Cyclic tetrapeptides from the marine strain Streptomyces sp. PNM-161a with activity against rice and yam phytopathogens

Article abstract of DOI:10.1038/s41429-019-0201-0

Two cyclotetrapeptides, henceforth named Provipeptides A (1) and B (2), along with five known diketopiperazines (3–7) were isolated from the liquid culture of marine Streptomyces sp. 161a recovered from a sample of sea grass Bryopsis sp. The structures of cyclotetrapeptides and diketopiperazines (DKPs) were established by 1D and 2D NMR data, MS, and by comparison with literature data. The absolute stereochemistry of compounds cyclo-(l-Pro-l-Leu-d-Pro-l-Phe) 1 and cyclo-(-Pro-Ile-Pro-Phe) 2 was established by the Marfey’s method. Compound 1 showed antibacterial activity against rice phytopathogenic strains Burkholderia glumae (MIC = 1.1 mM) and Burkholderia gladioli (MIC = 0.068 mM), compound 2 was active only against B. glumae (MIC = 1.1 mM), and DKP cyclo-[l-Pro-l-Leu] 5 showed to be active against B. gladioli (MIC = 0.3 mM) and B. glumae (MIC = 2.4 mM). Compounds 1 and 2 showed 65% and 50% inhibition of Colletotrichum gloeosporioides (yam pathogen) conidia germination, respectively at a concentration of 1.1 mM.

Full text of DOI:10.1038/s41429-019-0201-0

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0201-0
ARTICLE
Cyclic tetrapeptides from the marine strain Streptomyces sp. PNM-161a
with activity against rice and yam phytopathogens
Luz A. Betancur1,2 Abel M. Forero1 Adriana Romero-Otero1 Lady Yohanna Sepúlveda1
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Nubia C. Moreno-Sarmiento3 Leonardo Castellanos1 Freddy A. Ramos1
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Received: 21 July 2018 / Revised: 29 May 2019 / Accepted: 8 June 2019
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
Abstract
Two cyclotetrapeptides, henceforth named Provipeptides A (1) and B (2), along with five known diketopiperazines (3–7)
were isolated from the liquid culture of marine Streptomyces sp. 161a recovered from a sample of sea grass Bryopsis sp. The
structures of cyclotetrapeptides and diketopiperazines (DKPs) were established by 1D and 2D NMR data, MS, and by
comparison with literature data. The absolute stereochemistry of compounds cyclo-(L-Pro-L-Leu-D-Pro-L-Phe) 1 and cyclo-
(-Pro-Ile-Pro-Phe) 2 was established by the Marfey’s method. Compound 1 showed antibacterial activity against rice
phytopathogenic strains Burkholderia glumae (MIC = 1.1 mM) and Burkholderia gladioli (MIC = 0.068 mM), compound
2 was active only against B. glumae (MIC = 1.1 mM), and DKP cyclo-[L-Pro-L-Leu] 5 showed to be active against
B. gladioli (MIC = 0.3 mM) and B. glumae (MIC = 2.4 mM). Compounds 1 and 2 showed 65% and 50% inhibition of
Colletotrichum gloeosporioides (yam pathogen) conidia germination, respectively at a concentration of 1.1 mM.
Introduction
This species is widely distributed in regions with high
tropical and subtropical rainfall rates and is used mainly for
human nutrition in places such as Africa, South Asia, the
Pacific Islands and the Colombian Caribbean region. It is a
basic product in the diet of the population for its high
nutritional content, being starch its main component a rich
source of carbohydrates; also for its high content of vitamin
C, with its antiscurvy properties, making it a valuable food
for humans and animals [2]. This crop is affected by
anthracnose, which is caused by the fungal phytopathogen
Colletotrichum gloeosporioides, generating production
losses of up to 75% [2]. In view of that, a search for efficient
strategies for the control of these phytophatogen is required.
During the second half of the twentieth century, one of the
major concerns in agriculture was focused on the pollution
originated by the extensive use of highly toxic agrochem-
icals such as pesticides [3, 4]. Studies since the 1970s have
shown that, besides the harmful effects at the public health
level, the use of pesticides has led to phytopathogen resis-
tance caused by the systematic use of agrochemicals [5],
evincing the importance of seeking environmentally
friendly compounds for the control of phytopathogens.
In recent years, marine microorganisms have gained
great relevance as a source of secondary metabolites with
potential to be used for the control of phytopathogens [6–9].
