Candida albicans MTCC 277, Cryptococcus gastricus MTCC 1715 and
Trichophyton rubrum MTCC 296 and agriculturally important fungi:
Fusarium oxysporum MTCC 284, Rhizoctonia solani MTCC 4634, and
Penicillium expansum MTCC 2006. All the test microorganisms were
purchased from Microbial Type Culture collection Centre, IMTECH,
Chandigarh, India. The test bacteria were maintained on nutrient
agar slants and test fungi were maintained on potato dextrose agar
slants.
Entomopathogenic nematodes carrying symbiotic bacteria rep-
resent one of the best non-chemical strategies supporting insect
control [36,59]. In the soil, the infective juveniles of these nema-
todes (Heterorhabditis or Steinernema genera) actively seek the
host, penetrate through insect’s natural openings, travel to the
hemocoel and release symbiotic bacterial cells (Photorhabdus and
Xenorhabdus spp.). The bacteria multiply and release a number
of virulence factors, including complexes of toxins, hydrolytic
enzymes, hemolysins, and antimicrobial compounds that kill the
insect host within 48 h [20], thus providing nutrients for the nema-
todes development and reproduction within the insect cadaver.
By doing so, the bacterial symbionts are believed to prevent
putrefaction of the insect cadaver and establish conditions that
favor the development of both the nematode and bacterial sym-
bionts. These metabolites have been used in biological pesticides
and therapeutic agents such as antibiotic, antimycotic, insectici-
dal, nematicidal, antiulcer, antineoplastical and antiviral for many
decades [55]. The antimicrobial nature of metabolites produced
by Xenorhabdus and Photorhabdus spp. is known, and several of
such compounds with antibiotic activity have been isolated and
identified [21].
2.3. Bacterial isolation
The bacterium was isolated from the haemolymph of G. mel-
lonella infected with IJs of Rhabditis (Oscheius). Dead G. mellonella
larvae were surface sterilized in 70% alcohol for 10 min, flamed
and allowed to dry in a laminar airflow chamber for 10 min. Then
larvae were opened with sterile needles and scissors, care being
taken not to damage the gut, and a drop of the oozing haemolymph
was streaked onto nutrient agar plates. After 24–48 h incubation
at 30 ◦C, single colonies on the nutrient agar plates were selected
and aseptically transferred to fresh nutrient agar medium in slant
tubes.
During our studies on EPN, a new entomopathogenic nema-
tode belonging to the genus Rhabditis and subgenus Oscheius was
isolated from soil samples collected from Central Tuber Crops
Research Institute (CTCRI) farm, Thiruvananthapuram [41]. The
nematodes can be cultured on laboratory reared Galleria mellonella
larvae and maintained alive for several years. The specific symbi-
otic bacteria were found to be associated with the nematode [41]
and can be isolated from the haemolymph of nematode infested
G. mellonella larvae. Based on molecular characteristics, nematode
resembles Rhabditis (Oscheius) at D2 and D3 (nucleotide sequence
region) expansion segments of 28S rDNA [18]. The cell free cul-
ture filtrate of the bacteria was found to inhibit several pathogenic
bacteria and fungi [41], suggesting that it could be a rich source of
biologically active compounds. In the present study we reported
the taxonomic study of the symbiotic bacteria, purification and
structure elucidation of five tryptophan containing diketopiper-
azines, its antimicrobial activity with special reference to medically
important bacteria. The present study also reported the antimicro-
bial tryptophan containing diketopiperazines for the first time from
Comamonas testosteroni.
2.4. 16S rRNA sequencing and phylogenetic analysis
Genomic DNA was extracted from bacterial cultures through
enzymatic hydrolysis [43]. The complete 16S rRNA gene
(1.4–1.5 kp) was amplified via PCR, using universal bacterial
primers 27 F (5ꢀ-AGA GTT TGATCC TGG CTC AG-3ꢀ) and 1541R (5ꢀ-
AAG GAG GTG ATC CAG CCG CA-3ꢀ). The amplification was carried
out on a DNA thermal cycler (BIORAD, USA). The 50 ␮l PCR reactions
contained 4 ␮l of 2.5 U/␮l Taq DNA polymerase (Genei, Bangalore,
India), 5 ␮l of 10× buffer (Genei), 1 ␮l of 20 mM dNTPs (Genei),
37 ␮l of SDW, 1 ␮l of 50 ␮M each primer, and 1 ␮l of template.
The PCR conditions were initial denaturation at 94 ◦C for 4 min,
30 amplification cycles of 94 ◦C for 1 min, 56 ◦C for 1 min, and 72 ◦C
for 2 min, and a final elongation at 72 ◦C for 10 min. The PCR product
was purified using a QIAquick Gel extraction kit (QIAGEN, Tokyo,
Japan) and sequenced in both directions using the same primers as
for the PCR amplification. The nucleotide sequence obtained was
processed to remove low quality reads, transformed into consen-
sus sequences with Geneious Pro software version 5.6. The resulted
high quality sequences were analyzed with BLASTn (NCBI) to con-
firm the authenticity of the bacterium. The sequences of related
species and genus were downloaded from the Genbank database
and a phylogenetic study was carried out with the program MEGA
version 5 [51]. Sequences were aligned using the computer package
ClustalW [53] and were analyzed to determine the relationships
between isolates by the neighbor-joining method [44] using the
Maximum Composite Likelihood model. Bootstrap values were
generated using 2000 replicates.
