A new siderophore isolated from streptomyces sp. TM-34 with potent inhibitory activity against angiotensin-converting enzyme

Article abstract of DOI:10.1002/ejoc.201100189

A new siderophore named tsukubachelin was isolated from an iron-deficient culture medium of newly isolated strain Streptomyces sp. TM-34. The chemical structure of tsukubachelin was established by the interpretation of 2D NMR and TOF-Mass spectroscopic da

Full text of DOI:10.1002/ejoc.201100189

FULL PAPER
DOI: 10.1002/ejoc.201100189
A New Siderophore Isolated from Streptomyces sp. TM-34 with Potent
Inhibitory Activity Against Angiotensin-Converting Enzyme
Shinya Kodani,*[a][‡] Mayumi Ohnishi-Kameyama,[b] Mitsuru Yoshida,[b] and Kozo Ochi[c][‡‡]
Keywords: Siderophores / Natural products / Gallium / Inhibitors / Structure elucidation
A new siderophore named tsukubachelin was isolated from
an iron-deficient culture medium of newly isolated strain
Streptomyces sp. TM-34. The chemical structure of tsukuba-
chelin was established by the interpretation of 2D NMR and
TOF-Mass spectroscopic data. The structure of tsukubach-
elin consists of six amino acid residues, including three ser-
ine, and one each of N-α-methyl-N-δ-hydroxy-N-δ-formyl-
ornithine, N-α-methyl-N-δ-hydroxyornithine, and cyclic N-
hydroxyornithine. Because the structurally related sidero-
phore, desferri-foroximithine, was reported to have potent
angiotensin-converting enzyme inhibition activity, the in-
hibitory activity of desferri-tsukubachelin and desferri-forox-
imithine were tested for structure–activity comparison.
Desferri-tsukubachelin showed 14 times more potent inhibi-
tory activity than desferri-foroximithine. This result indicates
that desferri-tsukubachelin may become a promising agent
for angiotensin-converting enzyme inhibition.
Introduction
Siderophore is defined as a low molecular weight com-
pound that is secreted by microorganisms to uptake ferric
ions efficiently under low-iron stress. Ferric iron has very
low solubility at neutral pH and, therefore, cannot be uti-
lized by microorganisms in this form. Siderophore chelates
the ferric ion with high affinity and establishes a soluble
complex that can be taken up by a specific membrane trans-
porter.[1] It has been reported that actinomycetes produce a
wide variety of siderophores.[2] Desferroxiamine was iso-
lated from Streptomyces pilous and has been used as an ef-
fective medical agent to cure hemochromatosis.[3] It has
been found that streptomycetes produce a group of struc-
turally related siderophores, including coelichelin,[4] and
foroxymithine (1; Figure 1).[5] Among them, it is of interest
that foroxymithine was originally isolated as an angioten-
sin-converting enzyme (ACE) inhibitor in 1985.
Figure 1. Chemical structures of foroxymithine (1) and tsukubach-
elin (2).
[a] Food Biotechnology Division, National Food Research
Institute, NARO,
2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
[b] Analytical Science Division, National Food Research Institute,
NARO,
2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
[c] Food Biotechnology Division, National Food Research
Institute, NARO,
ACE, which catalyses the conversion of angiotensin I
into angiotensin II by cleaving the C-terminal histidyl–leu-
cine dipeptide, is a key molecule in the rennin–angiotensin
system.[6] Angiotensin II works as a potent vasoconstrictor
throughout the human body,[7] and it also has other impor-
tant biological functions, such as a constrictor of glomeru-
lar arterioles in the kidney.[8] Therefore, ACE has been an
attractive target for screening trials to find suitable inhibi-
tors.
2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
[‡] Current address: Graduate School of Science and Technology,
Shizuoka University,
836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
Fax: +81-54-238-5008
E-mail: askodan@ipc.shizuoka.ac.jp
Several promising ACE inhibitors, including ancov-
enin,[9] muraceins,[10] L-681,[11] L-176,[11] I5B2,[12] and
phenacein[13] have been isolated from actinomycetes to date.