Marine-derived metabolites become prototypes for the
In Colombia, rice is the third most important crop in the
country [1] and has been affected by several bacterial
phytopathogens including Burkholderia spp., causing bac-
terial panicle blight disease. On the other hand, Colombia
produces more than 385,000 tons per year of yam (Dios-
corea spp.), which is a monocotyledon plant characterized
by having fleshy tubers called yams containing abundant
starch.
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-019-0201-0) contains supplementary
material, which is available to authorized users.
* Freddy A. Ramos
faramosr@unal.edu.co
1
Universidad Nacional de Colombia, Sede Bogotá, Departamento
de Química, Carrera 30 No. 45-03, Edificio de Química of 427,
Bogotá 111321, Colombia
2
Universidad de Caldas, Departamento de Química, Edificio
Orlando Sierra, Bloque B, Sede Palogrande Calle 65 No. 26-10,
Manizales, Caldas, Colombia
3
Universidad Nacional de Colombia, Sede Bogotá, Instituto de
Biotecnología, Grupo de Bioprocesos y Bioprospección,
Bogotá 111321, Colombia

L. A. Betancur et al.
development of new substances with a putative insecticidal
and antimicrobial potential, making them excellent candi-
dates for use in the agricultural sector [10]. Actinobacteria
are ubiquitous in the marine environment and one of the
most prolific source of natural products among marine
microorganisms and continue to surprise with the versatility
of their biosynthetic machinery. The structural novelty and
diversity of actinobacteria secondary metabolites still make
them a prolific source of new leads for antibiotics discovery
and development [6, 11, 12]. In addition, about two-thirds
of all biologically active compounds produced by actino-
bacteria are produced by bacteria belonging to the genus
Streptomyces [13].
In this paper, as a part of our search for bioactive
compounds for the control of phytopathogens [14], we
present the isolation of antimicrobial compounds from the
strain Streptomyces PNM-161a isolated from a sample of
Bryopsis sp. (Chlorophyta) as a source of active com-
pounds against Burkholderia glumae, Burkholderia gla-
dioli (rice pathogens), and a strain of C. gloeosporioides
(yam pathogen) [14]. Overall, we describe isolation,
structural elucidation and biological activities of two
cyclic tetrapeptides along with 5 DKPs from Streptomyces
sp. PNM-161a.
ISP-2 medium contained 4 g of malt extract, 10 g of
dextrose, and 4 g of agar per liter of tap water. Luria-Bertani
(LB) medium contained, 5 g yeast extract, 10 g NaCl, 10 g
tryptone, per liter of tap water, adjusting pH at 7.2 using
NaOH 3 M. King’s B medium (KB) contained 10 g pep-
tone, 1.5 g anhydrous KH2PO4, 15 g glycerol, and 1.5 g
MgSO4 per liter of tap water.
Fermentation and extraction
Streptomyces sp. PNM-161a was originally isolated from a
sample of Bryopsis sp. (Chlorophyta) [14]. DNA 16S
rRNA nucleotide sequence for this strain was deposited
in GenBank/EMBL/DDBJ, under accession number
KX641396 [14]. For the present study, the isolate was
recovered from its cryopreserved stock and cultured into
ISP-2 plates at 28 °C for one week. A two-step culture was
performed for extract production, consisting of an initial
inoculum of Streptomyces sp. PNM-161a into a primary
culture of 100 ml in LB medium in 500 ml conical flasks
by shaking at 130 rpm at 25 °C for 5 days. This 100 ml
preculture was transferred to a 5 l flask containing 900 ml
of LB medium to complete 1 l of culture per flask
(40 flasks for a total of 40 l culture). The flask was shaken
at 130 rpm and 25 °C for 10 days. The biomass of each
flask was removed by centrifugation at 5000 rpm for
15 min. The culture supernatant was extracted three times
with ethyl acetate (1:1). The organic layers of the 40 cul-
ture flasks were pooled and then evaporated under vacuum
to obtain 3.0 g of crude extract.
