Synthesis and iron coordination properties of schizokinen and its imide derivative

Article abstract of DOI:10.1039/c9dt02731a

The iron(iii) affinity constants for schizokinen and its imide derivative are reported for the first time. Surprisingly, schizokinen possesses a higher affinity for iron(iii) than desferrioxamine B; log?KFeIII (FeL), 36.2 and 30.6, respectively. This incr

Full text of DOI:10.1039/c9dt02731a

Dalton
Transactions
View Article Online
View Journal | View Issue
PAPER
Synthesis and iron coordination properties of
schizokinen and its imide derivative†
Cite this: Dalton Trans., 2019, 48,
17395
Hataichanok Chuljerm,a,b Yu-Lin Chen,a Somdet Srichairatanakool,b
a
Robert C. Hider a and Agostino Cilibrizzi
*
The iron(III) affinity constants for schizokinen and its imide derivative are reported for the first time.
Surprisingly, schizokinen possesses a higher affinity for iron(III) than desferrioxamine B; log KFeIII (FeL), 36.2
and 30.6, respectively. This increase in value is associated with the substitution of one hydroxamate func-
tion by an α-hydroxycarboxylate grouping. By virtue of the similarity of siderophore–iron(III) complexes
and siderophore–gallium(III) complexes, schizokinen (which is a Gram positive siderophore) has potential
for 68Ga PET-based imaging.
Received 1st July 2019,
Accepted 13th November 2019
DOI: 10.1039/c9dt02731a
rsc.li/dalton
Siderophores bind gallium(III) in a similar fashion to that of
iron(III)15–17 and 68Ga-siderophore complexes have been investi-
Introduction
Siderophores, are low-molecular-weight compounds (500–1500 gated for their potential to detect infections in a range of clini-
Da) which possess a high affinity for iron(III) (Kf > 1030). They cal circumstances.18–20 However, most of the siderophores so
are secreted by bacteria, fungi and graminaceous plants for far investigated for this property are associated with Gram-
scavenging iron from the environment.1 The majority bind negative organisms. Schizokinen (1) is produced by Bacillus
iron(III) in a hexadentate fashion and the most common megaterium, a Gram-positive organism, and thus has potential
ligands are hydroxamate and catechol. However there is an for the imaging of Gram-positive organisms in the clinical situ-
increasing number of siderophores that have been identified ation. A knowledge of the affinity constant of schizokinen for
that use a mixture of hydroxamate and α-hydroxycarboxylate iron(III) will provide strong indications of the affinity constant
ligands in order to bind iron(III) (Fig. 1), for instance, schizoki- for gallium(III).1
nen (1),2 aerobactin (2),3 acinetoferrin (3),4 nannochelin A (4),5
arthrobactin (5),6 rhizobactin (6),7 ochrobactin A (7)8 and snye-
chobactin A (8).9 In addition to these 8 siderophores, there are
Results and discussion
Synthesis of the schizokinen (1) and its cyclic imide
many siderophore families that also utilise the same ligand
composition, for instance, the ornibactins,10 the aquache-
derivative (9)
lins,11 the loihichelins12 and the sodachelins.13 The advan-
The synthesis of schizokinen (1)21–23 (as well as the closely
tages of forming an iron(III) complex with mixed ligands is
related acinetoferrin (3))23,24 has been previously reported. In
unclear, indeed there have been no affinity constant measure-
this work, we have adopted the procedure reported by Fadeev
ments made on such compounds, in contrast to the tri-hydro-
et al. (Scheme 1) with several modifications.23 The full syn-
thetic design is based on a di-orthogonal strategy using tert-
butyl and benzoyloxy protections on the carboxylic and amino
groups, respectively. This approach allowed us to determine
the most appropriate de-protection order to obtain the two
chelators 1 and 9. The starting materials 13 and 14 were syn-
thesised through methods reported by Guo et al. (for 13)22 and
Wang and Phanstiel (for 14).24
A key feature of the synthesis adopted in this investigation
is the coupling of 3-tert-butyl citrate 13 with 1-N-benzoyloxy-
1,3-diaminopropane 14 (Scheme 1) under microwave
irradiation (40 °C for 30 min), in order to limit the generation
of by-products due to degradation and side reactions.
xyamide and tri-catecholate siderophores.1 For this reason it
was decided to determine the affinity constant of both schizo-
kinen (1) and its imide derivative (9) which lacks the
α-hydroxycarboxylate function. Both compounds are produced
by Bacillus megaterium.14
aInstitute of Pharmaceutical Science, King’s College London, London, SE1 9NH, UK.
