Isolation and structure identification of novel brominated diketopiperazines from Nocardia ignorata - A lichen-associated actinobacterium

Article abstract of DOI:10.3390/molecules22030371

Actinobacteria are well known for their potential in biotechnology and their production of metabolites of interest. Lichens are a promising source of new bacterial strains, especially Actinobacteria, which afford a broad chemical diversity. In this contex

Full text of DOI:10.3390/molecules22030371

molecules
Article
Isolation and Structure Identification of Novel
Brominated Diketopiperazines from
Nocardia ignorata—A
Lichen-Associated Actinobacterium
Alba Noël, Solenn Ferron, Isabelle Rouaud, Nicolas Gouault, Jean-Pierre Hurvois
and Sophie Tomasi *
CORINT, UMR CNRS ISCR 6226, UFR Sciences Pharmaceutiques et Biologiques, Université Bretagne Loire,
2 Av. du Professeur Léon Bernard, 35043 Rennes, France; alba.noel@univ-rennes1.fr (A.N.);
solenn.ferron@univ-rennes1.fr (S.F.); isabelle.rouaud@univ-rennes1.fr (I.R.);
nicolas.gouault@univ-rennes1.fr (N.G.); jean-pierre.hurvois@univ-rennes1.fr (J.-P.H.)
* Correspondence: sophie.tomasi@univ-rennes1.fr; Tel.: +33-223-234-817
Academic Editor: Derek J. McPhee
Received: 25 January 2017; Accepted: 23 February 2017; Published: 28 February 2017
Abstract: Actinobacteria are well known for their potential in biotechnology and their production
of metabolites of interest. Lichens are a promising source of new bacterial strains, especially
Actinobacteria, which afford a broad chemical diversity. In this context, the culture medium of
the actinobacterium Nocardia ignorata, isolated from the terrestrial lichen Collema auriforme, was
studied. The strain was cultivated in a BioFlo 115 bioreactor, and the culture medium was extracted
using an XAD7HP resin. Five known diketopiperazines: cyclo (L-Pro-L-OMet) (
), cyclo (D-Pro-L-Tyr) ( ), cyclo (L-Pro-L-Val) ( ), cyclo (L-Pro-L-Leu) ( ), and one auxin derivative:
indole-carboxaldehyde ( ) were isolated, along with two new brominated diketopiperazines: cyclo
(D-Pro-L-Br-Tyr) ( ) and cyclo (L-Pro-L-Br-Tyr) ( ). Structure elucidation was performed using HRMS
1), cyclo (L-Pro-L-Tyr)
(
2
3
4
5
8
6
7
and 1D and 2D NMR analysis, and the synthesis of compounds
confirm their structure.
6 and 7 was carried out in order to
Keywords: diketopiperazines; Nocardia; lichen-associated actinobacterium
1. Introduction
In the field of research on new active compounds from nature, the exploration of original sources
of natural products has become more attractive. In this context, the investigation on lichens is an
interesting challenge. The symbiotic feature of this organism classically described between a green
algae (or cyanobacteria) and a fungus led to the production of interesting metabolites [
a long-lasting environment which can host various microorganisms [ ]. A plethora of bacteria have
been listed from this ecological niche using culture-independent (reviewed by Suzuki et al. [ ]) or
culture-dependent approaches [ ]. The three main bacterial phyla found associated with lichens are
Proteobacteria, Firmicutes, and Actinobacteria [ ]. The chemistry of some of these bacteria have been
1], but it is also
2–5
6
2,7
2,5,8
already studied, highlighting their ability to produce active compounds [6,9].
In our ongoing research to study lichen-associated bacteria, we focused this study on an
actinobacterium, relative to Nocardia ignorata (16s RNA sequence similarity of 98.58%), previously
isolated from a terrestrial lichen Collema auriforme [
biotechnological properties, and especially for the production of compounds of therapeutic interest,
such as antibiotics (e.g., streptomycin) or anticancer agents (e.g., mithramycin) [10 11]. The chemistry
of Nocardia ignorata was not yet described, and a preliminary assay on different cell lines revealed an
2]. Actinobacteria are well-known for their
,
Molecules 2017, 22, 371; doi:10.3390/molecules22030371
www.mdpi.com/journal/molecules

Molecules 2017, 22, 371
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interesting cytotoxic activity of its extract against B16 (murine melanoma) cell lines (IC50 23
±
3
µ
g/mL).
In the present study, we have isolated two new brominated diketopiperazines and six known
compounds from our strain (Figure 1). The aims of this study were to characterize these compounds
and to evaluate their cytotoxicity against HaCaT and B16 cell lines.