Materials and methods
General
1H and 13C NMR (1D and 2D) spectra were recorded on a
1
Bruker Advance 400 spectrometer (400 MHz for H and
Isolation of compounds
100 MHz for 13C) using CDCl3 (7.26 ppm and 77.0 ppm),
CD3OD (3.35 ppm and 49.0 ppm) and DMSO-d6 (2.50 ppm
and 39.5 ppm) as solvents, and residual solvent signals were
used as internal standards. High-resolution mass data were
collected on an accurate-mass quadrupole Time-of-Flight
(q-TOF) (Agilent Technologies) mass spectrometer, ESI
The crude extract was fractionated using a solid phase
extraction (SPE) cartridge (Phenomenex Strata C-18, 5 g,
20 ml) with a MeOH/H2O discontinuous gradient solvent
of 10, 30, 50, 70 and 100% MeOH (30 ml each) to obtain
five fractions F1–F5. Fraction F4 (860 mg) obtained from
the 70% MeOH showed antimicrobial activity (vide infra)
and was chromatographed on silica gel 60 using a dis-
continuous gradient solvent from CHCl3 100% to MeOH
100% to obtain 170 fractions of 10 ml pooled in 11
fractions F4.1–F4.11 by their TLC profiles. Fraction F4.1
(100% CHCl3) yielded compound 1 (138 mg). Fraction
F4.2 (430 mg, eluted with CHCl3/MeOH 98:2) was sub-
jected to HPLC using a Kromasil C8 column (10 × 250
mm and 10 µm) with an isocratic solvent system of MeCN
15% and water 85% (2 ml min−1) to yield compound 1
(280 mg), compound 2 (29,2 mg) and compound 5 (5 mg).
Fraction F4.3 (51 mg, eluted with CHCl3/MeOH 96:4)
was subjected to HPLC using a Kromasil C8 column
(10 × 250 mm and 10 µm) and eluted with a gradient of
MeCN (A) and water (B), as follows: 10% A with a linear
positive mode. Nebulizer 50 (psi); Gas Flow 10 l min−1
;
Gas Temp 350 °C. Fragmentor 175V, Skimmer 75V,
Vpp 750V.
Column chromatography was carried out under vacuum
using silica gel 60G Merck (0.063–0.200 mm), and flash
chromatography was carried out on silica gel 60 (70-230,
Macherey-Nagel®). HPLC-ELSD was performed on a
Thermo Dionex ultimate 3000 system coupled to an ELSD
Sedex 85 (Sedere, France) detector (Gain detector 10 and
temperature 80 °C). Optical rotations were measured on a
Polartronic E, Schmidt+Haensch polarimeter in 1 ml ×
5 cm cells. For reading the antibacterial bioassay in 96-well
plates, an AccuReader Metertech (Taipei, Taiwan) was used
reading at 600 nm. All solvents used were HPLC (High
Performance Liquid Chromatography) grade.

Cyclic tetrapeptides from the marine strain Streptomyces sp. PNM-161a with activity against rice and. . .
increase of up to 25% A in 20 min, followed by 10 min at
25% A; then a linear increase of up to 30% A in 10 min,
followed by a linear increase of up to 50% A in 10 min,
and a final linear increase of up to 100% A in 3 min fol-
lowed by 3 min at 100% A to yield compound 3 (5 mg),
compound 4 (1 mg), and compound 6 (4 mg). Fraction
F4.4 (59 mg, eluted in CHCl3/MeOH 94:6) was fractio-
nated by HPLC. The chromatographic analysis used the
same chromatographic column mentioned above at a flow
rate of 2.0 ml min−1. The mobile phase consisted of a
gradient of MeCN (A) and water (B), as follows: a linear
increase from 5% to up to 25% A in 30 min, followed by a
linear increase of up to 70% A in 3 min and then by 5 min
at 70% A. This fractionation yielded compound 7 (10 mg).
Antimicrobial activity of fraction and pure
compounds
Strains of Burkholderia spp and C. gloeosporioides 26B
were kindly provided by the Biotechnology Institute of the
Universidad Nacional de Colombia. The antibacterial activity
analysis of the compounds isolated from Streptomyces sp.
PNM-161a, against bacterial phytopathogens B. glumae
(ATCC 33617) and B. gladioli (3704-1-FEDEARROZ), was
performed in 96-well plates using the broth dilution method
[16]. Briefly, 190 µl of KB medium and 10 µl of each bac-
terial phytopathogen (Absorbance λ600 nm = 0.25) were cul-
tured in each well. The amount of 200 µl of a solution of
1000 µg ml−1 of pure compounds and positive control
(oxolinic acid) previously dissolved in MeOH 10% was
applied in the first vertical line of the plate. Serial dilutions
were made to obtain five different concentrations in each
well: 500, 250, 125, 62.5, 32.25, 16.12 and 8.1 µg ml−1.
