N-acetyl-3,4-dihydroxy-L-phenylalanine, a second identified bioactive metabolite produced by Streptomyces sp. 8812

Full text of DOI:10.1038/ja.2012.2

The Journal of Antibiotics (2012) 65, 219–221
2012 Japan Antibiotics Research Association All rights reserved 0021-8820/12
www.nature.com/ja
&
NOTE
N-acetyl-3,4-dihydroxy-L-phenylalanine, a second
identified bioactive metabolite produced by
Streptomyces sp. 8812
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Jolanta Solecka , Aleksandra Rajnisz , Magdalena Postek , Joanna Zajko , Robert Kawecki , Vladimir Havlicek ,
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Elzbieta Bednarek and Lech Kozerski
The Journal of Antibiotics (2012) 65, 219–221; doi:10.1038/ja.2012.2; published online 1 February 2012
Keywords: antimicrobial activity; DD-peptidase inhibitor; JS-2; Streptomyces sp.
The emergence of multi-drug-resistant bacteria and the lack of new NMR data are shown in Table 1 and in the Supplementary materials
antibiotics in the antibiotic drug development pipeline is a major (Supplementary Figures 2S, 3S, 4aS, 4bS). NMR spectra were recorded
1
health concern. Therefore, there is a constant need to search for novel at 298 K on Varian INOVA 500 MHz spectrometer (Varian, Palo Alto,
1
antibacterial compounds. Streptomyces species are the richest source of CA, USA) operated at 499.8 MHz for H. The 2D experiments were
bioactive metabolites, including antibacterial compounds.2
run by using the standard Varian software, except the HMBC experi-
,9
In this paper, we report the isolation of a second bioactive metabolite, ment.8
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N-acetyl-3,4-dihydroxy-L-phenylalanine, named JS-2 (Figure 1), from the
The H NMR spectrum consists of seven signals: an ABM system in
supernatant of a Streptomyces sp. 8812 culture.3,4 In the course of our the aromatic part at 6.85, 6.70 and 6.79p.p.m. (3H); an ABX aliphatic
screening program for antimicrobial compounds from actinomycetal system at 2.80, 3.06 and 4.38 p.p.m. (3H); and a singlet from the CH3
metabolites for specific inhibitors of DD-peptidases (EC 3.4.16.4), we used group at 1.93p.p.m. (3H). Dynamic OH, COOH and NH protons are
1
exocellular DD-carboxypeptidase/transpeptidase (DD-peptidase) 64–575 II in fast exchange with bulk water and are not observed in the H NMR
from Saccharopolyspora erythraea 64–575 II.5 JS-2 exhibited slight spectrum. The assignment of protons was accomplished following the
inhibitory properties on the DD-peptidases 64–575 II and R39 from general rule of chemical-shift dispersion, using the proton–proton
6
Actinomadura R39 (formerly Streptomyces R39). It showed antimicrobial coupling pattern and the COSY spectrum.
activity against Bordetella bronchiseptica (B. bronchiseptica), Staphylococcus Carbon resonances were obtained from the H-{ C} HSQC (Sup-
epidermidis (S. epidermidis) and S. aureus (methicillin-resistant (MRSA) plementary Figures 4aS, 4bS) (six signals of proton-bearing carbon
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or vancomycin resistant (VRSA).
Streptomyces sp. 8812 fermentation culture was conducted as atoms) spectra. The H { C} HMBC spectrum was used also as a final
atoms) and H { C} HMBC (five signals of the quaternary carbon
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reported previously. Isolation and purification of JS-2 was performed, tool to make assignment of carbon resonance and to confirm the
guided by DD-peptidase 64–575 II assay.3,7 Purification steps were structure of JS-2.