2. Materials and methods
2.1. Chemicals and media
All the chemicals used for extraction, column chromatogra-
phy and high performance liquid chromatography (HPLC) were
from Merck Limited, Mumbai, India. Silica gel (230–400 mesh)
used for column chromatography and precoated silica gel 60 GF254
plates used for Thin Layer Chromatography (TLC) were from Merck
Limited, Germany. Microbiological media were from Hi-Media Lab-
oratories Limited, Mumbai, India. All other reagents and chemicals
used in this study were of the highest purity. The standard antibi-
otics ciprofloxacin, ampicillin and amphotericin B were purchased
from Sigma Aldrich (USA). The software used for the chemical struc-
ture drawing was Chemsketch Ultra, Toronto, Canada.
2.5. Fermentation and extraction
The bacterial fermentation was carried out using modified Tryp-
tic soya broth (TSB) (tryptone 17 g/l, soytone 3.0 g/l, glucose 2.5 g/l,
NaCl 5.0 g/l, meat peptone 10 g/l, water 1000 ml) supplemented by
0.1% tryptophan. A single colony of Comamonas testosteroni from

50
S. Nishanth Kumar et al. / Peptides 53 (2014) 48–58
the agar plate was inoculated into the flask containing 100 ml
sterile media. The flasks were incubated in a gyrorotatory shaker
(120 rpm) at 28 ◦C in dark for 24 h. When the optical density of
the culture at 600 nm was approximately 1.7, the bacterial cul-
tures were transferred aseptically into 400 ml sterile medium and
incubated in the gyrorotatory shaker at 28 ◦C in dark for 72 h.
After fermentation, the culture media was centrifuged (10,000 × g,
20 min, 4 ◦C) followed by filtration through a 0.22 ␮m filter, to
obtain cell free culture filtrate. Thirty liters of cell free culture fil-
trate were neutralized with concentrated hydrochloric acid and
extracted with an equal volume of ethyl acetate thrice. Then the
ethyl acetate layers were combined, dried over anhydrous sodium
sulphate, and concentrated at 35 ◦C using a rotary flash evaporator.
2.9.4. Optical rotations
Optical rotation of the compounds was measured using a
Rudolph Research Autopol III polarimeter (Hackettstown, NJ, USA)
at 25 ◦C in acetone.
2.9.5. Differential scanning calorimetry
The melting point of the compounds was measured using a
differential scanning calorimeter in a Mettler Toledo DSC 822e
instrument (Mettler-Toledo, Schcoerfenbach, Switzerland). Tem-
perature ranges from 25 ◦C to 300 ◦C were employed.
2.10. Absolute configuration determination of compounds
A solution of compounds (1.5 mg) in 6 M HCl (1 ml) was heated
to 120 ◦C for 24 h. The solution was then evaporated to dryness and
the residue redissolved in H2O (100 ␮l) and was then placed in a
1 ml reaction vial and treated with a 2% solution of FDAA (200 ␮l)
in acetone followed by 1.0 M NaHCO3 (40 ␮l). The reaction mix-
ture was heated at 47 ◦C for 1 h, cooled to room temperature, and
then acidified with 2.0 M HCl (20 ␮l). In a similar fashion, standard
D- and L-amino acids were derivatized separately. The derivatives
of the hydrolysates and standard amino acids were subjected to
analytical HPLC analysis (Shimadzu LC-20AD, C18 column; 5 ␮m,
4.6 mm × 250 mm; 1.0 ml/min) at 30 ◦C using the following gradi-
ent program: solvent A, water + 0.2% TFA; solvent B, MeCN; linear
gradient 0 min 25% B, 40 min 60% B, 45 min 100% B; UV detection
at 340 nm [39].
2.6. Fractionation of the crude ethyl acetate extract
Activated silica gel (230–400 mesh) was packed onto a glass
column (600 × 30 mm) using n-hexane solvent and eluted suc-
cessively with 200 ml of 100% hexane, 200 ml of linear gradient
hexane: dichloromethane (v/v, 75:25 to 25:75), 200 ml of 100%
dichloromethane, 200 ml of linear gradient dichloromethane:ethyl
acetate (v/v, 95:5 to 5:95), 200 ml of 100% ethyl acetate and finally
with 200 ml of 100% methanol. Two fractions (100 ml each) were
collected from each combination. About 82 fractions measuring
100 ml each were collected and concentrated by using the rotary
evaporator.
2.7. Thin-layer chromatography (TLC)
2.11. Antibacterial activity
An aliquot of the pure compounds were loaded on the activated
silica gel TLC plates (20 cm × 20 cm). The plates were developed
using 40% benzene in acetone. Spots were located by exposing the
plate to iodine fumes.
2.11.1. Minimum inhibitory concentration (MIC)
The minimum inhibitory concentration of the compounds was
determined according to the method described by the Clinical and
Laboratory Standards Institute [14], with some modifications. Two
fold serial dilutions of the antibiotics and peptide compounds were
made with Mueller Hinton broth (MHB) to give concentrations
ranging from 0.12 to 1000 ␮g/ml. Hundred microliters of test bac-
terial suspension were inoculated in each tubes to give a final
concentration of 1 × 105 CFU/ml. The tubes were incubated for 24 h
at 37 ◦C. The control tube did not have any antibiotics or test com-
pounds, but contained the test bacteria and the solvent used to
dissolve the antibiotics and compounds. The growth was observed
both visually and by measuring OD at 600 nm. The lowest con-
centration of the test compound showing no visible growth was
recorded as the MIC. Triplicate sets of tubes were maintained for
each concentration of the test sample. Ciprofloxacin and ampicillin
were used as positive control.
2.8. Analytical HPLC
HPLC analysis of pure compounds was carried out on a LC-10AT
liquid chromatography (LC; Shimadzu, Singapore) equipped with a
C-18 column (5 ␮m, 4.6 mm × 250 mm) (150 mm × 3.9 mm, 5 mm).
Mobile phase: 100% methanol. Flow rate: 1.0 ml/min. Detection:
220 nm (diode array detector).