Twenty microliters of the ethyl acetate extract previously
dissolved in MeOH 10% of the LB media culture was used
as negative controls, respectively. The plates were incubated
at 37 °C for 24 h, growth inhibition was measured by
absorbance at λ = 600 nm [16].
For the antifungal testing, the phytopathogenic fungus
C. gloeosporioides 26B obtained from yam plants with
classical symptoms of anthracnose [17] was tested. Phyto-
pathogenic fungi were kindly provided by the Instituto de
Biotecnología—Universidad Nacional de Colombia. The
antifungal activities of compounds 1, 2 and 3 were deter-
mined using liquid cultures in 96-well plates using a mod-
ification of the broth microdilution method [16]. The
medium volume per well was 100 μl. These fungi were
inoculated in RPMI-1640 medium. As for the antifungal
activity evaluation of pure compounds, the final con-
centrations of pure compounds in the wells were 500, 250,
125, 62.5 µg ml−1 and 32.25, 16.12, and 8.1 µg ml−1. All
tested compounds were previously dissolved in MeOH 10%
and applied in the first vertical line of the plate. The
microorganism suspensions (10 μl) of each pathogenic
fungus were added to a 96-well microtiter plate. The culture
media (20 µl of MeOH 10%) and a solution of 1% clo-
trimazole (5 μl well−1) were used as negative and positive
control, respectively. The plates were incubated at 25 °C
for 48 h, growth inhibition was measured by absorbance at
λ = 600 nm.
Marfey analysis
The absolute configurations of the amino acids present in
1 and 2 were determined by the Marfey’s method [15, 16].
Briefly, solutions at 50 μM of each L-amino acid (proline,
leucine, isoleucine and phenylalanine) were prepared. Then,
50 μl of each solution was treated with a 1-fluoro-2,4-dini-
trophenyl-5-L-alaninamide (L-FDAA) at 1% in acetone
(100 μl) and sodium bicarbonate 1 M (20 μl). The mixture
was heated for 1 h at 50 °C. Subsequently, the mixture was
cooled to room temperature, and a solution of HCl 1 M
(10 μl) was added. The obtained residue was dried in a
desiccator over NaOH pellets and dissolved in DMSO
(1 ml). The same procedure was repeated employing
1-fluoro-2,4-dinitrophenyl-5-D-alaninamide
(D-FDAA).
HPLC analyses were performed in a HPLC-DAD (agilent
1260), symmetry C-18 column (100 × 4.6 mm, 3.5 μm)
eluting with a first step gradient (MeCN–HCOOH 0,1%)
from 10:90 up to 18:82 in 30 min, followed by a second
gradient from 18:82 up to 40:60 in 15 min, and maintaining
40:40 for other 10 min for a total run time of 55 min, at a
flow rate of 2 ml min−1 and 25 °C, and detecting on a DAD
detector.
For the hydrolysis of 1 and 2, the compounds (1 mg
each) were treated with a 1.5 M HCl solution (1 ml) and
heated for 24 h at 110 °C. The mixture was dried in a
desiccator over NaOH pellets and the residue was dis-
solved in water. The obtained solution of each compound
(100 μl) was derivatized with L- or D-FDAA reagent 1% in
acetone. The mixture was heated at 50 °C for 24 h, and
then cooled to room temperature. Then, HCl 2 M (20 μl)
was added to the mixture. The obtained solution was
dissolved in formic acid 0.01 M/MeCN (1:3) to complete
a final volume of 1 ml. This sample was analyzed by
HPLC employing the conditions described above.
Retention times and UV spectra of each chromatographic
peak were compared with those obtained for the amino
acid derivatives previously obtained.
The minimum inhibitory concentration (MIC) for both,
the antibacterial and the antifungal tests, was determined as
the lowest concentration that inhibited visible growth of
phytopathogens. The growth inhibition of each dilution was
calculated using the following formula: % inhibition =
100 × [1 − OD of treated well/OD of negative control well].
The dilutions of the tested compounds were independently
performed at least three times.

L. A. Betancur et al.
Results and discussion
Isolation and purification of bioactive compounds
In our previous work, the ethyl acetate extract from Strep-
tomyces sp. PNM-161a showed broad spectra of anti-
microbial activity against rice and yam phytopathogens
[14]. The phylogenetic analysis showed that its closest
neighbors were Streptomyces griseochromogenes, Strepto-
myces violascens, Streptomyces resistomycificus, Strepto-
myces exfoliates, Streptomyces albidoflavus, all of them
with a 99.86% of similarity [14], making these species a
prolific source of bioactive compounds. To identify the
production of bioactive compounds, Streptomyces sp.