ꢀ
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done as follows: anion exchange resin IRA-400 (AcO ), Backerbond
IR absorption at 3366 and 1724–1608 cm suggested the presence
SPE (PolarPlus C18 column, J.T. Baker, Phillipsburg, NJ, USA) and of hydroxyl and carbonyl groups in the structure (Supplementary
HPLC (Atlantis, dC18, Waters, Milford, MA, USA) (Supplementary Figure 5S). The UV spectrum showed a typical pattern for an aromatic
Figure 1S; Supplementary Purification of JS-2).
compound. Strong absorption at 280 nm confirmed a phenolic moiety
The structure of JS-2 compound was determined using 1- and 2D (Supplementary Figure 6S). According to the interpretation of these
NMR spectroscopy, UV, IR and MS measurements. Physico-chemical data, compound JS-2 presents the structure of N-acetyl-3,4-dihy-
properties of the compound are shown in Table 1.
The molecular formula of JS-2 was determined to be C H NO
5
on the basis of HRESI-MS.
droxy-L-phenylalanine.
Unequivocal confirmation of the JS-2 structure has been obtained
by its comparison with a synthetic sample prepared by acetylation of
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Laboratory of Biologically Active Compounds, National Institute of Public Health—National Institute of Hygiene, Warsaw, Poland; Siedlce University, Siedlce, Poland; Institute of
Microbiology, Laboratory of Molecular Structure Characterization, Prague, Czech Republic; 4National Medicines Institute, Warsaw, Poland and 5Institute of Organic Chemistry,
Polish Academy of Sciences, Warsaw, Poland
Correspondence: Dr J Solecka, Laboratory of Biologically Active Compounds, National Institute of Public Health—National Institute of Hygiene, Chocimska 24, Warsaw 00-791,
Poland.
E-mail: jsolecka@pzh.gov.pl
Received 19 September 2011; revised 5 January 2012; accepted 10 January 2012; published online 1 February 2012

bioactive metabolite
J Solecka et al
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L-Dopa with excess of acetic anhydride in hot water. The analytical compounds was weaker for DD-peptidase 64–575 II than for DD-
sample was obtained by preparative HPLC (RPC18, 5% acetonitrile in peptidase R39.
0.1% aq. AcOH). The spectral data of the obtained product fully
agreed with the above analysis.
The IC50 values of b-lactam antibiotics for DD-peptidase 64–575 II
and R39 were compared with the IC50 values of JS-2 for both
enzymes.5,13 IC50 values of many b-lactam antibiotics (ranging from
Under the procedures described previously,10–12 the inhibitory 1.6 mM to 0.015 mM) for DD-peptidase 64–575 II, as well as for DD-
effect of DD-carboxypeptidases activity was measured. Thioester peptidase R39 (ranging from 5 to 0.03mM) were lower than the IC50
C H -CO-NH-(D)CH(CH )-CO-S-CH -COOH was used as a sub- value of the investigated compounds for these enzymes. JS-2 exhibited
6
5
3
2
strate10,12 (Supplementary Bioassay). JS-2 and structurally related weak inhibitory effects on the DD-peptidases 64–575 II and R39.
compounds, 3,4-dihydroxy-L-phenylalanine (Sigma, St Louis, MO, However, so far, no protoalkaloid inhibitors of DD-peptidases have
USA), 3,4-dihydroxy-D-phenylalanine (Sigma) and N-acetyl-3,4-dihy- been described.
droxy-D-phenylalanine (synthesized analogously as L- form) were
examined. The IC50 values of these compounds for DD-peptidases tion assays were performed for a range of common proteolytic
4–575 II and R39 are presented in Table 2. enzymes: pancreatic elastase (type IV) from porcine pancreas, carboxy-
Results showed that IC50 values ranged from 3.96 to 17.79 mM. The peptidase A from bovine pancreas, trypsin from porcine pancreas, and
To examine the specificity spectrum of the JS-2 compound, inhibi-
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IC50 values of JS-2 for DD-peptidase 64–575 II and for DD-peptidase papain from Papaya latex, according to the procedures described in our
3
R39 were compared with the IC50 values of L-Dopa, D-Dopa and N- previous paper, and leucine aminopeptidase (type IV-S) from porcine
acetyl-D-Dopa for these enzymes. The IC50 values of JS-2 for DD- kidney microsomes as reported by Cahan et al.14 It was determined
peptidases 64-575 II and R39 were the lowest. In each case, L-isomer of that at a concentration of 1.4 mM, the JS-2 compound causes 50%
the compound inhibited both DD-peptidases in lower concentration inhibition of carboxypeptidase A, whereas the rest of the enzymes
than the D-isomer. Furthermore, the inhibitory activity of evaluated remained active at higher concentration, 1.84 mM.