2.9. Structure determination of antimicrobial compound
2.9.1. UV spectrophotometer
UV–visible spectrum of the pure compounds was recorded on
a Systronics double beam spectrophotometer 2201, India at room
temperature (scanning range 190–800 nm).
2.11.2. Minimum bactericidal concentration (MBC)
Minimum bactericidal concentration was determined according
to the method of Smith-Palmer et al. [48]. About 100 ␮l from the
tubes not showing bacterial growth in the MIC test were serially
diluted and plated on Nutrient agar. The plates were incubated at
37 ◦C for 24 h. Minimum bactericidal concentration is defined as
the concentration at which bacteria failed to grow on nutrient agar
inoculated with 100 ␮l test bacterial suspensions.
2.9.2. Nuclear magnetic resonance (NMR)
The structure of the compounds was determined using nuclear
magnetic resonance (NMR) spectroscopy (Bruker DRX 500 NMR
instrument, Bruker, Rheinstetten, Germany) equipped with a 2.5-
mm microprobe. NMR Spectrometer using CDCl3 was deployed to
measure 1H and 13C and 2D NMR. All spectra were recorded at 23 ◦C.
One-dimensional 1H NMR experiments as well as two-dimensional
1H–1H correlation spectroscopy, 1H–13C heteronuclear multiple
bond correlation, and 1H–13C heteronuclear multiple quantum
coherence (HMQC) experiments were performed according to
Bruker standard pulse sequences. Chemical shifts are reported rel-
ative to the solvent peaks. (CDCl3: 1H ı 7.24 and 13C ı 77.23).
2.11.3. Antibacterial assay by disk diffusion technique
The antimicrobial activity of the pure compounds was deter-
mined by the disk diffusion method [15] against bacteria. The test
cultures maintained in nutrient agar slant at 4 ◦C were sub-cultured
in nutrient broth to obtain the working cultures approximately
containing 1 × 106 CFU/ml. The MIC concentration of the peptide
compounds was incorporated in a 6 mm sterile disk. Mueller Hin-
ton (MH) agar plates were swabbed with each bacterial strain
and the test disks were placed along with the control disks.
2.9.3. High resolution mass spectrophotometer (HRMS)
HRMS was performed on a Thermo Scientific Exactive Orbitrap
LC-Mass Spectrometer with an electrospray ionization mode.

S. Nishanth Kumar et al. / Peptides 53 (2014) 48–58
51
Ciprofloxacin disks (5 ␮g/disk) were used as positive control. Plates
2.14. Statistical analysis
were incubated overnight at 37 ◦C for 24 h. Clear, distinct zone
of inhibition was visualized surrounding the discs. The antimi-
crobial activity of the test agents (compounds and antibiotics)
was determined by measuring the zone of inhibition expressed in
mm.
All statistical analyses were performed with SPSS (Version 17.0;
SPSS, Inc., Chicago, IL, USA). Data for disk diffusion assay was pre-
sented as means standard deviations.
2.15. Sequence submitted to GenBank
2.12. Antifungal assay
Partial sequence data for the 16S rRNA gene of Comamonas
testosteroni have been deposited in the GenBank (NCBI) nucleotide
database under the ac. no. (HQ200410).
2.12.1. Minimum inhibitory concentration (MIC)
The MIC was performed by broth microdilution methods as
per the guidelines of Clinical and Laboratory Standard Institute
[10,13], with RPMI 1640 medium containing L-glutamine, with-
out sodium bicarbonate and buffered to pH 7.0 (both from Sigma).
Twofold serial dilutions of the peptide compounds were prepared
in media in amounts of 100 ␮l per well in 96-well microtiter
plates (Tarson, Mumbai, India). The test fungal suspensions were
further diluted in media, and a 100 ␮l volume of this diluted inocu-
lums was added to each well of the plate, resulting in a final
inoculum of 0.5 × 104 to 2.5 × 104 CFU/ml for Candida species and
0.4 × 104 to 5 × 104 CFU/ml for other fungi. The final concentra-
tion of peptide compounds ranged from 1 to 1000 ␮g/ml. The
medium without the agents was used as a growth control and the
blank control used contained only the medium. Amphotericin B
served as the standard drug controls. The microtiter plates were
incubated at 35 ◦C for 48 h for Candida species and 30 ◦C for 72 h.
for other fungi. The plates were read using ELISA, and the MIC
was defined as the lowest concentration of the antifungal agents
that prevented visible growth with respect to the growth con-
trol.
3. Results
3.1. Identification of bacterium
The bacteria isolate (Comamonas testosteroni strain) was identi-
fied based on 16S rRNA gene sequencing. PCR amplification yielded
∼1500 bp amplicon. Blast analysis showed 99% similarity to C.
testosteroni sequence available in the Genbank database and thus
the bacterium was identified as C. testosteroni. Partial sequence
data for the 16S rRNA gene have been deposited in the GenBank
(NCBI) nucleotide database under the ac. no. (HQ200410). The phy-
logram clearly portrayed the relationships of the isolates used for
the analysis. The present bacteria isolate (C. testosteroni strain)
was successfully grouped along with other C. testosteroni isolates
obtained from the Genbank database confirming the authenticity
of the isolate (Fig. 1). The strain was currently deposited in IMTECH
(Institute of Microbial Technology, Chandigarh; India).
3.2. Isolation and purification of bioactive compounds
2.12.2. Antifungal assay by disk diffusion technique
The ethyl acetate extract of the cell free culture filtrate of the
bacterium showed significant antibacterial activity against B. sub-
tilis. Silica gel column chromatography of this extract yielded five
crystal compounds. The column solvent and yield were shown in
Table 1. These crystal compounds were further purified by crystal-
lization using hexane and benzene. Bioactivity of these compounds
was confirmed by testing against the indicator test microorganism
B. subtilis. TLC of these purified crystal compounds recorded sin-
gle spots and RF value is presented in Table 1. HPLC analysis of
the compounds was performed by reverse phase and compounds
were eluted as single peaks (Table 1 and Fig. 2). The purity of the
compounds reached greater than 90% according to the peak area.