E-mail: agostino.cilibrizzi@kcl.ac.uk
bDepartment of Biochemistry, Faculty of Medicine, Chiang Mai University,
Chiang Mai, 50200, Thailand
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9dt02731a
This journal is © The Royal Society of Chemistry 2019
Dalton Trans., 2019, 48, 17395–17401 | 17395

View Article Online
Paper
Dalton Transactions
Fig. 1 Chemical structures of schizokinen (1), α-hydroxycarboxylate siderophore analogues (2–8), the imide derivative of schizokinen (9), desfer-
rioxamine B (10), acetylschizokinen (11) and rhodotrulic acid (12). The chirality of the central citrate carbon atom has not been established for sidero-
phores 2, 6–8 and 9.
Scheme 1 Di-orthogonal approach for the synthesis of schizokinen (1) and its cyclic imide derivative (9). Reagents and conditions: (a) HATU, DIPEA,
anhydrous DMF, microwave, 150 watts, 30 min, 40 °C; (b) acetyl chloride, anhydrous CH2Cl2, 3 h, reflux; (c) TFA/water 95 : 5, 2 h, rt; (d) 7 N NaOH,
30 min, rt.
Intermediate 15 was obtained in 20% yield. In our hands, the diacetyl derivative 16a were detected. Column chromato-
when reacted with acetyl chloride, the major product resulting graphy was performed to isolate pure 16b in good yield (92%).
from the acetylation reaction was the tri-acetyl compound 16b. tert-Butyl removal was carried out under standard conditions
The diacetyl compound 16a (with the acetyl groups located on (TFA/water, 95 : 5). Intermediate 17 was subsequently reacted
the nitrogen of the hydroxamic functions) was previously with 7 N NaOH to unmask the hydroxamic acid functions and
reported to be the main product.23 Under the conditions simultaneously hydrolyse the acetyl ester on the citrate moiety,
adopted in this study, only traces (by both TLC and LC-MS) of yielding a mixture of schizokinen (1) and its cyclic imide
17396 | Dalton Trans., 2019, 48, 17395–17401
This journal is © The Royal Society of Chemistry 2019

View Article Online
Dalton Transactions
Paper
derivative (9) in racemic form (as indicated by NMR peaks, see ditions, schizokinen imide (9) was slowly converted to the
spectral data in section 6 of ESI†), which generates due to parent schizokinen, over this same time period (Fig. 3b).
spontaneous dehydration reaction of 1.25 After removal of the When the samples were stored at −20 °C, 1 demonstrated
hydroxamate protection groups, all glassware used to handle good levels of stability (Fig. 3c and d), but 9 was slowly con-
the free chelators was pre-treated with concentrated HCl, to verted to 1 (Fig. 3d). Both the compounds were found to be
prevent possible metal contamination,26 and reversed-phase sufficiently stable for titration studies. We have evaluated the
HPLC was performed to purify the final compounds. The for- half-life (t1/2) for the hydrolysis of schizokinen imide (9) at pH
mulation and purity of siderophores 1 and racemate 9 was con- 7.4 and room temperature as 4.4 days (see section 8 in ESI†).
firmed by NMR and mass spectrometry (see Experimental
section and sections 5 and 6 in ESI† for spectral data).
Spectrophotometric titration of schizokinen (1) in the pres-
ence of iron yielded an isosbestic point and, after curve fitting,
a log KFeIII value of 36.2 (FeL) was determined (Table 1).
Titration of schizokinen imide (9) in the presence of iron
yielded a log affinity constant of 73.5 (Fe2L3). These two con-
stants corresponded to the pFeIII values ([Fe]total = 1 µM; [L]total
= 10 µM; pH = 7.4) of 26.8 for schizokinen and 27.8 for schizo-
kinen imide (Table 1). These values compare favourably with
corresponding pFeIII value for desferrioxamine (10), namely 25.1
Schizokinen (1) binds iron(III) at pH 7.4 almost two orders
of magnitude higher than desferrioxamine B (10) (Table 1).
