Structure elucidation of a new natural diketopiperazine from a Microbispora aerata strain isolated from Livingston Island, Antarctica

Article abstract of DOI:10.1080/14786419.2012.665911

A new natural diketopiperazine (1) was obtained from the culture broth of Microbispora aerata strain imbas-11A, isolated from penguin excrements collected on the Antarctic Livingston Island. Compound 1 was purified consecutively by solvent extraction, silica gel column chromatography and preparative HPLC. The structure of the compound was elucidated by 1D and 2D NMR experiments and mass spectrometric investigations. The absolute configuration of compound 1 was determined by amino acid analysis and NOESY correlations. A low antiproliferative and cytotoxic effect of trans-cyclo-(D-tryptophanyl-L-tyrosyl) (1) was determined with L-929 mouse fibroblast cells, K-562 human leukemia cells and HeLa human cervix carcinoma. Trans-cyclo-(D-tryptophanyl-L-tyrosyl) (1) did not show antimicrobial activity at a concentration of 50 μg per disc against Bacillus subtilis, Staphylococcus aureus, Streptomyces viridochromogenes, Escherichia coli, Candida albicans and Mucor miehei.

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Structure elucidation of a new natural
diketopiperazine from a Microbispora
aerata strain isolated from Livingston
Island, Antarctica
a
b
a
Veneta Ivanova , Hartmut Laatsch , Mariana Kolarova
&
a
Krasja Aleksieva
a
Institute of Microbiology, Bulgarian Academy of Sciences, Acad.
G.Bonchev-str., Bl. 26, 1113 Sofia, Bulgaria
b
Institute for Organic and Biomolecular Chemistry, Georg-August-
University Göttingen, Tammannstrasse 2, D-37077 Göttingen,
Germany
Version of record first published: 15 Mar 2012.
To cite this article: Veneta Ivanova , Hartmut Laatsch , Mariana Kolarova & Krasja Aleksieva (2013):
Structure elucidation of a new natural diketopiperazine from a Microbispora aerata strain isolated
from Livingston Island, Antarctica, Natural Product Research: Formerly Natural Product Letters,
27:2, 164-170
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Natural Product Research, 2013
Natural Product Research
Vol. 27, No. 2, 164–170, http://dx.doi.org/10.1080/14786419.2012.665911
2
012, 1–7, iFirst
Structure elucidation of a new natural diketopiperazine from a
Microbispora aerata strain isolated from Livingston Island, Antarctica
a
b
a
a
Veneta Ivanova *, Hartmut Laatsch , Mariana Kolarova and Krasja Aleksieva
a
Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G.Bonchev-str., Bl. 26,
1
Go¨ttingen, Tammannstrasse 2, D-37077 Go¨ttingen, Germany
b
113 Sofia, Bulgaria; Institute for Organic and Biomolecular Chemistry, Georg-August-University
(
Received 19 May 2011; final version received 19 November 2011)
A new natural diketopiperazine (1) was obtained from the culture broth of
Microbispora aerata strain imbas-11A, isolated from penguin excrements collected
on the Antarctic Livingston Island. Compound 1 was purified consecutively by
solvent extraction, silica gel column chromatography and preparative HPLC. The
structure of the compound was elucidated by 1D and 2D NMR experiments and
mass spectrometric investigations. The absolute configuration of compound 1
was determined by amino acid analysis and NOESY correlations. A low
antiproliferative and cytotoxic effect of trans-cyclo-(D-tryptophanyl-L-tyrosyl) (1)
was determined with L-929 mouse fibroblast cells, K-562 human leukemia cells and
HeLa human cervix carcinoma. Trans-cyclo-(D-tryptophanyl-L-tyrosyl) (1) did not
show antimicrobial activity at a concentration of 50 mg per disc against Bacillus
subtilis, Staphylococcus aureus, Streptomyces viridochromogenes, Escherichia coli,
Candida albicans and Mucor miehei.
Keywords: Microbispora aerata; Antarctica; trans-cyclo-(D-tryptophanyl-
L-tyrosyl); dipeptide; diketopiperazine
1
. Introduction
Antarctica is the coldest, driest, windiest and most inaccessible continent on earth, with
inherent difficulties for scientific research, which make otherwise practical experiments
impossible. Only about 2% of continental Antarctica is ice-free (Chipev & Veltchev, 1996).