Compounds were screened for their antifungal activity against
test fungi by disk diffusion method [11,12]. The fungal cultures
were grown on potato dextrose broth. The mycelia mat of fungi
of 7-day old culture was suspended in normal saline solution and
test inoculum was adjusted to 5 × 105 CFU/ml. Inocula (0.1 ml) were
applied on the surface of the potato dextrose agar plate and spread
by using a cotton swab. Subsequently, filter paper discs (6 mm in
diameter, Hi-media) containing MIC concentrations of test com-
pounds were placed on the agar plates and incubated at 35 ◦C for
24–48 h. Afterwards, the diameter of the inhibition zone was mea-
sured.
3.3. Identification of bioactive compound
2.13. Cytotoxicity test
The five pure compounds were subjected to various spectro-
scopic analyses, i.e. UV, HR-MS and NMR. The structure of these five
compounds corresponded to different diketopiperazines (DKPs)
or cyclic dipeptides, The diketopiperazines identified are Cyclo-
(L-Trp-L-Pro) (1), Cyclo-(L-Trp-L-Tyr) (2), Cyclo-(L- Trp-L-Ile) (3),
Cyclo-(L-Trp-L-Leu) (4) and Cyclo-(L-Trp-L-Phe) (5), respectively
(Fig. 3).
The MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetra-
zolium bromide) assay was used to determine the cytotoxicity of
diketopiperazines. Lung cancer cell line (A 549) and colon cancer
cell line (HTL 116), FS normal fibroblast and African green mon-
key kidney cell line (VERO) were used for testing. Briefly, cells
(5 × 103 well−1) were seeded in 0.2 ml of the medium (DMEM with
10% PBS) in 96 well plates, treated with drugs for 72 h. and after
incubation, cytotoxicity was measured. For this after removing the
drug containing media, 25 ␮l of MTT solution (5 mg/ml in PBS) and
75 ␮l of complete medium were added to wells (untreated and
treated) and incubated for 2 h. At the end of incubation MTT lysis
buffer was added to the wells (0.1 ml well−1) and incubated for
another 4 h at 37 ◦C. At the end of incubation, the optical densities
at 570 nm were measured using a plate reader (Biorad ELISA reader
680, California, USA). The relative cell viability in percentage was
calculated (A570 of treated sample/A570 of untreated sample × 100)
[2].
Table 1
Isolation and purification details of pure compounds.
Compound
Column solvent
Yield (mg)
RF value
Retention
time (min)
1
2
3
4
5
25% DCM in hexane
100% DCM
10% ethyl acetate in DCM
30% ethyl acetate in DCM
65% ethyl acetate in DCM
16
18
36
30
23
0.33
0.39
0.27
0.44
0.37
2.579
4.167
2.927
3.234
3.719

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S. Nishanth Kumar et al. / Peptides 53 (2014) 48–58
Fig. 1. Phylogenic relationships of Comamonas testosteroni strain isolated from Rhabditis (Oscheius) sp. and known bacterial relatives based on 16S rRNA gene sequences
(neighbor-joining method).
DKP
1:
Cyclo(L-Trp-L-Pro),
(3S,8aS)-3-(1H-indol-3-
dd, J = 14.4, 4.1 Hz, H-8), 2.84 (1H, dd, J = 2.7, 0.8 Hz, H-12), 1.80 (1H,
m, H-15), 1.13 (2H, m, H-16), 0.69 (3H, d, J = 6.5 Hz, H-18), 0.58
(3H, t, J = 7.3 Hz, H-17); 13C NMR (CDCl3, 125 MHz): 172.3, 171.3,
138.2, 128.8, 126.8, 122.9, 120.7, 120.7, 113.5, 110.3, 59.2, 58.3, 39.9,
32.3, 27.2, 14.9, 12.0. Based on the spectral data the molecular for-
mula was determined to be C17H22N3O2 by HRMS (m/z 299.18143
[M + H]), calcd. for C17H21N3O2, 299.16344).
DKP 4: Cyclo-(L-Trp-L-Leu) (3S,6S)-3-(1H-indol-3-ylmethyl)-6-
(2-methylpropyl)piperazine-2,5-dione: was obtained as colorless
crystals. Melting point: 217.28 ◦C; [a]D −63 (c, 0.02, MeOH); UV
max: 208 nm (MeOH); 1H NMR (CDCl3, 500 MHz) ä 7.59 (1H, dd,
J = 8.1, 1.0 Hz, H-7), 7.32 (1H, dd, J = 8.1, 1.0 Hz, H-4), 7.01 (1H, ddd,
J = 8.1, 7.0, 1.0 Hz, H-6), 6.99 (1H, s, H-2), 6.96 (1H, ddd, J = 8.1,
7.0, 1.0 Hz, H-5), 4.22 (1H, dd, J = 4.1, 3.8 Hz, H-9), 3.41 (1H, dd,
J = 14.6, 3.8 Hz, H-8), 3.23 (1H, dd, J =14.6, 4.1 Hz, H-8), 1.57 (1H,
m, H-12), 1.44 (2H, dd, J = 7.8, 4.6 Hz, H-15), 1.43 (1H, m, H-16),
0.74 (3H, d, J = 6.2 Hz, H-18), 0.58 (3H, d, J = 6.2 Hz, H-17); 13C
NMR (CDCl3, 125 MHz): 172.3, 172.1, 138.8, 129.1 126.0, 123.7,
120.7, 119.9, 112.9, 110.1, 58.1, 54.3, 42.4, 31.7, 25.8, 23.5, 22.4.