PNM-161a was cultured in LB medium (40 l), extracted in
ethyl acetate and fractionated by SPE (see methodology).
Fraction F4 (70% MeOH), selected because of its potent
antibacterial activity against B. glumae and B. gladioli
(Table 1S, Fig. 1S, see supplementary information), allowed
isolating seven compounds (Fig. 1).
23
D
Compound 1 was isolated as a solid with ½αŠ -15.04
(c 0.1, MeOH) and showed a sodium adduct ion at m/z
477.2478 by HRESIMS (High-resolution Electrospray
ionisation mass spectrometry), consistent with the mole-
cular formula C25H34N4O4 (calcd for C25H34N4O4 Na m/z
Fig. 1 Compounds isolated from Streptomyces sp. PNM-161a cultured
in LB medium. cyclo-(L-Pro-L-Leu-D-Pro-L-Phe) 1, cyclo-(Pro-Ile-Pro-
Phe) 2, cyclo-(L-Phe-D-Pro) 3, cyclo-(L-Phe-L-Pro) 4, cyclo-(L-Pro-L-
Leu) 5, cyclo-(L-Met-L-Pro) 6, cyclo-(L-Tyr-L-Pro) 7
1
= 477.2483, Δ 1.04 ppm, 0.49 mmu). H NMR (400 MHz,
CD3OD, Table 1) showed characteristic signals for a pep-
tide with a single major conformation, with signals for an
NH amide proton at δH 7.91 (1H, bs), a monosubstituted
aromatic ring at δH 7.31–7.22 (5H, m), four α methine
protons at δH 4.45 (1H, dt, J = 4.9, 1.6 Hz), 4.25 (1H, dd,
J = 7.7, 1.5 Hz), 4.13 (1H, m) and 4.07 (1H, dd, J = 6.5,
1.7 Hz), suggesting that 1 was comprised of four amino acid
residues. In addition, eight methylenes at δH 3.54 (1H, m)
−δH 3.38 (1H, m); δH 3.52 (1H, m) −3.35 (1H, m); δH 3.17
(2H, dd, J = 4.9, 2.5 Hz); δH 2.30 (1H, m)−δH 1.94 (1H,
m); δH 2.11 (1H, m)−δH 1.27 (1H, m); δH 2.02 (2H, m),
1.92 (1H, m) −1.52 (1H, t, J = 7.7 Hz), δH 1.80 (2H, m)
were observed, along with one methyne at δH 1.89 (1 H, m),
and two methyles at δH 0.97 (3H, d, J = 6.3 Hz) and δH 0.95
(3H, d, J = 6.3 Hz). Spectra 13C NMR and HSQC (Hetero-
nuclear single quantum correlation experiment) (100 MHz,
CD3OD, Table 1) showed the presence of four amide car-
bonyl resonances at δC 172.8, 170.9, 168.9 and 166.9, along
with the four α-CH groups at δC 60.3, 60.1, 57.8, and 54.6,
confirming the presence of four amino acids. In addition,
the spectra showed one aromatic ring with signals at δC
137.3, 131.0 (×2), 129.5 (×2) and 128.1; eight methylenes
at δC 46.5, 45.9, 39.4, 38.7, 29.4, 29.3, 23.6, 23.3, one
methine at δC 25.7 and two aliphatic methyl carbons at δC
22.7 and δC 22.2.
multiple bond correlation experiment), and HSQC) were
consistent with a tetrapeptide composed of one phenylala-
nine, one leucine and two proline residues. The amino acid
sequence was determined from the observed key HMBC
correlations (CD3OD), as follows (Fig. 2 and Table 1): the δ
proton of Pro-1 (δH 3.52) correlated to the Leu carbonyl
group (δC 168.9); the α (δH 4.07) and δ (δH 3.38) protons of
Pro-2 correlated to the Phe carbonyl group at δC 166.9. A
further HMBC experiment measured in DMSO-d6,
(Fig. 11S, supplementary information) showed correlations
between the proton signal, assigned to N–H Phe (δH 7.98)
with the carbonyl carbon of Phe (δC 165.1) and with the
carbonyl carbon of Pro-1 (δC 170.4); and α Pro-1 (δC 58.5)
(Fig. 2). A cyclic structure for compound 1 is proposed by
correlations of N–H from Leu (δH 8.01) to α Pro-1 (δC 58.4)
and to the carbonyl of Pro-2 (δC 169.0), thus confirming the
structure of compound 1 as presented in Fig. 1.