[‡‡]Current address: Faculty of Applied Information Science,
Hiroshima Institute of Technology,
2-1-1 Miyake, Saeki-ku, Hiroshima, 731-5193, Japan
Eur. J. Org. Chem. 2011, 3191–3196
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3191

S. Kodani et al.
FULL PAPER
The potential of actinomycetes as a biological source ferri-tsukubachelin (Figure 3). Because the presence of fer-
prompted us to search for a new siderophore and to explore ric ion hampers NMR spectroscopic analysis, the conver-
the possibility that the siderophore may be a candidate for sion of the ferric siderophore into the corresponding gal-
an enzyme inhibitor. Here, we describe the exploration and lium ion complex via desferri-tsukubachelin was ac-
structure determination of a new siderophore, tsukubach- complished by a previously described method.[17] As a re-
elin (2; Figure 1), and detail its inhibitory activity against sult, 1.5 mg of gallium-chelating tsukubachelin was ob-
ACE.
tained after HPLC purification.
Results and Discussion
The new bacterial strain TM-34 was isolated from soil of
the Tsukuba Mountain in Japan using ISP2 agar me-
dium[14] along with 50 other bacterial strains. With the aim
of screening for siderophore, these strains were cultured in
iron-deficient liquid media for four days. After removal of
the cells, each culture medium was added to a FeCl3 solu-
tion to generate the ferric complex of siderophore. Each
solution was screened for siderophore by HPLC analysis
with UV/Vis detection at 435 nm. As a result, strain TM-
34 was found to produce a significant amount of sidero-
phore. To identify the genetic position of strain TM-34, se-
quencing analysis on the 16S rRNA coding gene was per-
formed. Based on the standard PCR method with universal
primers for bacterial 16S rRNA gene,[15] the complete
length of 16S rRNA gene was amplified. The sequence
analyses were accomplished using an automated DNA se-
quencer with eight universal primers. The obtained se-
quence was used to construct a phylogenetic tree with the
multiple-alignment program ClustalX.[16] As shown in Fig-
ure 2, the genetic position of TM-34 is located in the genus
of Streptomyces, and is closely related to Streptomyces pru-
nicolor with a high similarity of 99%.
Figure 3. HPLC separation of ferri-tsukubachelin. (A) Crude ferri-
tsukubachelin; (B) purified ferri-tsukubachelin.
To obtain information on the chemical structure of tsu-
kubachelin (2; Figure 1), NMR spectroscopic analyses, in-
cluding 2D NMR measurements such as DQF-COSY,
TOCSY, ROESY, HMBC, and HSQC were performed on
the gallium complex of tsukubachelin dissolved in 0.5 mL
of [D6]DMSO. The 1H NMR spectroscopic data revealed a
peptidic nature, with peaks of several amide residues over
the region δ = 8–9 ppm and α-protons over the range δ =
4–5 ppm (Figure 4). By interpretation of the DQF-COSY
and TOCSY proton spin systems, the connectivity of each
Figure 2. Phylogenetic position of strain TM-34.
To purify the new siderophore, large-scale cultivation of amino acid was constructed as shown by the bold line in
the TM-34 strain was performed with 1 L of iron-deficient Figure 5. The assignments of the C–H spin system were
media. To avoid contamination of ferric ion, flasks and fun- performed on the basis of the HSQC data (Table 1), and
nels made of polystyrene were used to culture and harvest revealed that the molecular structure of tsukubachelin con-
the cells. After cultivation for six days, the bacterial cells sisted of six amino acid residues, including three Serine and
were removed from the culture media by filtration. A solu- one each of N-α-methyl-N-δ-hydroxy-N-δ-formylornithine
tion of FeCl3 (1 m, 0.1 mL) was added to the culture media (N-Me hfOrn), N-α-methyl-N-δ-hydroxyornithine (N-Me
to generate the complex of siderophore with ferric ion. The hOrn), and cyclic N-hydroxyornithine (chOrn). As shown
culture media was concentrated to a volume of 50 mL by in Figure 5, the HMBC correlations from the α-proton or
rotary evaporation. The concentrated solution was centri- amide proton to the carboxyl carbon (one head arrow) were
fuged at 3000 rpm for 10 min and filtered using a mem- used to establish the connections between N-Me hfOrn1/
brane filter to remove insoluble compounds. The solution Ser2, Ser4/Ser5, and Ser5/chOrn6. The presence of a formyl
was subjected to HPLC purification to yield 2.5 mg of the residue was confirmed by the HMBC correlation from the
3192
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3191–3196

Siderophore Inhibitors of Angiotensin-Converting Enzyme
δ-protons (δ = 3.38 and 3.47 ppm) to the carbonyl carbon Table 1. NMR chemical shift values of Ga-tsukubachelin in [D6]-
DMSO.
with a characteristic chemical shift value of δ = 152.7 ppm.