Based on the spectral data the molecular formula was determined
to be C17H22N3O2 by HRMS (m/z 299.18142 [M + H]), calcd. for
ylmethyl)hexahydropyrrolo[1,2- a]pyrazine-1,4-dione: was obtained
as white crystalline powder; Melting point: 285.58 ◦C; [a]D −128
(c, 0.02, MeOH); UV max: 220 nm (MeOH); 1H NMR (CDCl3,
500 MHz) ␦ 1.79–2.15 (2H, m, H-4), 2.42–2.52 (2H, m, H-5), 2.90
(1H, dd J = 10.7 Hz/15.1 Hz, Ha-10), 3.54(2H, m, H-3), 3.71(1H, dd
J = 3.9 Hz/15.0 Hz, Hb-10), 4.28 (1H, t J = 8.0 Hz, H-6), 4.28 (1H, dd
J = 2.7 Hz/10.6 Hz, H-9); 5.84 (1H, brs, N H), 7.12–7.70 (5H, m,
Ar H), 8.32 (1H, brs, N H (Indole). 13C NMR (CDCl3, 125 MHz),
169.99, 46.30, 23.52, 29.23, 60.12, 166.22, 55.34, 27.79, 124.15,
110.76, 119.37, 120.17, 123.16, 112.12, 127.34, 136.18. Based
on the spectral data the molecular formula was determined to
be C16H18N3O2 by HRMS (m/z 284.34167 [M + H]), calcd. for
C16H17N3O2, 283.32345.
DKP 2: Cyclo-(L-Trp-L-Tyr) (3S,6S)-3-(4-hydroxybenzyl)-6-(1H-
indol-3-ylmethyl)piperazine-2,5-dione: was obtained as colorless
amorphous solid. Melting point: 265.58 ◦C; [a]D −118 (c, 0.02,
MeOH); UV max: 222 nm (MeOH); 1H NMR (CDCl3, 500 MHz): 2.64
(dd, J = 11.9, 8.4 Hz, 1H, H-15a), 1.54 (dd, J = 11.2, 8.3 Hz, 1H, H-15b),
3.24 (dd, J = 14.6, 7.9 Hz, 1H, H-8a), 2.83 (dd, J = 14.8, 7.9 Hz, 1H, H-
8b), 3.93 (m, 1H, H-12), 4.23 (m, 1H, H-9), 6.72 (d, J = 7.0 Hz, 2H,
H-18 and H-20), 6.54 (d, J = 7.0 Hz, 2H, H-17 and H-21), 7.21 (s, 1H,
H-2), 7.13 (dd, J = 7.4, 7.3 Hz, 1H, H-5), 7.23 (dd, J = 7.3, 7.3 Hz, 1H, H-
6), 7.34 (d, J = 7.4 Hz, 1H, H-7), 7.64 (d, J = 7.3 Hz, 1H, H-4). 13CNMR
(125 MHz in CDCl3): 169.9, 169.6, 156.7, 138.8, 132.3, 129.4, 128.9,
126.7, 123.8, 121.3, 120.8, 116.4, 112.7, 109.9, 58.0, 57.4, 40.8, 30.9.
Based on the spectral data the molecular formula was determined
to be C20H20N3O3 by HRMS (m/z 350.14584 [M + H]), calcd. for
C20H19N3O3, 349.14573).
C17H21N3O2, 299.16347).
DKP 5: Cyclo-(L-Trp-L-Phe). (3S,6S)-3-benzyl-6-(1H-indol-3-
ylmethyl)piperazine-2,5-dione: was obtained as colorless crystals.
Melting point: 270.28 ◦C; [a]D −153 (c, 0.02, MeOH); UV max:
220 nm (MeOH); IHNMR (500 MHz, DMBO-d6): 61.89 (1H, dd,
J = 13.49, 7.02 Hz, H-17), 2.48 (1H, dd, J = 13.49, 4.70 Hz, H-17),
2.63(1H, dd, J = 14.47, 5.68 Hz, H-10), 2.89 (1H, dd, J = 14.47,
4.46 Hz, H-10), 3.94 (1H, m, H-14), 3.98 (1H, m, H-11), 6.80
(2H, m, H-20, 22), 7.01 (1H, d, J = 2.20 Hz, H-2), 6.98 (1H, ddd,
J = 7.17, 7.45, 1.45 Hz, H-5), 7.18 (1H, ddd, J = 7.73, 7.45, 1.00 Hz,
H-6), 7.26 (2H, m, H-19, 23), 7.29 (1H, m, H-21), 7.32 (1H, dd,
J = 7.73, 7.45 Hz, H-7), 7.54 (1H, dd, J = 7.17, 1.00 Hz, H-4), 7.83
(1H, d, J = 2.00 Hz, H-15), 7.97 (1H, d, J = 2.00 Hz, H-12), 11.29
(1H, s, H-1)), 13CNMR (125 MHz, DMSO-d6): 30.69, 39.96, 56.21,
55.64, 109.15, 111.33, 119.47, 119.91, 121.83, 124.41, 126.36,
DKP 3: Cyclo-(L-Trp-L-Ile) (6S)-3-(butan-2-yl)-6-(1H-indol-3-
ylmethyl) piperazine-2, 5-dione: was obtained as colorless crystals.