O
O
O
O
S
O
N
H
N
H
N
H
N
H
HN
HN
HN
HN
HO
HO
O
O
O
4
O
2
3
1
O
O
O
H
O
10
12
N
H
11
2
N
9
7
13
3
N
8
HN
HN
6
16
HN
14
5
HO
N
15
HO
H
H
H
O
Br
O
Br
O
5
8
7
6
Figure 1. Structure of compounds 1–8 isolated from Nocardia ignorata.
2. Results and Discussion
2.1. Chemical Profiling of Extracts and Purification
Two different extracts were obtained from the culture of Nocardia ignorata. The first steps of the
process are to adsorb metabolites from the medium culture on Amberlite XAD7HP resin. Then, these
compounds were eluted from the resin using a mixture of MeOH/Acetone (1/1) leading to a resin
extract (RE). After depletion by the resin, the medium was extracted with EtOAc in order to collect
compounds not adsorbed on resin affording a supernatant extract (SE).
As shown in Figure 2, these extracts were analyzed using HPLC-UV, highlighting that chemical
profiles between RE and SE are different. While more polar compounds are present in SE (from 16.5 min
to 20.0 min), RE contained less polar metabolites (from 21.8 to 35.0 min). RE and SE extracts were then
fractionated with flash chromatography and semi-preparative HPLC to isolate pure compounds.
Compounds
from the resin extract. This extract was divided into 23 fractions by flash chromatography on normal
phase using a gradient of cyclohexane/CH2Cl2/EtOAc/MeOH. Compounds and were obtained
after purification of fractions 6 (compound ), 8 (compounds and ), 9 (compounds and ), and
21 (compound ) using semi-preparative HPLC on a C18 Prevail column with a gradient of H2O
1 (6.9 mg), 2 (5.7 mg), 3 (8.5 mg), 4 (1.1 mg), 5 (4.3 mg), and 8 (5.0 mg) were isolated
1–
5
8
8
4
5
2
3
1
and acetonitrile.
Compounds
6
(1.4 mg) and 7 (2.1 mg) were isolated from the supernatant extract, which was subjected
to flash chromatography on reversed phase to afford 14 fractions. The fifth fraction led to compounds
6 and 7 after purification by semi-preparative HPLC using a gradient of H2O/acetonitrile.
The culture of Nocardia ignorata was stopped after 4 days of growth during the death phase of the
bacterial growth. These two new compounds were only produced during this phase.
Six known compounds were isolated from culture media of Nocardia ignorata. Five of them
are diketopiperazines, including: cyclo (L-Pro-L-OMet) (
(D-Pro-L-Tyr) ( ) [13], cyclo (L-Pro-L-Val) ( ) [14], cyclo (L-Pro-L-Leu) (
determined as an auxin derivative: indole-carboxaldehyde ( ) [15]. The structure of these compounds
was elucidated using 1H- and 13C-NMR data from literature.
1) [12], cyclo (L-Pro-L-Tyr) (2) [13,14], cyclo
3
4
5
) [14 15] and the last one was
,
8

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Figure 2. Chemical profiling and isolation process of compounds 6 and 7. All samples were analyzed at
220 nm on Prevail reversed phase C18 column with a gradient of H2O (A)/acetonitrile (B) (10 min 100%
of A, 30 min from 0% of B to 100% of B, 10 min 100% of B). Supernatant extract (SE) was fractionated
using flash chromatography with a reversed phase to afford 14 fractions. Compounds 6 and 7 were
isolated from the fifth fraction using semi-preparative HPLC on Prevail C18 column. RE: resin extract.
2.2. Structural Determination of Diketopiperazines 6 and 7
2.2.1. Structural Analysis
Two new diketopiperazines were identified as cyclo (D-Pro-L-Br-Tyr) (6) and cyclo (L-Pro-L-Br-Tyr)
(7
). Compound was isolated as a white amorphous powder from the supernatant extract. Analysis by
7
HR-ESIMS (High Resolution ElectroSpray Ionisation Mass Spectrometry) revealed an isotope pattern
of a monobrominated compound with a molecular formula of C14H15BrN2O3 (m/z 339.0329 [M + H]+,
1
calcd. 339.0339). The H-NMR spectrum showed similar signals with those of cyclo (L-Pro-L-Tyr)
excepted for the aromatic protons.