During the course of our screening program for bioactive secondary metabolites, we
investigated an Antarctic isolate imbas-11A of Microbispora aerata and obtained a new
natural diketopiperazine (1).
Diketopiperazines are forming a class of cyclic organic compounds that result from
peptide bonds between two amino acids to form a bis-lactam. They are the smallest
possible cyclic peptides. Diketopiperazines are commonly biosynthesised from amino acids
by different organisms, including mammals, and are considered to be secondary
metabolites (Martins & Carvalho, 2007). Some proteases, such as dipeptidyl peptidases,
cleave the terminal ends of proteins to generate dipeptides, which naturally cyclise to form
diketopiperazines. Due to their rigid structure, chiral nature and varied side chains,
diketopiperazines are an attractive scaffold for drug design. For both natural and synthetic
*
Corresponding author. Email: venibiva@microbio.bas.bg
ISSN 1478–6419 print/ISSN 1478–6427 online
*
Corresponding author. Email: venibiva@microbio.bas.bg
�
http://dx.doi.org/10.1080/14786419.2012.665911
2012 Taylor & Francis
h© tt 2p 0: /1/ 3w Tw a wy l. to ar n&d f Fo rna lni nc ie s.com

2
V. Ivanova et al.
Natural Product Research
165
diketopiperazines, a wide variety of biological activities was reported, including antitumor
Graz et al., 2000; Milne, Hunt, Rostoll, Van Der Walt, & Graz, 1998; Nicholson et al.,
(
2
2
006), antiviral (Sinha, Srivastava, De Clercq, & Singh, 2004), antifungal (Houston et al.,
004) and antibacterial (Kwon, Park, Yun, Pyun, & Kim, 2000) activities. Fdhila,
Vazquez, Sanchez, and Riguera (2003) have found that especially the D, D-diketopiper-
´ ´
azines are bioactive, while other stereoisomers are less active or even inactive.
Unfortunately, the absolute configuration of simple diketopiperazines is seldom reported.
Graz, Hunt, Jamie, Grant, and Milne (1999) and Graz, Jamie, Versluis, and Milne
(
2001) established that cyclo-(Pro, Trp) and cyclo-(Phe, Pro) had broad spectrum
antibacterial properties, and cyclo-(Trp, Trp) and cyclo-(Trp, Pro) showed broad
antifungal activities. Kimura et al. (1996) isolated cyclo-(L-tryptophanyl-L-phenylalanyl)
from an unidentified Penicillium sp. The metabolite and its components, L-tryptophane
and L-phenylalanine, exhibited different biological activities against pine plants and tea
pollen, lettuce, cress and rice seeds. The metabolite cyclo-(L-tryptophanyl-L-phenylalanyl)
was also isolated from a strain of Aspergillus sp. SC319 (Barrow et al., 1993; Sedlock et al.,
1
994). Moreover, cyclo-(L-tryptophanyl-L-prolyl) was isolated as a fungal metabolite of
Penicillium brevicompactum (Bourke, 1994). Kobayashi et al. (1994) isolated the same
metabolite from a Vibrio sp. The activity of these cyclic dipeptides thus indicates potential
application as pharmacological agents.
In this article, we describe the isolation, structure elucidation and absolute configu-
ration of the related new diketopiperazine (1), using high-resolution EI-MS, ESI-MS and
extensive NMR spectroscopic analysis. The taxonomic study of the produced strain and
the growth conditions have been described in previous reports (Gushterova et al., 2002;
Ivanova et al., 2004).