This difference is clearly associated with the substitution of
Determination of the iron(III) affinity constants for
schizokinen (1) and its cyclic imide derivative (9)
The pKa values for the hydroxamate and carboxylic functions
were determined by spectrophotometric titration; the intrinsic
value being reported for the two hydroxamate functions
(Table 1). The pKa value of the hydroxyl function was an esti-
mated value, based on a previous study of citrate, malate and
lactate by 13C NMR.27 No pKa value was detected between pH
10 and pH 12.5. The composition (iron : ligand) of the two iron
(III) complexes was determined by Job’s plots at pH = 10.0,
being 1 : 1 for schizokinen (1) and 2 : 3 for the schizokinen
cyclic imide derivative (9) (Fig. 2).
one hydroxamate function in desferrioxamine
B by an
α-hydroxycarboxylate group in schizokinen. The speciation
plots of the two siderophores in the presence of iron(III) are
presented in Fig. 4. There is a marked difference in their
appearance over the pH range 2–5. Whereas the hydroxide
anion is able to compete with desferrioxamine B for iron(III)
under acid conditions, this is not the situation with schizoki-
nen, which dominates the competition with hydroxide over the
acidic pH range.
Schizokinen (1) was found to be stable at room temperature
(pH = 7.4) for a period of a week (Fig. 3a). Under the same con-
Table 1 Comparison of pKa, log KFeIII and pFeIII values between schizo-
kinen, schizokinen imide and desferrioxamine B
The imide derivative of schizokinen (9) behaves like acet-
ylschizokinen (11) in its mode of iron(III) coordination.29 The
acetylation of the hydroxy function removes the avid bidentate
α-hydroxycarboxylate function and, consequently, like 11,
schizokinen imide binds iron(III) as a 2 : 3 complex, as con-
firmed by the Job’s plot (Fig. 2). The pFe3+ value for 9
(i.e. 27.8) is much higher than that of the dihydroxamate 12,
namely 21.6.30
The limiting factor for schizokinen imide is its suscepti-
bility toward hydrolysis in aqueous solution at ambient
temperatures. In contrast, schizokinen, is stable in aqueous
solution at pH 7.0 and possesses the relatively low molecular
weight of 420, it is predicted to be orally active and therefore
possesses the potential to be an orally active iron chelator
suitable for the treatment of systemic iron overload.31
Furthermore, its ability to bind iron(III) more tightly than
desferrioxamine B suggests that it will also bind gallium(III)
with high affinity, in similar fashion to desferrioxamine.32
Indeed, the gallium–schizokinen complex has been studied
by 1H NMR17 and confirmed to be an octahedral complex
involving the 6 oxygen atoms associated with the two hydro-
xamate groups and the α-hydroxycarboxylate function. As
there is a close linear relationship between the affinity con-
stants of Ga(III) and Fe(III) for a wide range of ligands,15
the pGa value for schizokinen is predicted to be close to 27,
rendering schizokinen with potential for 68Ga PET-based
imaging.
Schizokinen Schizokinen Desferrioxamine B
(1)
imide (9)
(10)c
pKa (hydroxyl)a
pKa (hydroxamate)b
pKa (carboxylate)
log KFeIII (FeL)
log KFeIII (Fe2L3)
pFeIII d
14.5
9.0
4.1
—
8.9
—
—
9.0
—
36.2
—
26.8
—
73.5
27.8
30.6
—
25
a Estimated value. b Intrinsic value. c These measurements were made
in the present study. d [Fe]total = 1 µM, [L]total = 10 µM, pH = 7.4.
Fig. 2 Job’s plots for the determination of the stoichiometry of schizo-
kinen (1) (red line) and cyclic imide derivative (9) (i.e. dehydrated schizo-
kinen; purple dashed line) with iron(III).
This journal is © The Royal Society of Chemistry 2019
Dalton Trans., 2019, 48, 17395–17401 | 17397

View Article Online
Paper
Dalton Transactions
Fig. 3 Stability analysis of schizokinen (blue) and dehydrated schizokinen (i.e. cyclic imide) (orange) at pH = 7.4: schizokinen (a) and schizokinen
imide (b) samples at room temperature for 7 days; schizokinen (c) and schizokinen imide (d) samples stored at −20 °C and thawed daily for 7 days
(for UV spectra, see section 7 in ESI†).