The absolute configuration of the amino acids from
compound 1 was established by the Marfey’s method. The
mixture obtained after hydrolyzing compound 1 and fur-
ther derivatization with L-FDAA was analyzed by HPLC-
DAD. The chromatogram showed three peaks with reten-
tion times of 29.9, 33.9, 41.57 and 42.26 min, which were
consistent with the retention times and the UV spectra
obtained for L-Pro, D-Pro, L-Leu, and L-Phe standards and
2D NMR spectroscopic data from compound
1
(COSY (Correlated spectroscopy), HMBC (Heteronuclear

Cyclic tetrapeptides from the marine strain Streptomyces sp. PNM-161a with activity against rice and. . .
Table 1 1D NMR data of 1 and 2 in CD3OD (1H NMR at 400 MHz; 13C NMR at 100 MHz)
1
2
Residue
Leucine
Position
δ
1H, integr, mult,
δ
13C mult.
δ
1H, integr, mult, (J in Hz)
δ
13C mult.
(J in Hz)
α
β
4.13, 1H, m
54.6 CH
1.92, 1H, m
39.4, CH2
1.52, 1H, t, (7.7)
1.89, 1H, m
γ
δ
25.7, CH
22.7, CH3
22.2, CH3
168.9, C
0.97, 3H, d, 6.3
0.95, 3 H, d, 6.3
δ (CH3)
CO
NH
α
7.91, 1H, br s
Isoleucine
4.07, 1H, dd, (9.6, 1.9)
61.3, CH
37.1, CH
β
2.17, 1H, dqdd, (9.6, 7.0,
4.5, 2.4)
γ
1.45 1H, dqd (13.4,
7.5, 4.5)
25.4, CH2
1.33,1H, dqd (13.4,
7.5, 2.4)
δ
0.93, 3H, t, 7.5
1.07, 3H, d, 7.0
–
NDa
12.5, CH3
15.5 CH3
167.5, C
β (CH3)
CO
NH
α
Proline-1
4.25, 1H, dd (7.7, 1.5)
2.30, 1H, m
60.1, CH
29.3, CH2
4.20, 1H, dd, (6.9, 1.9)
2.32, 1H, m
60.1, CH
29.5, CH2
β
1.94, 1H, m
1.95, 1H, m
γ
δ
2.02, 2H, m
23.6, CH2
46.5, CH2
2.07, 1H, m
23.3, CH2
45.9, CH2
1.99, 1H, m
3.52, 1H, m
3.56, 1H, m
3.35, 1H, m
3.50, 1H, m
CO
α
βa
βb
1
–
172.8, C
57.8, CH
38.2 CH2
–
172.4, C
57.7, CH
38.2 CH2
Phenylalanine
4.45, 1H, dd (4.9, 1.6)
3.19, 1H, dd, (14.1, 5.0)
3.14, 1H, dd (14.1, 4.9)
4.45, 1H, dd, (4.9, 1.9)
3.19, 1H, dd, (14.5, 5.2)
3.15, 1H, dd (14.5, 5.0)
137.3, C
137.3, C
2–6
7.31–7.22, 2H + 2H +
1H, m
131.0 × 2, CH
7.31–7.22, 2H + 2H + 1H,
131.0 × 2, CH
mb
129.5 × 2, CH
128.1, CH
166.9, C
129.5 × 2, CH
128.1, CH
166.9, C
CO
NH
α
ND
NDa
Proline 2
4.07, 1H, dd, (6.5, 1.7)
2.11, 1H, m
1.27, 1H, m
1.80, 2H, m
3.54, 1H, m
3.38, 1H, m
–
60.3, CH
29.4, CH2
4.09, 1H, dd, (7.6, 1.9)
2.11, 1H, (m)
1.23, 1H, (m)
1.80, 2H, (m)
3.55, 1H, (m)
3.37, 2H, dd, (12.3, 6.3)
–
59.9, CH
29.3, CH2
β
γ
δ
23.3, CH2
45.9, CH2
22.7, CH2
46.2, CH2
CO
170.9, C
170.9
aND not detected
bSignals overlapped for the five protons of the aromatic ring
different from those obtained for D-FDAA (Fig. 12S, see
supplementary information). The entire analysis allowed
determining that compound 1 is a cyclic tetrapeptide
conformed by residues of L-Phe, L-Leu, and both L- and D-
Pro, and its structure is depicted in Fig. 2. This structure is
an stereoisomer of the the cyclic tetrapeptide cyclo-(L-
prolyl-L-leucyl-L-prolyl- L-phenylalanyl) produced by
a marine strain of Pseudomonas sp. isolated from the
sponge Halisarca ectofibrosa collected in Okinawa,
Japan [18]. The peptide sequence of 1 was established
as cyclo-(L-prolyl-L-leucyl-D-prolyl-L-phenylalanyl) by
HMBC (Fig. 2). A similar compound was isolated by
Rungprom [18] and synthetized by Dahiya [19], and the
NMR data published for the natural and the synthetic