2
. Results and discussion
Compound 1 was obtained as a colourless amorphous solid, which was soluble in lower
alcohols, dimethylsulfoxide, N, N-dimethylformamide and ethyl acetate, but insoluble in
’
water, ether and n-hexane. It gave a blue colour reaction with Ehrlich s reagent, suggesting
the presence of an indole skeleton in the molecule and also gave positive reactions with
Dragendorff’s reagent and 3% vanillin-sulfuric acid, but not with ninhydrine, Sakaguchi
and FeCl reagents. The UV-Vis spectra (Figure S1, Supplementary Online Material
3
(
SOM)) showed absorption maxima at 222, 278 nm by HPLC analysis (diode array
detector). The presence of two secondary amide groups in the molecule was suggested by
�
1
an infrared band at 1667 cm , together with signals at �7.71 (1H) and 7.90 (1H) in the
1
H NMR spectrum. The structure of 1 was finalised on the basis of electrospray (ESI-MS),
high-resolution electron impact mass spectrometry (HREIMS) and extensive NMR studies
1
13
1
1
1
H, C, APT, H– H COSY, H– H NOESY, HSQC and HMBC).
1
(
The (þ)-ESI mass spectrum (Figure S2) of 1 displayed pseudo-molecular ions at m/z
þ,
þ
þ
3
49.9 (M þ H) 372.0 (M þ Na) and 721.5 (2M þ Na) and in the negative ion mode at
�
m/z 348.3 (M � H) (Figure S3). The molecular formula of compound 1 was determined
þ
by high-resolution electron impact mass spectrometry (HREIMS) ([M] observed,
m/z 349.14331, Calcd 349.14584) to be C H N O . This formula indicated 13 degrees
2
0
19
3
3
of unsaturation. EI-MS (Figure S4) of 1 exhibited a strong ion peak at m/z 349, together
with fragment peaks at m/z 166, 163 (C H NO ), 130 (C H N), 107 and 77. The presence
of an indole ring was indicated by the characteristic fragment peak at m/z 130 in the mass
9
9
2
9
8
’
spectrum and by the positive reaction with Ehrlich s reagent.
1
In the H NMR spectrum (Figure S5) of compound 1, the low-field signals at � 7.02
H-2), 7.58 (H-4), 7.05 (H-5), 7.11 (H-6), 7.33 (H-7) were peculiar for an indole ring,
H
(

1
66
V. Ivanova et al.
Natural Product Research
3
exchangeable protons with signals at �H 10.85, 7.90, 7.71 (DMSO-d ) were due to NH
6
groups. Furthermore, two pairs of methylene protons were observed at �H 3.02/2.75 and
2.55/1.48, two methine multiplets at �H 4.18 and 3.85 and a 1H signal at �H 8.00 (OH).
A 1,4-disubstituted benzene ring was indicated by an AB pattern at � 6.43 and 6.61.
H
1
3
In the C NMR (Figure S6) and APT spectra of 1, all 20 carbon signals were visible
Table S1), which were assignable to two carbonyl carbons (�C 169.2 and 169.5),
(
1
methine carbons (� 57.1 and 57.9). The chemical shifts of all proton and carbon atoms of
4 aromatic carbons (9 CH and 5 C signals), two methylene (� 30.8 and 40.6) and two
q C
C
1
are summarised in Table S1.
For the structural assignment of compound 1, the H– H COSY and H– C long-range
heteronuclear coupled NMR spectra (HMBC) were of pivotal importance. According to the
1
1
1
13
1
1
H– H COSY spectra, the methylene group (H 8) at � 3.02/2.75 was linked to the methine
2
–
H
group (H-9) at � 4.18 and the methylene group (H 15) at � 2.55/1.48 was linked to the
2–
H
H
methine group (H-12) at � 3.85. The COSY experiment also demonstrated couplings from
H
the aromatic proton H-4 at � 7.58 to proton H-5 (� 7.05), from H-5 to H-6 (� 7.11), and
H
H
H
from H-6 to H-7 at � 7.33. The protons H-17/21 at � 6.43 of a p-substituted phenol gave
H
H
the expected correlations to protons H-18/20 (� 6.61) (Figure S7).
H
A heteronuclear multiple bond correlation (HMBC) experiment showed long-range
couplings from proton H-2 at �H 7.02 to the quaternary carbons C-3 at �C 109.6, C-3a
(� 128.8) and C-7a at � 137.9. Further correlations are reported in Table S1. Thus the
C C
planar structure of 1 was unequivocally determined as cyclo-(Trp, Tyr).