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The chemical shift of C-15 on 13C-NMR spectra and the characteristic isotopic pattern in the MS
1
spectrum confirmed the presence of a bromide atom in the structure. First, H-NMR signals and
chemical shift of H-3 and H-6 and the presence of two carbonyl functions (C-2
δ = 166.7, C-5 δ = 170.7)
highlighted a diketopiperazine structure. Then, COSY correlations between the H-6, H-7, H-8, and H-9
protons established the presence of an alkyl chain. This chain was connected to a carbonyl function
with an HMBC correlation between H-7 and C-5 corresponding to the proline group. Aromatic signals
in 1H-NMR data (H-12, H-13, and H-16) were characteristic of a meta- para-disubstituted phenyl group,
and chemical shifts of C-15 (δ: 154.5) and C-14 (δ: 110.6) showed the presence of a hydroxyl group and a
bromide atom, respectively. COSY correlations between H-10 and H-3 and HMBC correlations between
H-10 and C-3 and C-2 highlighted the linkage between the aromatic ring and the diketopiperazine
moiety. Finally, the structure of compound 7 was established as shown in Figure 3.
Figure 3. HMBC and COSY correlations on compound 7.
Compound
similar to that of compound
for compound , but with variations in the chemical shifts—especially for H-3, H-6, H-7, and H-10
6
was also obtained as a white amorphous powder, and its molecular formula was
7
. Its 1H-NMR spectrum displayed similar patterns with those reported
7
(Table 1). These data indicated that these compounds are two different diastereoisomers. To describe
the stereochemistry of compounds 6 and 7, their syntheses were realized.
1
Table 1. H-NMR and 13C-NMR spectroscopic data of compounds 6 and 7.
6
7
No.
δ
(J in Hz)
δC
δ
(J in Hz)
H
δC
H
1
2
3
4
5
-
-
-
-
-
-
167.3
59.8
-
166.7
57.7
-
4.14, t (4.7)
4.38, td (4.7, 2.0)
-
-
-
-
171.1
170.7
6
2.82, m
59.3
4.07, ddd (10.9, 6.3, 2.0)
60.0
1.70, m
2.12, m
1.25, m
2.13, m
7
29.9
29.5
1.95, m
1.70, m
8
9
22.5
46.2
39.7
1.84, m
22.6
45.9
37.1
3.36, m
3.56, m
3.38, m
3.57, m
2.88, dd (13.9, 4.9)
3.09, dd (13.9, 4.9)
2.99, dd (14.2, 4.7)
3.13, dd (14.2, 4.7)
10
11
12
13
14
15
16
-
128.9
131.3
117.2
110.8
155.0
135.5
-
129.6
131.4
116.9
110.6
154.5
135.4
6.98, dd (8.2, 2.1)
7.03, dd (8.3, 2.2)
6.83, d (8.2)
6.81, d (8.3)
-
-
-
-
7.29, d (2.1)
7.35, d (2.2)

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2.2.2. Synthesis
The synthesis of the cyclo (L-Pro-L-Br-Tyr)
7
and one of its diastereoisomer, cyclo (D-Pro-L-Br-Tyr)
6
started with the bromination of L-tyrosine with Br2 in acidic condition. Next, the amine function
was protected as its N-Boc derivative in the presence of sodium hydrogen carbonate to furnish
intermediate 11 in an excellent yield over two steps. The N-Boc-protected L-Br-Tyrosine (11) was then
condensed either with the L-Proline or the D-Proline in the presence of TBTU (O-(1H-Benzotriazol-1-
yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate) as the coupling reagent and the Hünig’s base
DIPEA (N,N-Diisopropylethylamine) to yield 13a (60%) and 13b (20%), respectively. Finally,
◦
ring-closure was carried out in hot water at 130 C during 4 h, affording compounds
6 and 7 in
moderate 64% and 36% yields, respectively. The synthesis of 6 and 7 is outlined in Scheme 1.
Scheme 1. Synthesis of cyclo (D-Pro-L-Br-Tyr) (
Benzotriazol-1-yl)-N,N,N ,N -tetramethyluronium tetrafluoroborate), DIPEA: (N,N-Diisopropyléthylamine).
6
) and cyclo (L-Pro-L-Br-Tyr) (
7). TBTU: (O-(1H-
0
0
1H-NMR data of the cyclo (D-Pro-L-Br-Tyr) and the cyclo (L-Pro-L-Br-Tyr) obtained in this way
were similar to those from isolated compounds and , confirming the stereochemistry of these
6
7
products. The presence of an amide bond was highlighted by the appearance of an N–H signal at
9.54 ppm for compound 6 in C5D5N and 8.72 ppm for compound 7 in (CD3)2CO.
2.3. Cytotoxic Activity
The cytotoxic activity of the two new synthesized diketopiperazines was evaluated against two
different cell lines: HaCaT (human keratinocyte) and B16 (murine melanoma). For both compounds,
the IC50 was >200 µg/mL.
Because the intermediates of synthesis 13a and 13b are newly described in this paper, their
cytotoxic activity were also determined. Only one of them, 13b, showed an interesting activity against
the two cell lines HaCaT (IC50 7 ± 2.5 µg/mL) and B16 (IC50 18 ± 5 µg/mL).