Antibacterial activity of JS-2 was tested by liquid microdilution
method (Spectrostar Omega, BMG Labtech, Offenburg, Germany),
under standard conditions according to the CLSI references.15
A panel of Gram-positive and Gram-negative bacterial strains was
used (Supplementary Bioassay). The JS-2 compound showed weak
antibacterial activity against B. bronchiseptica, S. aureus (MRSA and
VRSA) and S. epidermidis (Table 2). Moreover, JS-2 showed a 4-h
OH
1
2
N
H
O
O
1"
5
'
6'
4
'
1' 3
2"
HO
3
'
2'
ꢀ1
delay of growth of S. aureus (MSSA) at 256 mg ml , a 2.5-h delay of
HO
ꢀ
1
bacterial growth at concentration 128 mg ml , and a 2-h delay of
Figure 1 Chemical structure of JS-2.
bacterial growth at 64mg ml 1, although the general MIC value for
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Table 1 (a) Physico-chemical properties and (b) H and 13C NMR data of JS-2
1
Appearance
a)
Molecular weight
White-cream crystal
(
239
Molecular formula
HRESI-MS (m/z)
Calcd
C11H13NO5
ꢀ
238.07210 (M-H)
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Found
238.07226 (M-H)
UV (MeOH) lmax nm (e)
231 (2850), 281 (2290)
[
a]D24 +41.1O
IR (KBr) nmax (cm 1
(c¼1.11, H2O)
ꢀ
)
3366, 1724, 1608, 1527, 1445, 1375, 1286, 1115, 814, 589
Position
b)
dCa
dHa (J in Hz)
1H-13C correlations
dNa
(
1
2
3
180.9
59.0
39.6
4.38 (dd, 8.7, 4.9)
2.80 (dd, 14.0, 8.7)
C1’, C3
C1’, C6’, C2’, C2, C1
C1’, C6’, C2’, C2, C1
3.05 (dd, 14.0, 4.9)
¢
1
133.2
119.5
146.3
145.2
118.8
124.2
175.9
24.5
¢
2
6.79 (d, 2.1)
C4’, C6’, C3, C3’
¢
3
¢
4
¢
5
6.85 (d, 8.1)
C3’, C1’, C4’, C6’
C4’, C2’
¢
6
6.70 (dd, 2.1, 8.1)
0
0
1
0
0
2
1.93 (s)
C1’’
NH
ꢀ251
aThe 1H and 13C chemical shifts, d, given in p.p.m., were referenced against the internal reference TSPA-d4, solvent H2O/D2O (9:1).
The Journal of Antibiotics

bioactive metabolite
J Solecka et al
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Table 2 Biological activity of JS-2 and structurally related
Natural compounds themselves are highly important, as they
constitute also the basis for developing synthetic drugs. In the last
10 years among the launched antibiotics, there are more natural
product-derivative new antibiotic templates compared with those
synthetically derived.1 Protoalkaloid JS-2 shows weak antibacterial
activity; however, due to the lack of hemolytic properties and
genotoxicity, it can be considered as a candidate for chemical
modifications that can increase its antibacterial activity.
compounds: (a) inhibitory effect on DD-peptidases and (b) in vitro
antibacterial activity (MIC lg ml 1)
ꢀ
Inhibition, IC50 (mM)
JS-2
DD-peptidases
a)
(N-Ac-L-Dopa) L-Dopa N-Ac-D-Dopa D-Dopa
(
DD-peptidase 64-575 II
DD-peptidase R39
10.97
3.95
13.84
5.11
11.85
4.92
17.79
6.05
ACKNOWLEDGEMENTS
This study was supported in part by the European Union, Ministry of Regional
Economy, Grant UDA-POIG. 01.03.01-14-136/09.