The moiety of chOrn6 was determined from the HMBC
correlations between the α- and γ-protons to the carbonyl
carbon. The connection between N-Me hOrn3 and Ser4
was elucidated by the NOESY correlation between the α-
proton of N-Me hOrn3 and the amide proton of Ser4.
Residue
Position 1H NMR [δ] (J in Hz) 13C NMR [δ]
N-Me hfOrn1 N-CH3
2.50 (m)
8.74 (br.), 8.91 (br.)
31.5
NH
CO
α
167.5
59.5
25.3
3.89 (m)
1.49 (m)
1.87 (m)
1.32 (m)
1.58 (m)
3.38 (m)
3.47 (m)
8.05(s)
β
γ
17.5
49.8
152.7
δ
formyl
Ser2
NH
CO
α
8.99 (d, 8.21 Hz)
160.4
49.7
60.3
4.80 (q, 7.39 Hz)
3.52 (m)
β
3.69 (dd, 10.59,
7.51 Hz)
5.24 (br.)
OH
N-Me hOrn3 N-CH3
2.48 (m)
8.87 (br.), 9.15 (br.)
31.2
NH
CO
α
167.1
60.2
27.3
3.90 (m)
1.72 (m)
1.78 (m)
1.55 (m)
1.66 (m)
3.24 (ddd, 14.03, 8.78, 50.4
5.44 Hz)
β
1
Figure 4. H NMR spectrum of Ga-tsukubachelin in [D6]DMSO.
γ
δ
24.3
4.19 (td, 14.03,6.98 Hz)
Ser4
Ser5
NH
CO
α
β
OH
8.75 (d, 7.37 Hz)
169.2
4.62 (m)
3.52 (m)
5.11 (br.)
55.6
62.0
NH
CO
α
8.07 (d, 9.24 Hz)
169.8
55.3
62.0
4.30 (m)
3.55 (m)
β
3.75 (dd,
10.71,4.65 Hz)
4.91 (br.)
Figure 5. Selected correlations in 2D NMR spectra of tsukubach-
elin.
OH
chOrn6
NH
CO
α
β
γ
7.90 (d, 8.76 Hz)
The hydroxyl residues in N-Me hfOrn1, N-Me hOrn3,
and chOrn6 were not detected by H NMR analysis, how-
1
159.2
46.0
26.7
18.5
4.61 (m)
1.80 (m)
1.80 (m)
2.05 (m)
3.49 (m)
3.58 (m)
ever the result of an ESI-MS/MS experiment supported the
indicated positions of the hydroxyl residues (Figure 6). The
major fragmentation ion peaks at m/z 536.25, 449.23, and
305.14 were observed, which corresponded to the fragmen-
tations of N-Me hfOrn (172 Da), Ser (87 Da), and N-Me
hOrn (144 Da). The molecular formula of tsukubachelin
was determined as C27H49N9O13 by HRMS (ESI) analysis
δ
49.9
of desferri-tsukubachelin, with [M + H]+ at m/z 708.3526 with l-FDVA or d-FDVA. To obtain Orn and N-Me Orn,
corresponding to the molecular formula of C27H50N9O13 hydrogen iodide (HI) was used for complete hydrolysis. As
(protonated MW, m/z calcd. 708.3522). By combining the a result, 1 mol each of d-Orn and l-Ser, 2 mol of d-Ser,
above data, the structure of tsukubachelin 2 was deter- and 0.5 mol of N-Me-Orn were detected. Considering that
mined to be as depicted in Figure 1.
chOrn was converted into Orn by hydrolysis, the stereo-
To elucidate the absolute stereochemistries of the amino chemistry of chOrn was reasoned to be in the d-form. Re-
acids, the hydrolysate of desferri-tsukubachelin was derivat- garding l-Me-Orn, although the yield of l-FDVA derivati-
ized with N-α-(5-fluoro-2,4-dinitrophenyl)-l-valinamide (l- zation was low, only the l-form was detected, which deter-
FDVA),[18] and the derivative was subjected to HPLC mined the stereochemistries of the 2 mol of N-Me-Orn to
analysis to compare with standard amino acid derivatives be in the l-form. Although 2 mol of l-Ser and 1 mol of d-
Eur. J. Org. Chem. 2011, 3191–3196
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3193

S. Kodani et al.
FULL PAPER
found to exert potent ACE inhibitory activity. The mecha-
nism of inhibition was thought to arise through deprivation
of zinc ions from ACE, however, further study is needed to
clarify the inhibitory mechanism.