The cytotoxicity of the six known compounds was been evaluated on both cell lines.
Diketopiperazines (
for a mixture of diketopiperazines [16]. Indole-carboxaldehyde (
HaCaT (IC50 79 ± 6 µg/mL) and B16 (IC50 72 ± 6 µg/mL). All data are shown in Table 2.
1
–
5) showed an IC50 > 200
µg/mL, as already described in a previous study
8) had a weak cytotoxic activity on

Molecules 2017, 22, 371
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Table 2. Cytotoxic activity of compounds 1–8 and 13a–b.
Compounds
1
2
3
4
5
6
7
8
13a
13b
HaCaT >200
B16 >200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
79
72
±
±
6
6
>200
>200
7 ± 2.5
18 ± 5
IC50 (µg/mL)
3. Materials and Methods
3.1. General Experimental Procedures
All commercial reagents were purchased from Carlo Erba Reactifs and/or from Sigma Aldrich
(Val de Reuil, France and St. Quentin Fallavier, France). An EasyPure (Barnstead , ThermoFisher
™
Waltham, MA, USA) water purification system was used to obtain HPLC and LC/MS grade water
for chromatographic analysis. Deuterated solvents were purchased from Euriso-top (Gif-sur-Yvette,
France). All spectra were recorded on a Bruker DMX 300 spectrometer (300 MHz (1H) and 75 MHz
(13C)) and Bruker 500 cryo-spectrometer (500 MHz (1H) and 125 MHz (13C), Bruker, Billerica, MA, USA)
using adequate deuterium solvents. Chemical shift values were referenced to residual solvent signals
for CDCl3 (δH/δC, 7.26/77.16) and CD3OD (δH/δC, 3.31/49.00). HSQC, HMBC, COSY, or TOCSY data
were recorded using a Bruker DMX 500 cryo-spectrometer instrument. NMR data were processed
using the MestReNova version 9.1 software (Mestrelab Research, Santiago de Compostela, Coruña,
Spain). The systems used for exact mass determination was an HPLC U3000 dual gradient RSLC
(Rapid Separation Liquid Chromatography) coupled with a Q-Exactive Focus (LC-FT-MS/MS with a
HESI probe), at Platform Bio2mar, Banyuls-sur-mer, France and an Agilent 6510 Q-TOF at CRMPO
(Centre Regional de Mesures Physiques de l’Ouest), Rennes, France.
3.2. Microorganism
Nocardia ignorata, DP94 was isolated from the lichen Collema auriforme collected in Kesselfallklamm
◦
◦
in Austria (47 12’21.26” N, 15 23’57.27” E) in November 2012. The strain was identified by sequencing
its 16S rRNA gene using Sanger sequencing [ ]. These data were compared with sequences in the
2
Eztaxon server type strain database [17], and showed that the close phylogenetic neighbor of the strain
was Nocardia ignorata DQ659907 at 98.59% sequence identity. The bacteria were stored after growth
in ISP2 medium (International Streptomyces Project 2 medium) (4 g yeast extract (Sigma-Aldrich,
St. Louis, MO, USA), 10 g malt extract (Sigma-Aldrich, St. Louis, MO, USA), and 4 g Dextrose
(Sigma-Aldrich, St. Louis, Missouri) for 1 L) with 50% v/v glycerol or 5% v/v DMSO at
referenced as DP94 (PNSCM collection).
−
80 ◦C and
3.3. Fermentation of Nocardia ignorata DP94
DP94 was cultivated in 50 mL test tubes containing 30 mL of TY medium (10 g yeast extract,
Sigma-Aldrich, St. Louis, MO, USA), 16 g tryptone (Sigma-Aldrich, St. Louis, MO, USA), and 5 g NaCl
(Sigma-Aldrich) for 1 L. The test tube was shaken on an orbital shaker New Brunswick Innova 42®
◦
(110 rpm) at 25 C for 72 h. Five liters of liquid TY medium were then inoculated with 60 mL of the test
tubes culture in◦bioreactor Bioflo 115 (7.5 L, New Brunswick, Edison, NJ, USA). Growth parameters
were fixed at 25 C, aeration 0.4 vvm, dissolved oxygen percentage (DO) at 40% and stirring in cascade
mode with DO (limited between 200 and 400 rpm), and pH was not controlled. After 4 days of culture,
the medium was centrifuged at 3500 rpm (Thermo scientific Sorvall ST40R, ThermoFisher scientific,
Waltham, MA, USA) during 15 min at 4 ◦C. The bacterial residue was frozen at
use. Forty grams of sterile resin XAD-7-HP (Sigma-Aldrich) were added for 1 L of supernatant medium
before being shaken during 4 h using the same conditions (25 ◦C, 110 rpm).