L. A. Betancur et al.
Fig. 2 Key HMBC correlations
for the structural elucidation of
cyclic tetrapeptides 1 and 2.
Dashed arrows represents the
correlations observed in the
experiment measured in DMSO-
d6 and black represents those
observed in the experiment
measured in MeOH-d4
compound cyclo-(L-prolyl-L-leucyl-L-prolyl- L-phenylala-
nyl) are the same. However the NMR data of compound 1
are different from those published by Rungprom [18] and
Dahiya [19], suggesting that 1 corresponds to an stereo-
isomer of the published compound. The main difference
was the proton chemical shift of α Pro-2 at δH 2.62 for the
synthetic compound and δH 4.07 for compound 1. This fact
allowed us to propose D-Pro in position 2, and thus pro-
posing the structure of 1 as is presented in Fig. 1. This
proposal was confirmed by computational models of
compound 1 and the stereoisomer synthetized by Dahiya
[19], obtained by Free Maestro ver 11.4 software using
Universal Force Field (UFF), where the model obtained for
compound 1 do not present anisotropic effect from the Phe
aromatic ring to the α Pro-2 proton, while the compound
cyclo-(L-prolyl-L-leucyl-L-prolyl- L-phenylalanyl) does
present such anisotropic effect as can be seen in Fig. 13S
(see supplementary information).
the Pro-2 carbonyl (δC 170.9). The Phe was bonded to Pro-2
as established by the correlation from Pro-2 α-proton (δH
4.09) and δ-proton (δH 3.37) to the Phe carbonyl (δC 166.9).
The HMBC experiment in DMSO-d6 (Fig. 14S, supple-
mentary information) showed the correlations between the
Phe NH proton (δH 7.91) to its own carbonyl (δC 166.9) and
to the α-carbon of Pro-1 (δC 60.1), as displayed in Fig. 2,
allowing identifying compound 2 as cyclo-(Pro-Ile-Pro-
Phe), and its planar structure is presented in Fig. 1. To the
best of our knowledge, compound 2 has not been previously
reported in the literature.
In addition, in the present study, five diketopiperazines
were isolated and identified as cyclo-(L-Phe-D-Pro) 3 [19],
cyclo-(L-Phe-L-Pro) 4 [20], cyclo-(L-Pro-L-Leu) 5 [21],
cyclo-(L-Met-L-Pro) 6 [22], cyclo-(L-Tyr-L-Pro) 7 [23, 24],
on the basis of their NMR and MS data and by comparison
with those reported in the literature; the structures are
shown in Fig. 1.
Compound 2 was isolated as a solid with ½αŠ23 + 0.31 (c
D
0.1, MeOH) and showed a molecular ion peak at m/z
455.2649 for the pseudomolecular ion [M + H]+ by HRE-
SIMS, consistent with the molecular formula C25H34N4O4
(calcd for C25H35N4O4 m/z = 455.2663, Δ 3.07 ppm, 1.4
Antimicrobial activity evaluation
The evaluation of MIC for compounds 1 to 7 against rice
phytopathogen bacterial strains of B. glumae and B. gladioli
is shown in Table 2. Compounds 1 and 2 exhibited the
highest antibacterial activity against both bacteria with
values of MIC = 1.1 mM against B. glumae. Interestingly,
compound 1 showed to be active against B. gladioli (MIC
= 0.068 mM) with a very similar value to that of oxolinic
acid, whereas compound 2 was not active against the same
bacteria. In addition, compound 5 exerted selectivity to
inhibit B. gladioli (MIC = 0.29 mM). The remaining com-
pounds showed mild activities with values between 1.8 and
2.19 mM, and compound 7 was not active at the conditions
evaluated. Furthermore, compounds 1 and 2 were tested
against C. gloeosporioides at a concentration of 1.1 mM,
showing 65% and 50% germination inhibition of the C.