Fig. 4 Speciation plots. (A) Desferrioxamine B (10 μM), iron(III) (1 μM) and (B) schizokinen (10 μM), iron(III) (1 μM). Iron(III) hydrolysis constants were
taken from Baes and Mesmer.28
approach was developed where benzoyloxy and tert-butyl pro-
Conclusion
tecting groups were integrated into a di-orthogonal protecting
A multi-step synthesis involving microwave and batch reactions strategy, which proceeded smoothly, leading to good yields of
has been refined and implemented to prepare the siderophore all the intermediates. The overall mild conditions adopted
schizokinen (1) and its cyclic imide derivative (9). A synthetic suggest that this procedure could be useful in the general syn-
17398 | Dalton Trans., 2019, 48, 17395–17401
This journal is © The Royal Society of Chemistry 2019

View Article Online
Dalton Transactions
Paper
thesis of complex α-hydroxycarboxylate-based siderophores Axia column (100 mm × 30 mm, 5 micron) and H2O : CH3CN
and related analogues, associated with citric acid functionali- (gradient from 98 : 2 to 5 : 95, over 45 min) as the mobile
zation. The affinity constant data for iron(III) indicates that phase. The solvents were degassed and supplemented with
schizokinen (1) has considerable potential as a selective iron(III) 0.1% formic acid prior to use. The platform consisted of a
chelating agent, with potential as an orally active chelator31 Waters RP-HPLC system (Waters 2767 autosampler for
and as a suitable moiety for antibiotic therapy.21 Schizokinen sample injection and collection; Waters 2545 and 515 pumps
also has potential for positron–emission tomography (PET) to deliver the mobile phase to the source) coupled to a
imaging with 68Ga.
Waters Micromass ZQ mass spectrometer (with ESI in posi-
The introduction of the α-hydroxycarboxylic function tive and negative modes) and a Waters 2996 Photodiode Array
into the structure enhances the affinity for tribasic metal (with detection between 190–800 nm). 3-tert-Butyl-citrate
cations over and above that of tri-hydroxamate siderophores. (13)16 and 1-N-benzoyloxy-1,3-diaminopropane dihydrochlor-
Schizokinen imide (9), although possessing an extremely high ide (14)16,17 were prepared by literature procedures (for
pFe3+, is unsuitable for therapeutic application due to its ten- 1H NMR spectra, see section 1 in ESI†).
dency to hydrolyse in aqueous systems.
tert-Butyl 4-((3-((benzoyloxy)amino)propyl)amino)-2-(2-((3-
((benzoyloxy)amino)propyl)amino)-2-oxoethyl)-2-hydroxy-4-
oxobutanoate (15). 3-tert-Butyl-citrate 13 (0.5 g, 2.0 mmol,
1 equiv.) was dissolved in DMF (3.0 mL). HATU (1.9 g, 5.0 mmol,
2.5 equiv.) and N,N-diisopropylethylamine (1.75 mL, 10.0 mmol,
5 equiv.) were added to the solution, followed by 1-N-benzoyloxy-
Experimental details
General considerations
All reagents were purchased from chemical suppliers and 1,3-diaminopropane dihydrochloride 14 (0.98 g, 5.0 mmol,
used without further purification, unless otherwise men- 2.5 equiv.). The reaction was carried out under microwave
tioned. Room temperature refers to ambient temperature. irradiation (150 watts, 40 °C, 30 min). The solution mixture
Yields refer to chromatographically and spectroscopically was extracted with water three times. The organic layer was
pure compounds unless otherwise stated. Where possible, concentrated by rotary evaporation. Flash column chromato-
reactions were monitored by thin layer chromatography (TLC) graphy (eluent: ethyl acetate/MeOH 95 : 5) was used to obtain
performed on commercially available glass plates pre-coated 135 mg (20.0% yield) of pure 15. NMR and LC-MS results (for
with Merck silica gel 60 F254 or Merck silica gel 60 RP-18 spectra, see section 2 in ESI†) are in agreement with previously
F
254s. Visualisation was achieved by the quenching of UV reported characterisation data.17
fluorescence (λmax = 254/365 nm) and by staining with iodine, tert-Butyl 2-acetoxy-4-((3-(N-(benzoyloxy)acetamido)propyl)
ninhydrin, vanillin or potassium permanganate. All flash amino)-2-(2-((3-(N-(benzoyloxy)acetamido)propyl)amino)-
chromatography was carried out using slurry packed Merck 2-oxoethyl)-4-oxobutanoate (16b). Intermediate 15 (135 mg,
9325 Keiselgel 60 or Aldrich C18-reverse phase silica gel. 0.23 mmol, 1 equiv.) was dissolved in anhydrous dichloro-
Nomenclatures do not follow the IUPAC naming system. 1H, methane (3.0 mL). The solution was heated to reflux and acetyl
13C and DEPT NMR spectra were recorded using an internal chloride (68 μL, 0.945 mmol, 4.2 equiv.) in 1.0 mL of an-
deuterium lock at ambient probe temperatures on a Bruker hydrous CH2Cl2 was added. The reaction was refluxed for 3 h.