−
18 ◦C before further
3.4. Mass Spectrometry Analysis
Mass spectrometry analysis were carried out on a HPLC system—Diode Array Detector (LC-DAD)
(Shimadzu, Marne La Vallée, France) and a mass spectrometer with a single quadrupole analyzer

Molecules 2017, 22, 371
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(Advion expression CMS, Ithaca, NY, USA). Ionization was made by electrospray in negative or positive
mode (ESI) for low resolution analysis. High resolution analysis were performed either on HPLC
U3000 dual gradient RSLC (Rapid Separation Liquid Chromatography) coupled with a Q-Exactive
Focus (LC-FT-MS/MS with a HESI probe) or on Agilent 6510 Q-TOF mass spectra as already described.
A Prevail C18 column (5
µ
m, 250
×
4.6 mm, GRACE, Columbia, MD, USA) was used for HPLC, and a
gradient system was applied: A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile).
The following gradient was applied at a flow rate of 0.8 mL/min in the HPLC system: initial: 100% (A);
from 0 to 5 min: 100% (A); from 5 to 35 min: 100% (A)/0% (B) to 0% (A)/100% (B); from 35 to 45 min:
100% B; from 45 to 50 min: 100% (A)/0% (B) to 0% (A)/100% (B); from 50 to 55 min: 100% (A). A split
to 0.2 mL/min was applied before mass spectrometry system. Twenty microliters were injected.
The Xcalibur 1.0 software was used for data analyses.
3.5. Extraction and Isolation
After filtration of the medium, the resin was extracted three successive times with acetone/MeOH
(50/50, v/v) and the medium filtrate was kept for further use. Acetone/MeOH extracts were dried
in vacuo, redissolved, and three further successive extractions with EtOAc/H2O (1/1, v/v) were
realized. The organic phase (EtOAc extract) was collected and dried on anhydrous sodium sulfate.
The organic phase was evaporated under vacuum to yield 657.3 mg of resin extract (RE). The medium
previously depleted with the resin was extracted two times with EtOAc (1/2, v/v) in order to collect
compounds not adsorbed on resin. The organic layer was desiccated with anhydrous sodium sulfate
and dried under vacuum giving 137.6 mg of this supernatant extract (SE). These two extracts (resin
and supernatant) were fractionated using various methods (Flash liquid chromatography (Puriflash
Interchim) and semi-preparative HPLC).
For Flash liquid chromatography of the supernatant extract, the stationary phase was a C18
reversed phase pre-packed column (Chromabond Flash 25, 26 g, Macherey-Nagel, Hoerdt, France),
and the mobile phase was a gradient: H2O (A)/acetonitrile (B) (80:20 to 100:0 in 190 min). Fourteen
fractions were collected, and the fifth fraction (16.5 mg) was purified using semi-preparative HPLC
with a Prevail C18 column and a gradient of water and acetonitrile (70:30). Five fractions were obtained:
compound 6 in the second fraction and compound 7 in the third fraction.
For the resin extract, the flash chromatography was realized with a silica column pre-packed
normal phase (SiO2) as stationary phase, and the mobile phase was a gradient: cyclohexane
(A)/CH2Cl2 (B) (100:0 to 0:100 in 60 min), CH2Cl2 (B)/EtOAc (C) fractions (100:0 to 0:100 in 315 min)
and EtOAc (C)/MeOH (D) (100:0 to 80:20 in 50 min) with a 10-min run per step. Twenty-three fractions
were collected.
The sixth fraction (12.1 mg) was purified by semi-preparative HPLC with a Prevail C18 column
and a gradient of H2O/acetonitrile (5 min 30% of B, 5 to 25 min 30:70 to 0:100, 25 to 30 min 100% B),
yielding compound 8.
The eighth fraction (235.3 mg) was firstly fractionated by flash chromatography using a silica
gel column pre-packed normal phase (SiO2) and a gradient of cyclohexane, CH2Cl2, EtOAc, and
MeOH in 150 min with a 10 min run per step. Ten fractions were collected, and compound
5 was
purified from the fourth fraction by semi-preparative HPLC on a Prevail C18 column with a gradient
of H2O (A)/acetonitrile (B) (100:0 to 40:60 in 20 min and 40:60 to 0:100 in 2 min and 100% B in 10 min).
Compound
4 was purified from the sixth fraction by semi-preparative HPLC on a Prevail™ C18 column
with a gradient of H2O/acetonitrile 70:30.
The ninth fraction (143.1 mg) was also fractionated by flash chromatography using a silica gel
column pre-packed normal phase (SiO2) and a gradient of cyclohexane, CH2Cl2, EtOAc, and MeOH in
150 min with a 10-min run per step. Eleven fractions were collected, and compound
from the third fraction by semi-preparative HPLC with the Prevail C18 column with a gradient of
H2O/acetonitrile 70:30. Compound was purified from the seventh fraction with the same system
(Prevail C18 column) and a gradient of H2O/acetonitrile 75:25.