So far, a series of similar siderophores: foroximithine, co-
elichelin, and tsukubachelin, have been isolated from strep-
tomycetes. Considering the variety of secondary metabolite
biosynthetic genes in streptomycetes, this class of sidero-
phore may be broadly distributed in streptomycetes. Fur-
ther extensive screening for siderophores may lead to the
discovery of more potent ACE inhibitors.
Experimental Section
Figure 6. ESI MS/MS spectrum of desferri-tsukabachelin.
NMR and Mass Spectroscopic Measurements: The gallium complex
of tsukubachelin (1 mg) was dissolved in [D6]DMSO (0.6 mL), and
NMR spectra including 1H NMR at 800.23 MHz, 13C NMR at
Ser were determined to exist in the molecule, the specific
positions of l- and d-Ser in the molecule were not eluci-
dated.
1
201.24 MHz, H-1H correlation 2D NMR (DQF-COSY, TOCSY,
1
ROESY, and NOESY), and H-13C correlation 2D NMR (HSQC
With respect to the inhibitory activity against ACE,[19]
desferri-tsukubachelin showed the most potent inhibitory
activity, with an IC50 value of 1.4 μg/mL. For structure–
activity comparison, the IC50 value of authentic desferri-
foroxymithine was also determined to be 20.1 μg/mL in the
same experiment. Because the inhibitory activity of
desferri-tsukubachelin was 14 times more potent, tsukuba-
chelin may be promising reagent as an ACE inhibitor. The
X-ray crystallographic analysis of human ACE revealed
that it has a structural similarity to zinc metalloprote-
ases,[20,21] so there was the possibility that the inhibition
was caused by deprival of zinc from ACE. Umezawa et al.
reported that the ACE inhibition activity of foroxymithine
was completely abolished upon addition of FeCl3 and par-
tially abolished with ZnCl2.[5] In this experiment, ferri-tsu-
kubachlin did not show inhibitory activity at a concentra-
tion of 100 μg/mL.
As shown in Figure 1, tsukubachelin (2) and foroximi-
thine (1) both have three hydroxyamate moieties, which
have the ability to chelate ferric iron in the molecule. The
N-terminus part (hfOrn1-Ser2-hOrn3), which contains two
hydroxyamate moieties, was very similar in both tsukubach-
elin and foroxymithine, however, the third hydroxyamate
group, in the cyclic Orn of tsukubachelin was slightly fur-
ther away from the other two hydroxamate moieties than in
foroxymithine. Because the three hydroxamate moieties are
critical for ferric ion chelation, the distances between these
groups are very important. Moreover, the diketopiperazine
structure in foroxymithine was thought to be less flexible
than a normal peptide bond. These structural features may
influence the affinity of these peptides towards zinc ions
and may affect the inhibitory activities.
and HMBC) were measured with an AVANCE 800 spectrometer
with a CryoProbe (Bruker Biospin, Karlsruhe, Germany) at 303 K.
An electrospray ionization (ESI) Fourier-transform ion cyclotron
resonance (FT-ICR) mass spectrometer (Apex II, Bruker Dalton-
ics, Billerica, MA, USA) was used for accurate mass analyses.
Bacterial Strain and Culture Media: Soil samples were collected
from the ground of Tsukuba Mountain, Ibaraki Prefecture, Japan.
The samples were suspended in sterile water (10 mL), and spread
over ISP2 agar medium. After 4–5 d of incubation at 30 °C, devel-
oped colonies were isolated and stored at –80 °C. The iron-deficient
medium consisted of K2SO4 (2 g), K2HPO4 (3 g), NaCl (1 g), and
NH4Cl (5 g) in deionized water (1 L). To remove ferric ions, the
solution was stirred with 50 g of chelex-100 Na form (Bio-Rad) for
16 h. The solution was filtered through filter paper (Whatman
No.1) and combined with the stock solutions: 100 μL of thiamine
(20 mg/mL), 100 μL of ZnSO4·7H2O (20 mg/mL), 20 μL of
CuSO4·7H2O (0.5 mg/mL), 20 μL of MnSO4·4H2O (3.5 mg/mL),
followed by autoclaving. The separately sterilized solutions (10 mL
each) of CaCl2·H2O (10 mg/mL), glucose (250 mg/mL), and 0.5%
yeast extract (Difco) were added to the medium.