Ascend™-400 (400 MHz) instrument. Chemical shifts (δ) are After evaporating the solvent, the crude mixture was purified
quoted in ppm, to the nearest 0.01 ppm (for 1H NMRs), or by flash column chromatography (eluent: ethyl acetate/MeOH
0.1 ppm (for 13C NMR), and are referenced to the residual 95 : 5). Yield after purification was 90% (150 mg). 1H NMR
non-deuterated solvent peak. Coupling constants (J) are (400 MHz, CDCl3): δ 8.08 (d, 4H, Ar, J = 8.4 Hz), 7.68 (t, 2H, Ar,
reported in Hertz (Hz) to the nearest 0.5 Hz. Data are reported J = 7.6 Hz), 7.52 (t, 4H, Ar, J = 7.6 Hz), 3.80–3.93 (m, 4H,
as follows: chemical shift, multiplicity, integration, assign- CONHCH2), 3.25–3.35 (m, 4H, ONCH2), 2.94 (AB-quartet, 4H,
ment and coupling constant(s). Assignments were deter- COCH2, J = 53.2 Hz, J = 13.6 Hz), 2.07 (s, 6H, 2 × COCH3), 2.05
mined either on the basis of unambiguous chemical shift or (s, 3H, COCH3), 1.80–1.85 (m, 4H, CH2CH2CH2), 1.44 (s, 9H,
coupling pattern, or by analogy to fully interpreted spectra for 3 × OCCH3). 13C NMR (100 MHz, CDCl3) δ 134.8 (2CH), 130.2
related compounds. Analytical HPLC-MS experiments were (4CH), 129.1 (4CH), 41.3 (2CH2), 38.8 (CH3), 36.7 (2CH2), 27.9
performed on Agilent 1100 Series coupled to a Thermo LCQ (3CH3), 27.2 (2CH2), 22.1 (2CH2), 21.3 (CH3), 20.4 (CH3).
DecaXP (operating in ES+ or ES− mode). A Phenomenex Luna LC-MS (ESI) calcd for C36H46N4O12 726.78 (MW), found m/z
C
18-(2)-HST (50 × 2 mm, 2.5 μm) column was used and the 727.02 [M + H]+, 749.05 [M + Na]+, tR = 12.6.
mobile phase, in linear gradient, was composed of solvent A 2-Acetoxy-4-((3-(N-(benzoyloxy)acetamido)propyl)amino)-2-
(H2O, +0.1% formic acid +0.05 TFA) and solvent B (aceto- (2-((3-(N-(benzoyloxy)acetamido) propyl)amino)-2-oxoethyl)-
nitrile, +0.1% formic acid +0.05 TFA). The sample solutions 4-oxobutanoic acid (17). Intermediate 16b (150 mg,
were prepared at a concentration of 0.1–0.01 mg/1 mL. The 0.21 mmol) was suspended in 6 mL of TFA/water 95 : 5 (v/v)
injection volume was 10–50 µL, the flow rate was 0.3 mL and stirred for 2 h at room temperature. After the deprotection
min−1, the column temperature was 40 °C. The values of was complete, the reaction mixture was evaporated under
retention time (tR) are given in minutes. Preparative HPLC vacuum and purified by flash column chromatography (eluent:
purifications were performed using a Gemini 5μ C18 110A CHCl3/EtOH 80 : 20). The yield after purification was 96%
This journal is © The Royal Society of Chemistry 2019
Dalton Trans., 2019, 48, 17395–17401 | 17399

View Article Online
Paper
Dalton Transactions
1
(135 mg). H NMR (400 MHz, CDCl3 + CD2Cl2): δ 10.67 (exch room temperature storage conditions (i.e. samples at room
br s, 1H, OH), 8.08 (d, 4H, Ar, J = 7.6 Hz), 7.69 (t, 2H, Ar, J = temperature were monitored daily up to 7 days). Stability
7.6 Hz), 7.53 (t, 4H, Ar, J = 7.6 Hz), 3.89 (t, 4H, CONHCH2, J = under freeze/thaw conditions was also monitored for 7 days
6.0 Hz), 3.32–3.41 (m, 4H, ONCH2), 3.06 (AB-quartet, 4H, (samples were stored at −20 °C and thawed daily). The half-life
COCH2, J = 37.2 Hz, J = 14.0 Hz), 2.07 (s, 9H, 3 × CH3), 1.85 (t1/2) value of the hydrolysis was calculated by plotting the per-
(q, 4H, CH2CH2CH2, J = 6.0 Hz). LC-MS (ESI) calcd for centages of peak area against incubation time (section 8 in
C32H38N4O12 670.67 (MW), found m/z 671.08 [M + H]+, 693.04 ESI†).