As no cross peaks between the two amino acid residues were visible in the NOESY
spectra (Table S2), the absolute configuration of the amino acids of compound 1 was
determined by acid hydrolysis and analysis of the amino acids. The configuration of the
resulting amino acids was determined as L-tyrosine and D-trypthophane by HPLC, using a
chiral CR þ Crompak Daicel column without further derivatisation. The retention times
of the amino acids (tR 7.69 and 21.02 min) were compared with those of authentic
standards (D-Tyr tR 5.84/L-Tyr tR 7.51; D-Trp tR 20.39/L-Trp tR 23.50). On the basis of
these data, the structure of compound 1 (Figure 1) was finally elucidated as the
diketopiperazine trans-cyclo-(D-tryptophanyl-L-tyrosyl). A low antiproliferative and cyto-
toxic effect of 1 was determined with L-929 mouse fibroblast cells, K-562 human leukaemia
�
1
�1
cells (GI ¼ 50.0 mg mL ) and HeLa human cervix carcinoma (CC ¼ 47.0 mg mL ).
5
0
50
Trans-cyclo-(D-tryptophanyl-L-tyrosyl) (1) did not show antimicrobial activity at a
concentration of 50 mg per disc against Bacillus subtilis, Staphylococcus aureus,
Streptomyces viridochromogenes, Escherichia coli, Candida albicans and Mucor miehei.
O
H
4
8
18
OH
3
a
5
1
4
NH
3
2
9
1
2
16
6
1
N
H
HN
11
20
7a
7
15
H
O
Figure 1. Structure of trans-cyclo-(D-tryptophanyl-L-tyrosyl) (1).

4
V. Ivanova et al.
Natural Product Research
167
3
3
. Experimental
.1. General experimental procedures
Electrospray mass spectra (ESI-MS) were recorded on a LCQ Finnigan Mass
Spectrometer. ESI-HR mass spectra were recorded on a APEX IV, 7T, FT-ICR MS
Bruker Daltonik. Electron Impact Mass-spectra (EI-HRMS) were recorded on a double
focusing sector field mass spectrometer AMD-402 (AMD Inectra, Harpstedt, Germany).
1
13
H (300 and 600 MHz) and C (75.5 and 125.7 MHz) NMR spectra were measured on a
Bruker AMX 300 and on a Varian Inova 600 (599.740 MHz) spectrometer in DMSO-d6
and CD OD. The chemical shifts are expressed in � values with TMS as an internal
3
1
1
1
1
standard. H– H COSY, H– H NOESY, HSQC, and HMBC were obtained by
conventional methods. UV-Vis spectroscopy was recorded on a Specord 2000 instrument
(Analytik Jena, Germany). IR spectra were recorded on a Beckman DU 601 and
Shimadzu IR scanning spectrophotometers.
3
.2. Producing organism and growth conditions
Microbispora aerata strain imbas-11A was isolated from penguin excrements collected on
the Antarctic Livingston Island. Sampling had been performed in the course of the Third
Bulgarian Expedition to Antarctica (1995–1996). The strain imbas-11A of M. aerata was
deposited in the collection of the Institute of Microbiology of the Bulgarian Academy of
Sciences. Based on the effect of temperature on its growth rate, it is thermophilic with an
�
optimal growth temperature at 50 C. The mature slant culture of M. aerata imbas-11A
was inoculated into Erlenmeyer flasks (500 mL), containing 50 mL of seed medium
consisting of 0.5% peptone, 0.5% cornsteep liquor, 1.0% starch, 0.5% NaCl and 0.5%
CaCO , pH 7.2 after autoclaving. The flasks were cultivated on a rotary shaker at 325 rpm
3
�
for 18 h at 50 C. The seed culture (5%) was inoculated into Erlenmeyer flasks (1000 mL),
containing 400 mL of the production medium. The fermentation medium contained the
�
same ingredients as the seed medium. The cultivation was carried out for 48 h at 50 C on a
rotary shaker (Gushterova et al., 2002; Ivanova et al., 2004).