Screening for Siderophore Using HPLC: Each bacterial strain was
cultured using a shaker (180 rpm) set at 30 °C for 4 d in a conical
flask containing 10 mL of the iron-deficient culture medium. Each
cultured medium was centrifuged at 3000 rpm, and 5 mL of the
supernatant was collected in a new conical flask. FeCl3 (1 m,
0.05 mL) was added to the supernatant to generate the ferric sider-
ophore. After centrifugation at 10000 rpm for 5 min, 50 μL of the
solution was subjected to HPLC analysis. The HPLC analysis was
performed using an analytical C18 column (Nacalai tesque, Cos-
mosil 5C18MS-II), eluted with solvent A (distilled water containing
0.05% TFA) and solvent B (MeCN containing 0.05%TFA) using
time program: 0 to 10 min with 100% solvent A, then 10 min to
60 min increasing solvent B with the ratio of 1%/min. The UV/Vis
detector was set at the wavelength of 435 nm.
Polymerase Chain Reaction (PCR) Amplification, Sequencing, and
Phylogenetic Analysis of 16S rRNA Genes: The extraction of total
DNA from the cells of TM-34 was performed according to the
previous paper.[19] The 16S rRNA-encoding sequence was ampli-
fied from the total DNA by PCR using two universal primer pairs:
9F (5Ј-GAGTTTGATCCTGGCTCAG-3Ј) and 1541R (5Ј-
CTCCTACGGGTGAGTAACAC-3Ј). The reaction mixture for
Conclusions
ACE is a very promising target for screening agents that
have the potential to be used against kidney disease caused
by high blood pressure or diabetes. In the present study, a
new siderophore named tsukubachelin was discovered and PCR was prepared by adding 0.5 μL of total DNA of TM-34 (100
3194 Eur. J. Org. Chem. 2011, 3191–3196
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Siderophore Inhibitors of Angiotensin-Converting Enzyme
ng), 0.5 μL of LA taq DNA polymerase (Takara, Japan), 25 μL of
X2 GC buffer (Takara, Japan), 4 μL of 2.5 mm dNTPmix solution
(Takara, Japan), and 20 μL of distilled water into the PCR reaction
tube. PCR amplification was carried out with a thermal cycler
using the following program: initial denaturation for 2 min at
95 °C, followed by 30 cycles consisting of denaturation for 10 s at
95 °C, annealing for 10 s at 55 °C, and DNA synthesis for 3 min at
72 °C. A final extension of 3 min at 72 °C was added at the end of
the 30 cycles. The PCR product was purified with QIAGEN PCR
PURE kit following the manufacturer’s instruction. The reactions
for sequencing were performed in a solution containing 2 μL of
purified PCR product, 1 μL of ABI Prism BigDye (Applied Biosys-
tems), 7.4 μL of sterile distilled water, and 1.6 μL of each primer
with the following PCR protocol: 96 °C for 3 min and 25 cycles of
95 °C for 40 s, 55 °C for 40 s, and 60° for 4 min. The eight primers
used for the reaction: 9F (5Ј-GAGTTTGATCCTGGCTCAG-3Ј),
min using solvent A (distilled water containing 0.05% TFA) and
solvent B (MeCN containing 0.05%TFA) with a linear gradient
mode from 0 min to 60 min increasing solvent B at 1%/min. The
UV/Vis detector was set at the wavelength of 340 nm. Retention
times for l- and d-FDVA derivatized amino acids were as follows:
l-Ser-l-FDVA (35.3 min), d-Ser-d-FDVA (36.2 min), l-Orn-l-
FDVA (30.2 min), l-Orn-d-FDVA (31.8 min), N-Me Orn-l-FDVA
(32.3 min), N-Me Orn-d-FDVA (31.30 min).