Melting point: 205.58 ◦C; [a]D −102 (c, 0.02, MeOH); UV max:
214 nm (MeOH); 1H NMR (CDCl3, 500 MHz): 7.59 (1H, dd, J = 8.7,
1.0 Hz, H-7), 7.29 (1H, dd, J = 8.6, 1.0 Hz, H-4), 6.99 (1H, ddd, J = 8.3,
7.0, 1.0 Hz, H-6), 7.01 (1H, s, H-2), 6.92 (1H, ddd, J = 8.1, 7.0, 1.0 Hz,
H-5), 4.26 (1H, m, H-9), 3.70 (1H, dd, J = 14.4, 4.2 Hz, H-8), 3.16 (1H,

S. Nishanth Kumar et al. / Peptides 53 (2014) 48–58
53
Fig. 2. Analytical HPLC profile of diketopiperazines. (A) Cyclo-(L-Trp-L-Pro), (B) Cyclo-(L-Trp-L-Tyr), (C) Cyclo-(L- Trp-L-Ile), (D) Cyclo-(L-Trp-L-Leu) and (E) Cyclo-(L-Trp-L-
Phe).
127.54, 128.03, 129.70, 136.07, 136.56, 166.22, 167.18. Based on
the spectral data the molecular formula was determined to be
C20H20N3O2 by HRMS (m/z 334.38765 [M + H calcd, for C20H19N3O2,
333.14752).
respectively. The FDAA derivatives of Trp and Ile in the hydrolysate
of DKP 3 were 37.188 and 25.123 min (Fig. S3), respectively. The
FDAA derivatives of Trp and Leu in the hydrolysate of DKP 4 were
37.456 and 27.567 min (Fig. S4), respectively. The FDAA derivatives
of Trp and Phe in the hydrolysate of DKP 5 were 37.346 and
26.448 min (Fig. S5), respectively.
3.4. Absolute configuration determination of compounds
The modified Marfey’s method was successfully applied
to the determination of the absolute configuration of com-
pounds. Regarding the absolute stereochemistry, all the
compounds contain L amino acids except DKP 1, which con-
tain L-proline and D-tryptophan (Supplementary Figs. S1–S5).
All the derivatized obtained from the hydrolysis of the dike-
topiperazines were compared with the retention times of the
derivatized standard D and L-amino acids. The retention times
for the FDAA derivatives of standard amino acids are pre-
sented in Table 2. The FDAA derivatives of Trp and Pro in the
3.5. Bioactivity
3.5.1. Antibacterial activity
The isolated compounds were tested for antibacterial activity
against ten bacterial strains using standard methods. MIC and MBC
values were also determined and are shown in Table 3. The microor-
ganism that presented highest sensitivity toward DKP 1 was B.
subtilis and S. aureus. DKP 2 was active against all the test bacteria
and best activity of this compound was recorded against S. epider-
mis (8 ␮g/ml), followed by P. aeruginosa and S. typhi (16 ␮g/ml). DKP
3 presented highest activity against E. coli and S. typhi (4 ␮g/ml).
But DKP 4 is active only against four test bacteria and highest
activity was recorded against S. aureus (125 ␮g/ml). Interestingly
hydrolysate of DKP
1 were 40.344 and 18.453 min, respec-
tively (Fig. S1). The FDAA derivatives of Trp and Tyr in the
hydrolysate of DKP 2 were 37.188 and 25.123 min (Fig. S2),

54
S. Nishanth Kumar et al. / Peptides 53 (2014) 48–58
Table 2
HPLC reversed-phase retention times of the L and D-amino acid standard derivatives.
Amino acid derivative
Retention time (min)
Amino acid derivative
Retention time (min)
L-Trp
L-Pro
L-Tyr
L-Ile
L-Leu
L-Phe
37.342
17.451
20.514
25.234
27.248
26.897
D-Trp
D-Pro
D-Tyr
D-Ile
D-Leu
D-Phe
40.232
18.344
33.084
23.567
24.045
31.456
Table 3
MIC and MBC of diketopiperazines against bacteria.
Test bacteria
MIC (␮g/ml)
DKP 1
DKP 2
MIC
DKP 3
DKP 4
MIC
DKP 5
Ampicillin
MIC
Ciprofloxacin
MIC
MBC
MBC
MIC
MBC
MBC
MIC
MBC
MBC
MIC
MBC
B. subtilis
S. aureus
E. coli
P. aeruginosa
S. epidermis
P. mirabilis
V. cholerae
K. pneumonia
S. simulans
S. typhi
16
16
500
125
–
–
–
250
64
–
32
32
500
250
–
–
–
500
125
–
32
64
32
16
8
32
125
64
32
16
64
64
32
16
16
64
250
64
64
32
16
8
4
32
32
64
32
16
32
4
32
8
4
32
64
128
32
16
64
8
250
125
500
–
–
–
–
–
125
–
500
250
1000
–
–
–
–
–
125
–
4
8
8
16
2
2
0.5
16
16
1
8
8
16
16
2
2
1
16
32
1
100
250
100
500
125
64
250
–
–
100
250
250
1000
125
125
500
–
2
2
1
2
2
4
2
2
4
4
2
2
2
4
4
8
4
4
8
4
–
–
–
Values represent mean of three replications. –, no MIC upto 1000 ␮g/ml.
DKP 5 recorded significantly good activity against test pathogens
in impressive low concentration and best activity was recorded
against V. cholerae (0.5 ␮g/ml) followed by S. typhi (1 ␮g/ml).
It appeared that effective MIC also represents the effective
bactericidal concentration of the bacteria tested. The activity of the
test compounds was higher than the standard antibiotic ampicillin
but lower than that of ciprofloxacin. But the activity of DKP 5 against
V. cholerae and S. typhi was significantly better than ciprofloxacin
(Table 3).
3.5.2. Antifungal activity
Antifungal activity against eight fungi and corresponding MIC
values are indicated in Table 4. Five compounds recorded good
antifungal activity against all the tested fungi. DKP 1 and 2 exhib-
ited best MIC value against P. expansum (8 ␮g/ml and 32 ␮g/ml).