3
.3. Isolation of trans-cyclo-(Trp, Tyr) from the culture broth of M. aerata imbas-11A
�
After 48 h of cultivation at 50 C, the culture broth (10.0 L) of a peptone/cornsteep liquor/
starch medium of M. aerata imbas-11A was centrifuged. The supernatant fluid was
extracted twice with each 5 L of ethyl acetate. The mycelium (ꢀ100 g) was extracted thrice
by 100 mL acetone and the combined acetone extracts were concentrated to the aqueous
residue. This water phase was re-extracted twice by 300 mL of ethyl acetate. The combined
ethyl acetate extracts from the mycelium and the culture filtrate respectively, were filtered,
dried over Na SO and evaporated to dryness under reduced pressure yielding 240 mg of
2
4
crude product, which was dissolved in chloroform and chromatographed on a silica gel 60
column (70–230 mesh) with chloroform : methanol (1 : 1 v/v). The final separation and
purification by preparative HPLC on Lichrospher RP-18 (column 250 ꢁ 10 mm, 10 mm),
using a gradient of 12–90% acetonitrile in 2 mM NH OAc buffer, pH 5.0 and monitoring
4
ꢂ1
at 220 nm (flow rate, 5.0 mL min , t ¼ 7.23 min) delivered the pure diketopiperazine (1),
R
(
10 mg) as colourless solid.
3
.4. Analytical HPLC
HPLC analysis of pure diketopiperazine (1) was carried out on a Symmetry C18 column
150 ꢁ 3.9 mm, 5 mm). Mobile phase: gradient of 20–90% acetonitrile in 2 mM NH OAc
(
4

1
68
V. Ivanova et al.
Natural Product Research
5
�
1
buffer, pH 5.0. Flow rate: 1.0 mLmin . Detection: 220 nm (diode array detector).
Compound 1 was eluted with a retention time (t ) of 7.83 min.
R
3
.5. Thin-layer chromatography
Thin-layer chromatography (TLC) was carried out on silica gel plates (Merck 60, F254)
with chloroform : methanol (8.0 : 2.0 v/v). The chromatographic spots were visualised by
’
spraying with a 0.5% solution of vanillin in methanol/sulfuric acid/acetic acid or Ehrlich s
ꢀ
reagent and heating at 120 C for 3–5 min. In this system compound 1 possessed an Rf
value of 0.38 and gave a blue-violet, rsp. a blue colour.
3
.6. Acid hydrolysis of compound 1
ꢀ
A sample (0.17 mg) of compound 1 was heated with 0.1 mL 6 N HCl at 110 C for 16 h. The
hydrolysate was evaporated to dryness and dissolved in H O. Analytical HPLC analysis of
2
the hydrolysate was carried out under isocratic conditions on a chiral CR þ Crompak
Daicel column (150 ꢁ 4 mm, 1.0, SUS 316, 5 mm) with bidist. water/HClO (pH 2); flow:
4
�1
0
.6 mLmin ; detection 254 nm. HPLC analysis of the 1-hydrolysate indicated the presence
of L-tyrosine and D-trypthophane: The retention times (tR, min) of the corresponding
amino acids matched those of authentic L-tyrosine (7.69) and D-trypthophane (21.02), and
were clearly different from the respective enantiomers.
3
.7. Antimicrobial activity of compound 1
Antimicrobial activity was determined by agar diffusion test according to European
Pharmacopoeia (1997). Test organisms were suspended in the melted nutrient agar (Serva)
and poured into petri dishes. Holes of 8 mm diameter were cut in the agar and filled with
�1
1
incubation for 18 h at 37 C for bacterial strains and 30 C for yeasts.
00 mL of a 100 mg L solution of the compound. Inhibition zones were read after
ꢀ
ꢀ
3
.8. Antiproliferative and cytotoxic assay of compound 1
Antiproliferative and cytotoxic activity was determined according to Ziehl, He, Dahse,
and Hertweck (2005).