gloeosporioides conidia, respectively. This test allowed
identifying tetrapeptides as the compounds responsible of
1
mmu). H NMR (400 MHz, CD3OD, Table 1) showed that
compound 2 was a tetrapeptide similar to 1, but bearing an
isoleucine residue with signals at δH 4.07 (1H, dd, J = 9.6,
1.9 Hz), δH 2.17 (1H, dqdd, 9.6, 7.0, 4.5, 2.4 Hz), δH 1.45
(1H, dqd 13.4, 7.5, 4.5 Hz)−δH 1.33 (1H, dqd, 13.4, 7.5,
2.4 Hz), 1.07 (3H, d, J = 7.0 Hz) δH 0.93 (3H, t, J = 7.5
Hz), instead of the leucine residue. 13C NMR (100 MHz,
CD3OD, Table 1) confirmed the presence of the Ile residue
with signals at 167.5 (C), 61.3 (CH), 37.1 (CH), 25.4 (CH2),
12.5(CH3), 15.5 (CH3).
The amino acid sequence was determined from the
observed key HMBC correlations shown in Fig. 1, where
the Ile unit was placed between the two Pro units by the
correlations from the δ-proton of Pro-1 (δH 3.50) to the Ile
carbonyl (δC 167.5) and from the α Ile proton (δH 4.07) to

Cyclic tetrapeptides from the marine strain Streptomyces sp. PNM-161a with activity against rice and. . .
Table 2 Antibacterial activity of compounds 1–7 against the rice
Rev Cienc Agrícolas. 2016;33:16. http://revistas.udenar.edu.co/
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Compound
B. glumaea
B. gladiolia
1
1.1
0.068
NAb
2.04
2.04
0.29
2.19
NAb
NAb
0.062
2
1.1
3
2.04
2.04
2.37
2.19
NAb
NAb
0.119
4
5
6
7
MeOH 10% (−)
Oxolinic acid (+)
aAntibacterial activity is expressed as MIC (mM), determined by the
broth microdilution method.
bNA: Non-active at the maximum examined concentration (500 μg ml−1
)
the bioactivity observed in the crude extract of Streptomyces
sp. PNM-161a.
In summary, two new cyclic tetrapeptides 1 and 2, along
with 5 known diketopiperazines 3–7, were isolated from the
strain Streptomyces sp. PNM-161a cultured in LB broth.
The bioactivity assays evinced that compound 1 (B. gladioli
MIC = 0.068 mM; B. glumae MIC = 1.1 mM), compound 2
(B. glumae MIC = 1.1 mM) and the diketopiperazine 5
(B. gladioli MIC = 0.3 mM; B. glumae MIC = 2.4 mM)
were the compounds responsible for the antimicrobial
activity against bacterial and fungal phytopathogens here
tested.
Acknowledgements This study was conducted with the financial
support of COLCIENCIAS (grant Cod. 1101-659-44402 CT.537/14)
and International Foundation for Science IFS, Uppsala-Sweden (grant
Cod. 5023–2). Universidad Nacional de Colombia DIEB grant 37151.
L.A.B thanks the Programa Doctoral Becas Colciencias (567) for the
granted scholarship (2012), Universidad de Caldas for the leave of
absence for doctoral studies. The ANLA (Agencia Nacional de
Licencias Ambientales) and the Ministerio de Ambiente y Desarrollo
Sostenible granted permission to collect samples and perform this
research (Permission No 4 of 10/02/2010, Anexo 2, Contrato de
Acceso a Recurso Genético No 108).
13. Newman DJ, Cragg GM. Natural products as sources of new
drugs over the last 25 years. J Nat Prod. 2007;70:461–77.
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Moreno-Sarmiento NC, Maldonado LA, Suarez-Moreno ZR, et al.
Marine actinobacteria as a source of compounds for phytopatho-
gen control: an integrative metabolic-profiling/bioactivity and
taxonomical approach. Virolle M-J, editor. PLoS ONE. 2017;12:
e0170148. http://dx.plos.org/10.1371/journal.pone.0170148.
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49, Carlsberg Res. Commun. 1984. https://link.springer.com/
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Compliance with ethical standards
16. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating
antimicrobial activity: A review. J Pharm Anal. 2016;6:71–9. http://
linkinghub.elsevier.com/retrieve/pii/S2095177915300150.
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Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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