MIC mg ml 1)
ꢀ
(
Microorganism
JS-2
L-Dopa N-Ac-D-Dopa D-Dopa
1
Butler, M. S. & Cooper, M. A. Antibiotics in the clinical pipeline in 2011. J. Antibiot.
64, 413–425 (2011).
(b)
S. aureus (MSSA)
S. aureus (MRSA)
S. aureus (VRSA)
4256
256
128
128
128
32
640
640
80
40
2
3
Berdy, J. Bioactive microbial metabolites. J. Antibiot. 58, 1–26 (2005).
Solecka, J., Rajnisz, A. & Laudy, A. E. A novel isoquinoline alkaloid, DD-carboxypepti-
dase inhibitor, with antibacterial activity isolated from Streptomyces sp. 8812. Part I:
Taxonomy, fermentation, isolation and biological activities. J. Antibiot. 62, 575–580
(2009).
256
320
40
S. epidermisis
64
320
40
4
Solecka, J. et al. A novel isoquinoline alkaloid, DD-carboxypeptidase inhibitor,
B. bronchiseptica
40
20
320
40
with antibacterial activity isolated from Streptomyces sp. 8812. Part II:
Physicochemical properties and structure elucidation. J. Antibiot. 62, 581–585
(2009).
Solecka, J. & Kurzatkowski, W. Affinity of exocellular DD-carboxypeptidase/transpepti-
dase from Saccharopolyspora erythraea PZH 64-575 to beta-lactam compounds. Med.
Dosw. Mikrobiol. 51, 151–165 (1999).
Fr e` re, J.- M., Moreno, R. & Ghuysen, J. M. Molecular weight, amino acid composition
and physicochemical properties of the exocellular DD-carboxypeptidase-transpeptidase
of Streptomyces R39. Biochem. J. 143, 233–240 (1974).
Fr e` re, J. M., Leyh-Bouille, M., Ghuysen, J. M., Nieto, M. & Perkins, H. R. Exocellular
DD-carboxypeptidases/transpeptidases from Streptomyces. Methods Enzymol. 45,
Acinetobacter baumanii
Proteus vulgaris
4640
4640
4640
4640
4640
4640
80
4640
640
640
160
160
320
640
40
160
160
640
640
160
5
6
7
8
9
P. mirabilis
4640
4640
4640
640
Burkholderia cepacia
Pseudomonas aeruginosa
Stenotrophomonas maltophilia
6
10–636 (1976).
ꢀ
1
this bacteria was higher than 256 mg ml (Supplementary Figure 7S).
Additionally, for comparison, the antimicrobial activity of com-
pounds: 3,4-dihydroxy-L-phenylalanine, 3,4-dihydroxy-D-phenylala-
nine and N-acetyl-3,4-dihydroxy-D-phenylalanine were examined.
These compounds showed a slightly higher activity than JS-2
Meissner, A. & Sørensen, O. W. Economizing spectrometer time and broadband
excitation in small-molecule heteronuclear NMR correlation spectroscopy. Broadband
HMBC. Magn. Res. Chem. 38, 981–984 (2000).
Ko z´ mi n´ ski, W., Bednarek, E., Bocian, W., Sitkowski, J. & Kozerski, L. The new HMQC-
based technique for the quantitative determination of heteronuclear coupling constans.
Application for the measurement of 3J(H’i, Pi+1 ) in DNA oligomers. J. Magn. Reson.
1
60, 120 (2003).
(
Table 2).
Moreover, other activities of JS-2 were also examined, including
1
1
0 Adam, M., Damblon, C., Plaitin, B., Christiaens, L. & Frere, J.- M. Chromogenic
depsipeptide substrates for b-lactamases and penicillin-sensitive DD-peptidases.