3
.8.1. Trans-cyclo-(D-tryptophanyl-L-tyrosyl) (1)
Colorless amorphous solid. UV-Vis spectra by HPLC analysis in MeOH (diode array
1
detector): �max 222, 278 nm. H NMR (600 MHz in CD OD, ꢀ in ppm): 2.55 (dd, J ¼ 10.9,
3
H
8
.3 Hz, 1H, H-15a), 1.48 (dd, J ¼ 10.9, 8.3 Hz, 1H, H-15b), 3.02 (dd, J ¼ 14.6, 7.7 Hz, 1H,
H-8a), 2.75 (dd, J ¼ 14.6, 7.7 Hz, 1H, H-8b), 3.85 (m, 1H, H-12), 4.18 (m, 1H, H-9), 6.61
d, J ¼ 7.0 Hz, 2H, H-18 and H-20), 6.43 (d, J ¼ 7.0 Hz, 2H, H-17 and H-21), 7.02 (s, 1H,
H-2), 7.05 (dd, J ¼ 7.3, 7.3 Hz, 1H, H-5), 7.11 (dd, J ¼ 7.3, 7.3 Hz, 1H, H-6), 7.33
(
1
(
ꢀ
d, J ¼ 7.4 Hz, 1H, H-7), 7.58 (d, J ¼ 7.3 Hz, 1H, H-4). H NMR (300 MHz in DMSO-d ,
6
in ppm): 10.85 (s, 1H, H-1), 7.90 (d, J ¼ 2.0 Hz, 1H, H-10), 7.71 (d, J ¼ 2.0 Hz, 1H, H-13),
H
1
3
8
.00 (s, 1H, OH-19). C/APT NMR (125.7 MHz in CD OD, ꢀ in ppm): 169.5 (Cq, C-11),
3 C
1
69.2 (Cq, C-14), 157.4 (Cq, C-19), 137.9 (Cq, C-7a), 131.8 (CH, C-17 & C-21), 128.8
Cq, C-3a), 127.6 (Cq, C-16), 125.6 (CH, C-2), 122.6 (CH, C-6), 120.1 (CH, C-5), 119.8
(
(
(
(
(
CH, C-4), 116.2 (CH, C-18 & C-20), 112.4 (CH, C-7), 109.6 (Cq, C-3), 57.9 (CH, C-12), 57.1
þ,
CH, C-9), 40.6 (CH , C-15), 30.8 (CH , C-8). ESI-MS (positive): m/z 349.9 (M þ H) 372.0
2
2
þ
þ
�
M þ Na) and 721.5 (2M þ Na) ; ESI-MS (negative): m/z 348.3 (M � H) ; HREIMS
þ
[M] obs., m/z 349.14331, Calcd for C H N O , 349.14584).
3
2
0
19
3

6
V. Ivanova et al.
. Conclusions
Natural Product Research
169
4
The diketopiperazine trans-cyclo-(D-tryptophanyl-L-tyrosyl) (1) is a new natural product,
isolated from the culture filtrate of M. aerata strain imbas-11A. The organism was isolated
from penguin excrements, collected on the Antarctic Livingston Island. Based on the effect
of temperature on its growth rate, the strain is thermophilic with an optimal growth
�
temperature at 50 C. The structure of trans-cyclo-(D-tryptophanyl-L-tyrosyl) (1) was
elucidated by one- and two-dimensional NMR experiments and mass spectrometric
investigations. The absolute configuration of compound 1 was determined by hydrolysis
and amino acid analysis on a chiral column and NOESY correlations.
To the best of our knowledge, cyclo-(Trp, Tyr) has not been isolated from any other
natural source, however, it is known as a synthetic product (Sloper & Land, 1980; Zeng,
´
Li, Hanzlik, & Aube, 2005). The crystal structure of the synthetic L-tyrosyl-L-tryptophan
monohydrate was reported from Goerbitz and Hartviksen, (2008). In our hands,
compound 1 showed weak antiproliferative and cytotoxic effects against L-929, K-562
and HeLa cell lines.
Supplementary material
Supplementary material is available online: UV-Vis spectrum (Figure S1), ESI- mass
spectrum (positive ion mode) (Figure S2), ESI- mass spectrum (negative ion mode) (Figure
1
13
1
S3), EI- mass spectrum (Figure S4), H (Figure S5), C (Figure S6) NMR data, H– H
1
COSY (Figure S7), Table S1 and Table S2 of trans-cyclo-(D-tryptophanyl-L-tyrosyl) (1).
Acknowledgements
We thank Dr H.-M. Dahse (HKI, Jena, Germany) for the determination of cytotoxicity, and
¨
Mr Olaf Senge (University of Gottingen) for the HPLC measurements. Support of this work by
The German Academic Exchange Service (DAAD), Bonn, Germany, is gratefully acknowledged.
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