[M + Na]+, tR = 11.32.
Determination of affinity constants
2-Hydroxy-4-((3-(N-hydroxyacetamido)propyl)amino)-2-(2-
((3-(N-hydroxyacetamido)propyl) amino)-2-oxoethyl)-4-oxo-
butanoic acid (1, shizokinen) and N-hydroxy-N-(2-(2-(3-
hydroxy-1-(2-(N-hydroxyacetamido)ethyl)-2,5-dioxopyrrolidin-3-yl)
acetamido)ethyl)acetamide (9, cyclic imide derivative).
Intermediate 17 (135 mg, 0.20 mmol) was dissolved in 7 N
NaOH (5 mL) in a plastic Falcon™ tube in an ice-bath and
shaked for 30 minutes at room temperature. The pH of the
reaction was adjusted to 7 by the addition of 7 N HCl. The
course of the reaction was monitored by TLC and LC-MS ana-
lysis, which revealed the presence of schizokinen (1) as the
main species and the cyclic imide product (9) as a secondary
racemic product. After evaporation of the solvent by freeze-
drying, preparative HPLC was used to obtain the pure com-
pounds. Yields after purification were 65% and 19% for 1
(55 mg) and racemate 9 (15 mg), respectively. NMR and LC-MS
results (for spectra, see sections 5 and 6 in ESI†) are in agree-
ment with previously reported characterisation data.14,23,25,33
The automated titration used for this study consists of a
Metrohm 765 Dosimat autoburette,
a
Mettler Toledo
MP230 pH meter with SENTEK pH electrode (P11), and an HP
8453 UV-visible spectrophotometer with a Hellem quartz flow
cuvette, with circulation driven by a Gilson Mini-plus #3 pump
(speed capability 20 mL min−1). A potassium chloride electro-
lyte solution (0.1 M) was used to maintain the ionic strength.
The temperature of the test solutions was maintained in a
thermostatic jacketed titration vessel at 25 0.1 °C, using a
Fisherbrand Isotemp water bath. The pH electrodes were cali-
brated using the software GLEE34 with data obtained by titrat-
ing a volumetric standard HCl (0.1 M) in KCl (0.1 M) with KOH
(0.1 M) under an atmosphere of argon. Analytical grade
reagent materials were used in the preparation of all solutions.
The solution under investigation was stirred vigorously during
the experiment. For pKa determinations, a cuvette path length
of 10 mm was used, while for metal stability constants deter-
minations, a cuvette path length of 50 mm was used (experi-
mental concentration was ca. 40 μM for iron complexes). All
instruments were interfaced to a computer and controlled by
an in-house program. The automated titration adopted the fol-
lowing strategy: the pH of a solution was increased in incre-
ments of 0.1 pH unit by the addition of potassium hydroxide
solution (0.1 M) from the autoburette. The pH readings were
judged stable if they varied by less than 0.01 pH unit after a
pre-set incubation period. For pKa determinations, an incu-
bation period of 1.5 min was adopted; for metal stability con-
stant determinations, an incubation period of 3 min was
adopted. The cycle was repeated until the predefined end
point pH value was achieved. Titration data were analysed with
the HypSpec2014 program (http://www.hyperquad.co.uk/).35,36
The fitting spectra range for iron complexes was 400–700 nm.