3 was purified
2

Molecules 2017, 22, 371
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The 21st fraction (6.9 mg) afforded compound 1.
3.6. Biological Assays: Cytotoxic Activities
Cytotoxic properties of pure compounds were determined with a standard tetrazolium assay [18].
B16 (6000 cells/well) and HaCaT (10,000 cells/well) cells were seeded in RPMI 1640 medium with 5%
fetal calf serum (FCS) and antibiotic at day 0 in well-plate. Incubation was performed at 37 ◦C in an
atmosphere of 5% CO2. After 24 h of incubation, compounds were added at diffe◦rent concentrations
(1, 10, 50, 100, and 200 µg/mL), and the plate was incubated 24 h more (37 C, 5% CO2). Cell
growth and viability were then measured at 540 nm, using a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) assay. Each experiment was repeated at least three times.
3.7. Preparation of L-Bromo-Tyrosine (10)
L-Tyrosine (5.0 g, 55.0 mmol) was dissolved into 15 mL of acetic acid. A solution of bromine
(4.3 g, 55.0 mmol) solubilized in 3 mL of acetic acid was added dropwise and the reaction mixture was
stirred at room temperature for 4 h. The precipitate was then filtrated and washed with acetic acid
and diethyl ether, affording 10 as a white solid. This compound was used in the next step without
further purification.
3-Bromo-L-tyrosine (10): white amorphous powder, 1H-NMR (CD3OD, 300 MHz), and 13C-NMR data
(CD3OD, 75 MHz) as described in the literature [19].
3.8. Protection of the Amine Group (11)
The brominated amino acid (10) (1 eq.) (4.5 g, 13.2 mmol) was poured in 60 mL of a mixture of
MeOH/acetone (1:1). A solution of sodium hydrogen carbonate (2.2 eq.) (2.4 g, 29.3 mmol) in 20 mL
of water and 1.6 eq. of (BOC)2O (4.8 g, 22.0 mmol) were added successively to the reaction medium
after stirring at room temperature for 4 h. The solvent was removed under vacuum, and the aqueous
residue was washed with cyclohexane and acidified with citric acid until reaching pH 2. The product
precipitated and was extracted from the aqueous layer with 3
layer was then dried with Na2SO4, and the solvent was removed in vacuo leading to 4.5 g (95%) of the
×
30 mL of ethyl acetate. The organic
protected product (11).
N-Boc-3-Bromo-L-tyrosine (11): white amorphous powder, 1H-NMR and 13C-NMR data (MeOD,
75 MHz) as described in the literature [19].
3.9. Condensation of the Amino Acids
General procedure: To a solution of intermediate 11 in (Dichloromethane/acetonitrile) (9/1) was
added successively 1.0 eq. of (D or L) proline methyl ester hydrochloride (12a or 12b), 2.2 eq. of DIPEA
and 1.2 eq. of TBTU. After stirring at room temperature for 2 h, the reaction was quenched by adding
NH4Cl and then washed with a solution of saturated NaHCO3 and brine. The organic residue was
then dried with Na2SO4 and concentrated under vacuum. The crude product was purified on silica
gel using CH2Cl2/EtOAc (60:40) as eluent to afford the final products 13a (L-Tyr, D-Pro) (60%) and 13b
(L-Tyr, L-Pro) (20%).
Methyl ((S)-3-(3-bromo-4-hydroxyphenyl)-2-((tert-butoxycarbonyl)amino)propanoyl)-D-prolinate
prepared from 11 (0.76 g, 2.1 mmol) and 12a (0.35 g, 2.1 mmol) in 20 mL of solvent (DIPEA 0.60 g,
4.6 mmol; TBTU 0.81 g, 2.5 mmol); white oil; [α]2D0 0.11 (c 0.77, CHCl3), 1H-NMR (300 MHz, CDCl3)
7.28 (d, J = 2.1 Hz, 1H, H-3), 7.04 (dd, J = 8.3, 2.1 Hz, 1H, H-5), 6.86 (d, J = 8.3 Hz, 1H, H-6), 5.42 (d,
J = 8.5 Hz, 1H, H-Boc), 4.56 (dd, J = 7.3, 6.3 Hz, 1H, H-8), 4.33 (dd, J = 8.0, 3.8 Hz, 1H, H-14), 3.71 (s,
3H, O-CH3), 3.55–3.65 (m, 1H, H-11a), 3.03–2.71 (m, 3H, H-11b, H-7a, H-7b), 2.01–1.79 (m, 3H, H12a,
H-12b, H-13a), 1.63 (m, 1H, H-13b), 1.41 (s, 9H, Boc C-(CH3)3), 13C-NMR (75 MHz, CDCl3)
172.2
(C-15), 170.1 (C-9), 155.0 (Boc = O), 151.3 (C-1), 132.8 (C-3), 130.4 (C-4), 130.2 (C-5), 115.9 (C-6), 109.8
(13a):
−
δ
N
δ
C

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(C-2), 79.9 (Boc
(Boc C-(
C
-(CH3)3), 58.8 (C-14), 53.5 (C-8), 52.3 (C-16), 46.9 (C-11), 38.9 (C-7), 29.0 (C-12), 28.3
C
H3)3), 24.5 (C-13), HRESIMS m/z 493.0945 [M + Na]+ (calcd. for C20H26BrN2O6Na, 493.0945).