DKP 3 showed significantly higher activity against R. solani and
P. expansum (4 ␮g/ml), followed by C. albicans, C. gastricus and C.
tropicalis (8 ␮g/ml). DKP 4 recorded significant activity against all
fungi except C. gastricus. Whereas DKP 5 was highly active against
all the eight test fungi and highest activity was recorded against
F. oxysporum and R. solani (2 ␮g/ml). Amphotericin B showed anti-
fungal activity at 16 ␮g/ml for T. rubrum, whereas DKP 5 recorded
activities at 4 ␮g/ml. This activity is significantly better than the
activity of Amphotericin B. DKPs recorded higher antifungal activ-
ity than the standard fungicide Bavistin against certain fungi
(Table 4).
3.5.3. Agar disk diffusion assay
The result of disk diffusion assay of DKPs against test bacteria
and fungi is presented in Table 5. Best activity was recorded by
DKP 5 against S. typhi (35 mm), followed by K. pneumonia (33 mm)
(Table 5 and Fig. 4). DKP 1 recorded best against T. rubrum (31 mm),
followed by P. expansum (26 mm). For DKP 2 and 3 best activity
was recorded against S. epidermis (34 and 32 mm). Where as DKP 4
recorded best activity against R. solani (27 mm).
3.6. Cytotoxicity activity
Cytotoxicity activity of DKPs against cancer and normal cell
lines was determined by MTT assay after 72 h of treatment and
was shown in Fig. 5. DKP 2 and 5 recorded activity against A549
cells and the IC50 was recorded at 100 and 50 ␮g/ml (Fig. 5IA).
Where as DKP 5 alone recorded activity against HTL 116 (Fig. 5IB).
Fig. 3. Structure of five tryptophan diketopiperazines. (A) Cyclo-(L-Trp-L-Pro), (B)
Cyclo-(L-Trp-L-Tyr), (C) Cyclo-(L- Trp-L-Ile), (D) Cyclo-(L-Trp-L-Leu) and (E) Cyclo-
(L-Trp-L-Phe).

S. Nishanth Kumar et al. / Peptides 53 (2014) 48–58
55
Table 4
MIC of diketopiperazines against fungi.
Test fungi
MIC (␮g/ml)
DKP 1
DKP 2
DKP 3
DKP 4
DKP 5
Bavistin
Amphotericin B
A. flavus
32
64
16
16
8
32
128
64
250
250
64
64
32
–
500
500
32
8
32
4
4
8
16
16
8
32
32
64
64
–
32
16
2
2
4
8
8
4
100
–
16
32
64
–
–
32
–
–
–
16
8
16
C. albicans
F. oxysporum
R. solani
P. expansum
C. gastricus
C. tropicalis
T. rubrum
8
16
–
–
Values represents mean of three replications. –, not tested.
Table 5
Antimicrobial activity of diketopiperazines against test bacteria and fungi.
Test organisms
Zone of inhibition (dia. in mm)
DKP 1 DKP 2
DKP 3
DKP 4
DKP 5
Ciprofloxacin
Bavastin
Amphotericin B
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
B. subtilis
S. aureus
E. coli
P. aeruginosa
S. epidermis
P. mirabilis
V. cholerae
K. pneumonia
S. simulans
S. typhi
17
18
20 1.52
21
–
–
–
22
16 1.2
–
15
23
25 0.57
24 1.73
26
20
18
1
1
23
17 0.57
29
32 0.57
34 0.57
21
22 0.57
29 0.57
24
19 1.2
21 1.2
25
22
21
27
–
17
0
18
0
17
20 0.57
21 1.52
–
–
–
–
–
–
–
22
26
21 1.52
27 1.15
15 1.52
22
21
17
1
25
27
26 1.15
30 1.15
20 0.57
24
23
33 1.52
30 1.73
35 1.73
25 1.52
23
29
27
30 0.57
25 1.15
0
1
26
27
32 1.15
30
29
31 0.57
31
0
1
16 0.57
19 1.15
30 1.15
32 1.15
15
22 1.73
21
–
22
27 1.73
15
19
25 1.15
23 1.15
22
20
21
0
1
0
1
0
1
1
0
0
0
0
1
28 1.52
25 0.57
0
30 1.52
a
A. flavus
0
1
1
0
24 1.52
a
a
a
a
a
a
a
a
C. albicans
F. oxysporum
R. solani
P. expansum
C. gastricus
C. tropicalis
T. rubrum
1
1
0
0
1
0
0
1
0
24 1.73
a
16
19 1.52
0
a
a
1
0
0
24 1.15
a
1
0
0
1
0
1
23 1.15
36 1.73
27
a
a
0
23
32
0
1
31 0.57
29 0.57
1
–, not tested as the MIC value is above 1000 ␮g/ml.
a
Not tested.
Fig. 4. Antibacterial activity of DKP 5 by disk diffusion method.
When exposed to DKPs in the range, 5–100 ␮g/ml, DKPs recorded
no cytotoxicity against VERO cells except DKP 4 (Fig. 51IA). DKP 4
recorded significant cytotoxicity in VERO cells at 100 ␮g/ml. Where
as DKP 3 and 4 recorded significant cytotoxicity against FS cells
(Fig. 5 1IB).
4. Discussion
Diketopiperazines are the smallest cyclic peptides known, com-
monly biosynthesised from amino acids by a large variety of
organisms, including mammals [45]. The ability of microorganisms

56
S. Nishanth Kumar et al. / Peptides 53 (2014) 48–58
Fig. 5. Cytotoxicity of five diketopiperazines against cancer and normal cell lines. (1) Cancer cell lines, (A) A549 and (B) HTL 116.
Normal cell lines (A) FS normal fibroblast and (B) VERO. 10, 25, 50 and 100 ␮M.