The associated hydrolysis constants used in the analysis were
collected from Martell’s critical stability constants.37 Metal
affinities of compounds in this study were determined in com-
petition with the metal hydrolysis species in a solution at a
high pH (titrated up to pH 12.0). Satisfactory fitting of both
the siderophore titrations with iron was achieved. Speciation
plots were calculated with the HYSS program,38 adopting the
iron(III) log hydrolysis constants, FeOH (−2.563), Fe(OH)2
(−6.205), Fe(OH)3 (−15.1), and Fe(OH)4 (−21.883), Fe2(OH)2
(−2.843).28
Job’s plots
The concentrations of the two components (iron(III) and side-
rophore) were continuously varied, while the total con-
centration was kept constant. Siderophore (1.8 mM) in
ammonium acetate (pH = 10.0, 20 mM) was added to FeIII–
NTA (1 : 2 mmolar ratio, 1.8 mM) also in ammonium acetate
(pH = 10, 20 mM) in different molar ratios ranging from 1 : 9
to 9 : 1 (siderophore : iron(III)) (see Table S1 in ESI†). The solu-
tions were allowed to stand at 20 °C for 1 h. The absorption
spectra of the solutions were recorded and the absorbances at
368 nm plotted vs. the molar ratio of siderophore : iron(III)
(Fig. 2).
Stability studies in aqueous solution
The stability of 1 and 9 was assessed by HPLC-UV on the HP
1050 Series equipment (for spectra, see section 7 in ESI†). For
all the analyses, an Agilent Zorbax SB-C18 column (2.1 mm ×
100 mm, 3.5 μm) was used and the mobile phase was com-
posed of solvent A (99.9% water, 0.1% TFA) and solvent B
(99.9% acetonitrile, 0.1% TFA) used in a linear gradient
(time = 0 min, 100%A and 0%B; time = 20 min, 10%A and
90%B; time = 23 min, 10%A and 90%B; time = 25 min, 100%A
and 0%B; total run time 31 min) at the flow rate of 0.3 mL
min−1. The sample solutions were prepared by dissolving pure
compounds in 5 mM ammonium acetate buffer at pH = 6.0,
which was then adjusted to pH = 7.4. The injection volume
was 10 µL, the column temperature was 40 °C and the UV
detector wavelength was fixed at 210, 214, 230, 254 and
Conflicts of interest
281 nm. The stability of siderophores was investigated under There are no conflicts to declare.
17400 | Dalton Trans., 2019, 48, 17395–17401
This journal is © The Royal Society of Chemistry 2019

View Article Online
Dalton Transactions
Paper
17 D. J. Berry, Y. Ma, J. R. Ballinger, R. Tavaré, A. Koers,
K. Sunassee, T. Zhou, S. Nawaz, G. E. Mullen, R. C. Hider
and P. J. Blower, Chem. Commun., 2011, 47, 7068–7070.
Acknowledgements
The authors gratefully acknowledge Dr Xiaole Kong (Hangzhou
Ai Yongmei Co. Ltd, Zhejiang, China) for his help with the spe- 18 M. Petrik, C. Zhai, H. Haas and C. Decristoforo, Clin.
ciation plot analyses. This work was supported by the Newton Transl. Imaging, 2017, 5, 15–27.
Fund 2017/2018 from British Council and the Research and 19 J. A. Ioppolo, D. Caldwell, O. Beiraghi, L. Llano, M. Blacker,
Researcher for Industries (RRi) (PHD59I0029), Thailand
Science Research and Innovation (TSRI).
J. F. Valliant and P. J. Berti, Nucl. Med. Biol., 2017, 52, 32–41.
20 M. Petrik, H. Haas, M. Schrettl, A. Helbok, M. Blatzer and
C. Decristoforo, Nucl. Med. Biol., 2012, 39, 361–369.
21 A. Ghosh and M. J. Miller, J. Org. Chem., 1993, 58, 7652–
7659.
22 H. Guo, S. A. Naser, G. Ghobrial and O. Phanstiel IV,
J. Med. Chem., 2002, 45, 2056–2063.
23 E. A. Fadeev, M. Luo and J. T. Groves, J. Am. Chem. Soc.,
2004, 126, 12065–12075.