Inhibition Test with Angiotensin-Converting Enzyme (ACE): ACE
inhibitory activity was determined by the modified method of
Cushman and Cheung.[22] The enzyme ACE was dissolved in dis-
tilled water at 8 mU/mL. The substrate hippuryl-l-histydyl-l-leu-
cine was dissolved in water at a concentration of 5 mm. The test
solution (30 μL) was added to 70 μL of the substrate solution, fol-
lowed by the addition of 100 μL enzyme solution. The mixture was
incubated at 37 °C for 30 min, then the enzyme reaction was ter-
minated by adding 300 μL of 0.5 n HCl. The hippuric acid was
extracted with 1.5 mL of ethyl acetate. The ethyl acetate layer
(0.5 mL) was evaporated by heating at 100 °C for 30 min. The hip-
puric acid was redissolved in 3 mL of 1 m NaCl and the UV ab-
sorbance at 228 nm was measured to calculate the inhibitory con-
centration.
339F
(5Ј-CTCCTACGGGTGAGTAACAC-3Ј),
1099F
686F
(5Ј-GCAAC-
(5Ј-
TAGCGGTGAAATGCGTAGA-3Ј),
GAGCGCAACCC-3Ј), 1541R (5Ј-AAGGAGGTGATCCAGCC-
3Ј), 1510R (5Ј-GGCTACCTTGTTACGA-3Ј), 926R (5Ј-
CCGTCAATTCCTTTGAGTTT-3Ј),
TACCGCGGCTGCTG-3Ј).
536R
(5Ј-GTAT-
Isolation of Tsukubachelin: Streptomyces sp. TM-34 was cultured
in 1 L of iron-deficient media for 5 d. The culture medium was
harvested by filtering through filter paper (No. 1 paper filter, What-
mann). The medium was added with 0.5 mL of 1 m FeCl3 and evap-
orated using a rotary evaporator to concentrate to 20 mL final vol-
ume. The concentrated solution was centrifuged at 3000 rpm for
10 min and filtered through a membrane filter (Millipore, 0.45 μm
pore size) to remove insoluble materials. HPLC purification was
performed to obtain 2.5 mg of ferri-tsukubachelin using C18 Semi
Prep column (10ϫ250 mm, Capcell Pak C18 UG80, Shiseido),
eluted with MeCN/water (2:98) containing 0.05% TFA and moni-
tored at a UV/Vis absorbance 435 nm.
Acknowledgments
This study was supported by research funds of the NOVARTIS
Foundation (Japan) and the Noda Institute for Scientific Research.
It was also supported in part by the Japan Society for the Pro-
motion of Science by Grants-in-aids (grant number 20880014).
[1] V. Braun, M. Braun, Curr. Opin. Microbiol. 2002, 5, 194–201.
[2] a) P. H. Oliveira, A. Batagov, J. Ward, F. Baganz, P. Krabben,
Lett. Appl. Microbiol. 2006, 42, 375–380; b) K. Schneider, I.
Rose, S. Vikineswary, A. L. Jones, M. Goodfellow, G. Nichol-
son, W. Beil, R. D. Sussmuth, H. P. Fiedler, J. Nat. Prod. 2007,
70, 932–935; c) B. Sontag, M. Gerlitz, T. Paululat, H. F. Rasser,
I. Grun-Wollny, F. G. Hansske, J. Antibiot. 2006, 59, 659–663.
[3] J. B. Porter, Br. J. Haematol. 2001, 115, 239–252.
[4] a) G. L. Challis, J. Ravel, FEMS Microbiol. Lett. 2000, 187,
111–114; b) S. Lautru, R. J. Deeth, L. M. Bailey, G. L. Challis,
Nat. Chem. Biol. 2005, 1, 265–269.
[5] H. Umezawa, T. Aoyagi, K. Ogawa, T. Obata, H. Iinuma, H.
Naganawa, M. Hamada, T. Takeuchi, J. Antibiot. 1985, 38,
1813–1815.
[6] T. Niu, X. Chen, X. Xu, Drugs 2002, 62, 977–993.
[7] S. A. Atlas, J. Manag. Care Pharm. 2007, 13, 9–20.
[8] J. E. Hall, Comprehensive Ther. 1991, 17, 8–17.
[9] N. Naruse, O. Tenmyo, K. Tomita, M. Konishi, T. Miyaki, H.
Kawaguchi, K. Fukase, T. Wakamiya, T. Shiba, J. Antibiot.
1989, 42, 837–845.