5,
10, 25,
50 and
100 ␮M. (11)
to produce diketopiperazines is widespread and published data
have shown that about 90% of Gram-negative bacteria produce
cyclic peptides [23]. Diketopiperazines have also been isolated from
Gram-positive bacteria [17,50], fungi [7] and higher marine orga-
nisms [28]. Although cyclic dipeptides are extensively obtained by
extracting from natural sources, they may be easily prepared by
conventional synthetic procedures, due to the relative structural
simplicity of their essential nucleus [23]. The conformationally con-
strained DKP scaffold is constituted of a six-membered ring that
orientates its substituents in a spatially defined manner and repre-
sents a significant pharmacophore in medicinal chemistry because
of its stable structural characteristics [17,50]. In addition, owing to
their restricted conformational freedom and structural simplicity,
DKPs have been extensively used as valuable models for conforma-
tional studies either in solution or in solid state, particularly with
regard to the consequences (i.e. cis, trans isomerism) deriving from
the relative configuration of amino acid residues [7,28].
Diketopiperazines (DKPs) are a relatively unexplored class of
bioactive peptides that may hold great promise for the future. In
recent years considerable interest has been focused on the cyclic
dipeptides, because of their remarkable bioactivity. Important bio-
logical activities of diketopiperazines are activities as antitumour
[32], antiviral [47], antifungal, antibacterial [54], antiprion [6], anti-
hyperglycemic [49] and glycosidase inhibitor [3] agents. A number
of studies are directed to important biological activities of dike-
topiperazines related to the inhibition of plasminogen activator
inhibitor-1 [25] and alteration of cardiovascular and blood-clotting
functions [9]. Recently, it was shown that diketopiperazines are
able to activate or antagonize LuxR-mediated quorum-sensing
systems of bacteria, and they are considered to influence cell-
cell signaling [1]. Furthermore, they have been reported as part
of antibacterial nucleosides [29]. Therefore, the DKPs scaffold is
considered a useful tool for the discovery of new lead compounds
and the suitable properties of DKPs make them attractive and
promising agents for the rational development of new therapeutic
agents [24,27].
DKPs are found to be ideal lead compounds for the rational
design of an agent capable of preventing many diseases and dis-
orders. Cyclic dipeptides provide excellent models for theoretical
studies as well as the development of new pharmaceutical com-
pounds due to their simplicity and limited conformational freedom.
In recent years there has been a growing awareness to understand
the specific function of each peptide and their structure. It is known
that the biological functions of the peptides in living systems are
related to their three-dimensional structures. Cyclic peptides are
more bioavailable and more stable against degradative peptidases
than linear peptides, the relevant research is fundamental to many
aspects of peptide chemistry. The Diketopiperazines are hetero-
cyclic compounds which exist in several stereoisomeric forms, and
also in DD, DL, LD, and LL forms. The stereochemistry appears to be
important for the biological activity of the cyclic dipeptides [22].
Cyclo(Pro-Trp) was previously reported from octocoral asso-
ciated bacterium Pseudoalteromonas sp. [40], marine B. subtilis
[38], Sulfitobacter Strain [37]. The antimicrobial activity of this
compound was reported against some bacterial strains [26].
Other biological activity reported by this was hepatotoxicity
[31]. Antifungal activity of this compound against filamentous
fungi is not reported earlier. But in our study cyclo(Pro-Trp)
recorded significant antifungal activity. The production cyclo(Trp-
Tyr) was previously reported from marine algous endophytic
fungus Aspergillus niger [58], Microbispora aerata strain [30], Strep-
tomyces sp. H7372 [8]. Cyclo(D-Trp-L-Tyr) isolated from endophytic
fungus A. niger did not show antimicrobial activity against B. sub-
tilis, S. aureus, Streptomyces viridochromogenes, E. coli, C. albicans and

S. Nishanth Kumar et al. / Peptides 53 (2014) 48–58
57
Mucor miehei [58], but low antiproliferative and cytotoxic effect
was recorded. But in the present study cyclo(L-Trp-L-Tyr) recorded
significant antimicrobial activity against the test microorganisms.
Cyclo-(D-Ile-L-Trp) and Cyclo-(D-Leu-L-Trp) having plant
growth regulators activity was reported from Penicillium brevi-
compactum [33]. To the best of our knowledge this is the first
report on the antimicrobial activity of Cyclo-(D-Ile-L-Trp), and
Cyclo-(D-Leu-L-Trp) and this is the second report that these com-
pounds were reported from a natural source. In the case of cyclo
(L-Trp-L-Phe), it was first isolated from an unidentified Penicillium
sp. and proved to be with the biological function of regulating
the growth of plants [34]. In 2008, cyclo (L-Trp-L-Phe) was iso-
lated from fungal EF8 which was isolated from the conchocelis
of Porphyra yezoensisit by Ding et al. [19] and proved to exhibit
a moderate cytotoxicity against 37 human tumor cell lines with
the average IC50 value of 3.3 ␮g/ml. In 2011 cyclo (L-Trp-L-Phe)
was reported from South China Sea sponge Holoxea sp. associ-
ated fungus Aspergillus versicolor strain TS08 [16]. But till now, few
reports on the production of DKPs by microbial cultivation have
been found [46]. This study was the first to find the cyclo (L-Trp-L-
Phe) from the bacterium-associated entomopathogenic nematode
and the antimicrobial activity of this compound is also reported for
the first time. Previously we have reported three DKPS [cyclo(L-Pro-
L-Leu), cyclo(D-Pro-L-Leu) and cyclo-(D-Pro-L-Tyr)] produced by a
bacterium associated with EPN having antifungal activity against
plant pathogenic fungi [35].
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Already a large diversity of natural diketopiperazines has been
reported but because only still a small fraction of organisms has
been explored and much more can be expected. The chemical diver-
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recent reports on novel diketopiperazines of unknown functions
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Acknowledgements
The authors are grateful to Indian Council Medical Research
(ICMR), Government of India for funding. We thank the Director,
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