References
1 R. C. Hider and X. Kong, Nat. Prod. Rep., 2010, 27, 637–
657.
2 M. Kreutzer, H. Koge and M. Nett, J. Am. Chem. Soc., 2012,
134, 5415–5422.
3 F. Gibson and D. I. Magrath, Biochim. Biophys. Acta, 1969,
192, 175–184.
4 N. Okujo, Y. Sakakibara, T. Yoshida and S. Yamamoto,
BioMetals, 1994, 7, 170–176.
5 B. Kunze, W. Trowitzsch-Kienast, G. Höfle and
H. Reichenbach, J. Antibiot., 1992, 45, 147–150.
6 W. D. Linke, A. Crueger and H. Diekmann, Arch. Mikrobiol.,
1972, 85, 44–50.
24 Q. X. Wang and O. Phanstiel, J. Org. Chem., 1998, 63, 1491–
1495.
25 E. P. Storey, R. Boghozian, J. L. Little, D. W. Lowman and
R. Chakraborty, BioMetals, 2006, 19, 637–649.
26 A. Cilibrizzi, V. Abbate, Y. L. Chen, Y. Ma, T. Zhou and
R. C. Hider, Chem. Rev., 2018, 118, 7657–7701.
27 A. M. Silva, X. Kong and R. C. Hider, BioMetals, 2009, 22,
771–778.
28 C. F. Baes and R. E. Mesmer, The Hydrolysis of Cations,
Wiley, New York, 1976.
7 M. Persmark, P. Pittman, J. S. Buyer, B. Schwyn, P. R. Gill
Jr. and J. B. Neilands, J. Am. Chem. Soc., 1993, 115, 3950– 29 J. E. Plowman, T. M. Loehr, S. J. Goldman and J. Sanders-
3956.
Loehr, J. Inorg. Biochem., 1984, 20, 183–197.
8 J. D. Martin, Y. Ito, V. V. Homann, M. G. Haygood and 30 C. J. Carrano, S. R. Cooper and K. N. Raymond, J. Am.
A. Butler, J. Biol. Inorg. Chem., 2006, 11, 633–641.
Chem. Soc., 1979, 101, 599–604.
9 Y. Ito and A. Butler, Limnol. Oceanogr., 2005, 50, 1918– 31 Z. D. Liu and R. C. Hider, Med. Res. Rev., 2002, 22, 26–64.
1923.
32 E. Banin, A. Lozinski, K. M. Brady, E. Berenshtein,
P. W. Butterfield, M. Moshe, M. Chevion, E. P. Greenberg
and E. Banin, Proc. Natl. Acad. Sci. U. S. A., 2008, 105,
16761–16766.
33 M. J. Milewska, A. Chmiak and Z. Glowacki, J. Prakt. Chem.,
1987, 329, 447–456.
10 H. Budzikiewicz, Prog. Chem. Org. Nat. Prod., 2004, 87, 81–
237.
11 S. Kishimoto, S. Nishimura and H. Kakeya, Chem. Lett.,
2015, 44, 1303–1305.
12 J. S. Martinz, G. P. Zhang, P. D. Holt, H.-T. Jung,
C. J. Carrano, M. G. Haygood and A. Butler, Science, 2000, 34 P. Gans and B. O’Sullivan, Talanta, 2000, 51, 33–37.
287, 1245–1247.
35 P. Gans, A. Sabatini and A. Vacca, Talanta, 1996, 43, 1739–
13 V. V. Homann, M. Sandy, A. Tincu, A. Templeton, B. Tebo
and A. Butler, J. Nat. Prod., 2009, 72, 884–888.
14 X. Hu and G. L. Boyer, BioMetals, 1995, 8, 357–364.
1753.
36 P. Gans, A. Sabatini and A. Vacca, Ann. Chim., 1999, 89,
45–49.
15 R. C. Hider and A. D. Hall, Prog. Med. Chem., 1991, 28, 41– 37 R. Smith and A. Martell, Critical stability constants, Plenum
173.
Press, New York, 1977–1979.
16 E. W. Price and C. Orvig, Chem. Soc. Rev., 2014, 43, 38 L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini and
260–290.
A. Vacca, Coord. Chem. Rev., 1999, 184, 311–318.
This journal is © The Royal Society of Chemistry 2019
Dalton Trans., 2019, 48, 17395–17401 | 17401