Methyl ((S)-3-(3-bromo-4-hydroxyphenyl)-2-((tert-butoxycarbonyl)amino)propanoyl)-L-prolinate
(13b):
prepared from 11 (1.8 g, 5.0 mmol) and 12b (0.83 g, 5.0 mmol) in 50 mL of solvent (DIPEA 1.42 g,
11.0 mmol; TBTU 1.90 g, 5.9 mmol); white oil; [α]2D0 +0.39 (c 0.77, CHCl3), 1H-NMR (500 MHz, CDCl3)
mixture of rotamers
δ 7.55 and 7.37 (d, J = 2.1 Hz, 1H, H-3), 7.25 and 7.10 (dd, J = 8.3, 2.2 Hz, 1H, H-5),
7.04 and 6.89 (d, J = 8.3 Hz, 1H, H-6), 4.82 and 4.60 (q, J = 6.6 Hz, 1H, H-8), 4.40 and 4.50 (dd, J = 8.4, 4.1
Hz, 1H, H-14), 3.62 and 3.75 (s, 3H, O-CH3), 3.40, 3.21 and 3.67–3.55 (m, 2H, H-11a and H-11b), 3.35
and 3.02 (dd, J = 13.9, 6.7 Hz, 1H, H-7a), 3.17 and 2.82 (dd, J = 13.9, 5.9 Hz, 1H, H-7b), 2.18 and 2.05 (m,
1H, H-12a), 1.96 and 1.81 (m, 1H, H-12b), 2.02–1.88 and 1.74 (m, 2H, H-13a and H-13b), 1.39 (s, 9H, Boc
C-(CH3)3), 13C-NMR (125 MHz, CDCl3)
δ
172.5 and 172.2 (C-15), 170.2 and 170.1 (C-9), 155.2 (Boc
C
= O), 148.5 and 148.2 (C-1), 134.7 and 133.1 (C-3), 129.7 (C-4), 130.3 and 130.1 (C-5), 123.3 and 116.4
(C-6), 109.8 and 109.7 (C-2), 80.1 (Boc C-(CH3)3), 59.2 and 53.1 (C-14), 54.6 and 52.8 (C-8), 52.5 and 52.4
(C-16), 47.2 (C-11), 38.3 and 37.3 (C-7), 30.7 and 29.2 (C-12), 28.4 (Boc C-(CH3)3), 25.0 and 22.4 (C-13),
HRESIMS m/z 493.0941 [M + Na]+ (calcd. for C20H26BrN2O6Na, 493.0945).
3.10. Deprotection and Cyclization
Compounds 13a (0.44 g, 0.9 mmol) and 13b (0.23 g, 0.5 mmol)) were poured into a reactor with
respectively 15 mL and 3 mL of water and heated to 130 ◦C during 4 h as described in literature for
a one-pot deprotection and cyclization [20]. After cooling, the reaction mixture was extracted three
times with 15 mL of ethyl acetate. The organic layer was desiccated with Na2SO4 and dried under
vacuum. The residues were purified on silica gel using different eluent systems: 100% ethyl acetate
for compound 13a and dichloromethane/ethyl acetate (60:40) for compound 13b. These purifications
yielded to the final products 6 (64%) and 7 (36%).
3.11. Analytical Data
All fractions were analyzed with HPLC-UV using a Prevail C18 column with a gradient of H2O
(A)/acetonitrile (B): 5 min 100:0, 30 min from 0% of B to 100% of B, 10 min 100% B followed by
equilibration of the column.
Cyclo (L-Pro-L-O-Met) (
1
): 6.9 mg, white amorphous powder; rt: 18.8 min, [α]D20
−
4.0 (c 1, MeOH),
1H-NMR (CDCl3, 500 MHz) and 13C-NMR data (CDCl3, 125 MHz) as described in the literature [12],
HRESIMS m/z 245.1277 [M + H]+ (calcd. for C10H17N2O3S, 245.0955).