Conversion of Ferri-Tsukubachelin into Ga-tsukubachelin via
Desferri-Tsukubachelin: Ferri-tsukubachelin (2.0 mg) was dissolved
in water (3 mL) and the solution was mixed with 8-quinolinol (1 m,
3 mL) and stirred at room temperature for 30 min. The reaction
mixture was extracted with CH2Cl2 (4ϫ 6 mL) to remove ferri-8-
quinolinol and the aqueous layer was immediately collected and
lyophilized by freeze-drying. After dissolving the dry material in
water (2 mL), HPLC purification was performed using a C18 Semi-
Prep column (10ϫ250 mm; Capcell Pak C18 UG80, Shiseido),
eluted with MeCN/water (3:97) containing 0.05% TFA, and moni-
tored at a UV/Vis absorbance of 215 nm, to yield desferri-tsukuba-
chelin (1.9 mg). Desferri-tsukubachelin (1.5 mg) was dissolved in
distilled water (2 mL), and gallium chloride (10 mg) was added to
generate Ga-tsukubachlin. After HPLC purification in the same
manner as described above, Ga-tsukubachelin (1.1 mg) was ob-
tained.
[10] P. D. Singh, J. H. Johnson, J. Antibiot. 1984, 37, 336–343.
[11] O. D. Hensens, J. M. Liesch, J. Antibiot. 1984, 37, 466–468.
[12] Y. Kido, T. Hamakado, M. Anno, E. Miyagawa, Y. Motoki, T.
Wakamiya, T. Shiba, J. Antibiot. 1984, 37, 965–969.
[13] W. C. Liu, W. L. Parker, S. S. Brandt, K. S. Atwal, E. P. Ruby,
J. Antibiot. 1984, 37, 1313–1319.
Modified Marfey Method: Desferri-tsukubachelin (0.2 mg) was
subjected to acid hydrolysis at 110 °C for 16 h with HI (6 n,
0.5 mL), and then the hydrolysates were dried under a stream of
N2 gas and resuspended in H2O (200 μL). To the hydrolysate, 10 μL
of a solution of either N-α-(5-fluoro-2,4-dinitrophenyl)-l-valinam-
ide (l-FDVA, Sigma–Aldrich) or d-FDVA (Sigma–Aldrich) in ace-
tone was added at a concentration of 10 mg/mL, followed by
NaHCO3 (1 m, 100 μL). The mixture was heated at 80 °C for 3 min,
then cooled, neutralized with HCl (2 n, 50 μL), and diluted with
MeCN (200 μL). About 10 μL of each solution of FDVA derivative
was subjected to HPLC analysis with a C18 column (4.6ϫ50 mm;
Cosmosil MSII); the elution was performed at a flow rate of 1 mL/
[14] E. B. Shirling, D. Gottlieb, Int. J. Syst. Bacteriol. 1966, 16,
313–340.
[15] G. C. Baker, J. J. Smith, D. A. Cowan, J. Microbial. Methods
2003, 55, 541–555.
[16] M. A. Larkin, G. Blackshields, N. P. Brown, R. Chenna, P. A.
McGettigan, H. McWilliam, F. Valentin, I. M. Wallace, A.
Wilm, R. Lopez, J. D. Thompson, T. J. Gibson, D. G. Higgins,
Bioinformatics 2007, 23, 2947–2948.
[17] G. J. Sharman, D. H. Williams, D. F. Ewing, C. Ratledge,
Chem. Biol. 1995, 2, 553–561.
Eur. J. Org. Chem. 2011, 3191–3196
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3195

S. Kodani et al.
FULL PAPER
[18] R. Bhushan, H. Bruckner, Amino Acids 2004, 27, 201–210.
[19] D. W. Cushman, H. S. Cheung, Biochem. Pharmacol. 1971, 20,
1637–1648.
[20] R. Natesh, S. L. Schwager, E. D. Sturrock, K. R. Acharya, Na-
ture 2003, 421, 551–554.
[21] A. Camara-Artigas, V. Jara-Perez, M. Andujar-Sanchez, An-
giotensin Converting Enzyme (ACE) Inhibitors, in: Drug Design
of Zinc-Enzyme Inhibitors: Functional, Structural, and Disease
Applications (Eds.: C. T. Supuran, J. Y. Winum), Wiley, Ho-
boken, 2009, pp. 751–788.
[22] S. Kodani, A. Imoto, A. Mitsutani, M. Murakami, J. Appl.
Phycol. 2002, 14, 109–114.
Received: February 10, 2011
Published Online: May 2, 2011
3196
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3191–3196