Biochem. J. 270, 525–529 (1990).
1 Pratt, R. F. Substrate specificity of bacterial DD-peptidases (penicillin-binding
proteins). Cell. Mol. Life Sci. 65, 2138–2155 (2008).
genotoxicity and hemolytic properties. For assessment of genotoxicity,
ꢀ
two genetically modified Bacillus subtilis strains, M45 rec and H17
+
12 Adam, M. et al. Acyltransferase activities of the high-molecular-mass essential
penicillin-binding proteins. Biochem. J. 279, 601–604 (1991).
1
rec , obtained from Dr Yoshito Sadaie (Department of Induced
Mutation, National Institute of Genetics, Shizuoka, Japan) were
3 Dusart, J. et al. DD-carboxypeptidase-transpeptidase and killing site of b-lactam
antibiotics in Streptomyces strains R39, R61 and K11. Antimicrob. Agents Chemother.
3, 181–187 (1973).
4 Cahan, R., Hetzroni, E., Nisnevitch, M. & Nitzan, Y. Purification and identification of a
novel leucine aminopeptidase from Bacillus thuringiensis israelensis. Curr. Microbiol.
55, 413–419 (2007).
examined by the disc-diffusion method according to Kada’s proce-
dure.3,16 The JS-2 compound did not inhibit the growth of the tested
B. subtilis strains; hence, it was determined as nongenotoxic.
1
The hemolysis test was carried out according to a method described
3
15 CLSI. Method for Dilution Antimicrobial Susceptibility Tests for Bacteria That
Grow Aerobically; Approved Standard-Eighth Edition M07-A8 [ISBN 1-56238-689-
1]. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA
in literature. No hemolytic activity of JS-2 was observed on human
erythrocytes.
(
2009).
In conclusion, N-acetyl-3,4-dihydroxy-L-phenylalanine (JS-2) had
been identified earlier as a Streptomyces akiyoshiensis metabolite17 and
obtained by semisynthesis using microorganisms; for example, Pseu-
1
6 Kada, T., Hirano, K. & Shirasu, Y. Bacillus subtilis rec-assay test. In Chemical
Mutagenesis Vol. 6 (eds de Sevres, F. E. & Hollaender, A.) 149 (Plenum Press,
New York, 1980).
17 Smith, K. C. & White, R. L. Isolation of N-acetyl-3,4-dihydroxy-L-phenylalanine from
Streptomyces akiyoshiensis. J. Nat. Prod. 58, 1274–1277 (1995).
8 Chibata, I., Kakimoto, T., Nabe, K. & Shibatani, T. 3,4-Dihydroxy-L-phenylalanine.
domonas striafaciens and Vibrio tyrosinaticus.18,19 Only its antitumour
properties were studied so far.17,20 DD-peptidase inhibitory activity and
antimicrobial properties of JS-2 have not been previously defined. JS-2
compound belongs to a group of protoalkaloids, b-phenylethylamines
1
Japanese Patent. 72 39,692 (Tanabe Seiyaku Co., Ltd), 08 Dec (1972).
9 Nakayama, K., Yoshida, H.
1
& Tanaka, Y. B-(3,4-Dihydroxyphenyl)-L-alanine
derivatives. Japanese Patent. 12,092 (Kyowa Hakko Kogyo Co, Ltd), 02 Feb
(1974).
0 Inoue, S., Ito, S., Imai, Y., Kasuga, T. & Fujita, K. Growth inhibition of melanoma cells
by N-protected dopa derivatives. Biochem. Pharmacol. 36, 3540–3543 (1987).
1 Bentley, K. W. b-Phenylethylamines and the isoquinoline alkaloids. Nat. Prod. Rep. 23,
444–463 (2006).
2
1
and -amides isolated usually as plant metabolites. Results of this
study demonstrate that b-phenylethylamides, like JS-2, may show
antimicrobial activities and inhibitory properties on some closely
related proteinases (for example DD-peptidases).
2
2
Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
The Journal of Antibiotics