Cyclo (L-Pro-L-Tyr) (
1H-NMR (CD3OD, 500 MHz) and 13C-NMR data (CD3OD, 125 MHz) as described in the literature [13
HRESIMS m/z 261.1225 [M + H]+ (calcd. for C14H17N2O3, 261.1234).
2
): 5.7 mg, white amorphous powder; r.t.: 20.7 min, [α]2D0 +15.7 (c 1, MeOH),
,14],
Cyclo (D-Pro-L-Tyr) (
3
): 8.5 mg, white amorphous powder; r.t.: 21.1 min, [α]D20
−
38.2 (c 1, MeOH),
1H-NMR (CD3OD, 500 MHz) and 13C-NMR data (CD3OD, 125 MHz) as described in the literature [13],
HRESIMS m/z 261.1225 [M + H]+ (calcd. for C14H17N2O3, 261.1234).
Cyclo (L-Pro-L-Val) (
4
): 1.1 mg, white amorphous powder; r.t.: 21.9 min, [α]D20
−
23.0 (c 1, CHCl3),
1H-NMR (CD3OD, 500 MHz) and 13C-NMR data (CD3OD, 125 MHz) as described in the literature [14],
HRESIMS m/z 197.1282 [M + H]+ (calcd. for C10H17N2O2, 197.1285).
Cyclo (L-Pro-L-Leu) (
1H-NMR (CD3OD, 500 MHz) and 13C-NMR data (CD3OD, 125 MHz) as described in the literature [14
HRESIMS m/z 211.1438 [M + H]+ (calcd. for C11H19N2O2, 211.1441).
5
): 4.3 mg, white amorphous powder; r.t.: 24.1 min, [α]D20
−
27.4 (c 1, MeOH),
,15],
Cyclo (D-Pro-L-Br-Tyr) (6
): 1.4 mg, white amorphous powder; r.t.: 23.4 min, [α]2D0 +38.4 (c 0.77, MeOH),
1H-NMR (CD3OD, 500 MHz) and 13C-NMR data (CD3OD, 125 MHz) as described in Table 1, HRESIMS
m/z 339.0329 [M + H]+ (calcd. for C14H16BrN2O3, 339.0339).

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Cyclo (L-Pro-L-Br-Tyr) (
7
): 2.1 mg, white amorphous powder; r.t.: 23.9 min, [α]D20
−
29.4 (c 0.77, MeOH),
1H-NMR (CD3OD, 500 MHz) and 13C-NMR data (CD3OD, 125 MHz) as described in Table 1, HRESIMS
m/z 339.0329 [M + H]+ (calcd. for C14H16BrN2O3, 339.0339).
Indole-Carboxaldehyde (8
): 5.0 mg, white amorphous powder; r.t.: 26.2 min, 1H-NMR (CD3OD, 500 MHz)
and 13C-NMR data (CD3OD, 125 MHz) as described in the literature [15], HRESIMS m/z 146.0598 [M + H]+
(calcd. for C9H8NO, 146.0600).
4. Conclusions
In this study two new diketopiperazines as well as five known diketopiperazines and one
known auxin derivative have been isolated from the culture medium of our strain Nocardia ignorata.
The synthesis of the new compounds was realized, and confirmed the structure elucidated by 2D NMR
data and HRMS. None of the compounds isolated—except the auxin derivative—presented cytotoxic
activity, but these compounds are well known for playing a role in bacterial communication. The role of
diketopiperazines in quorum sensing is currently described. These compounds could thus interact with
N-acylhomoserine lactone (AHL) biosensor and in this way they can modulate bacterial characters
such as bioluminescence (via LuxR from Vibrio fischeri) [21], swarming in Serratia liquefaciens [22],
pathogenicity of diverse bacteria [23,24], and could inhibit bacterial biofilm [25]. These types of
compounds involved in cell-to-cell communications activate specific bacterial behavior by regulating
the gene expression in response to bacterial density [26]. The two new compounds—only produced
during the death phase of the bacterial growth—probably have a role in decline of bacteria during
this stage. Moreover, the production of brominated compounds from our bacterium highlights the
presence of a halogenase in the enzymatic machinery of this strain.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: We thank the “Ligue contre le Cancer 35” for financial support for the acquisition of the
bioreactor NewBrunswick BioFlo 115, the Platform Bio2mar for the UPLC-HRMS analysis and the CRMPO for the
HRMS spectra.
Author Contributions: S.T. and A.N. conceived and designed the experiments; A.N. performed the experiments;
A.N. analyzed the data; S.F. and A.N. run the NMR experiments and analyzed them; I.R. realized the biological
assays; N.G., J.-P.H. and A.N. realized the synthesis experiments; A.N. and S.T. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 6, 7, 13a and 13b are available from the authors.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).