Formation of a tris(catecholato) iron(iii) complex with a nature-inspired cyclic peptoid ligand

Article abstract of DOI:10.1039/d1dt00091h

Siderophore-mimicking macrocyclic peptoids were synthesized. Peptoid3with intramolecular hydrogen bonds showed an optimally arranged primary coordination sphere leading to a stable catecholate-iron complex. The tris(catecholato) structure of 3-Fe(iii) was determined with UV-vis, fluorescence, and EPR spectroscopies and DFT calculations. The iron binding affinity was comparable to that of deferoxamine, with enhanced stability upon air exposure.

Full text of DOI:10.1039/d1dt00091h

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Formation of a tris(catecholato) iron(III) complex
with a nature-inspired cyclic peptoid ligand†
Cite this: Dalton Trans., 2021, 50,
3459
b
Jinyoung Oh,‡a Dahyun Kang,‡a Sugyeong Hong, b Sun H. Kim,
Received 11th January 2021,
Accepted 15th February 2021
a
Jun-Ho Choi a and Jiwon Seo
*
DOI: 10.1039/d1dt00091h
rsc.li/dalton
Siderophore-mimicking macrocyclic peptoids were synthesized. applied in, for example, iron chelation therapy9 and
Peptoid 3 with intramolecular hydrogen bonds showed an opti- sideromycin.10
mally arranged primary coordination sphere leading to a stable
For bacteria, backbone lability through enzymatic hydro-
catecholate-iron complex. The tris(catecholato) structure of 3-Fe lysis is a required aspect for intracellular release of iron;
(III) was determined with UV-vis, fluorescence, and EPR spectrosco- however, this lability often undermines the stability of these
pies and DFT calculations. The iron binding affinity was compar- iron complexes.11,12 To overcome this problem, synthetic iron
able to that of deferoxamine, with enhanced stability upon air chelators have been developed using iron-chelating moieties
exposure.
displayed on abiotic macrocyclic backbones13,14 or without a
backbone.15,16 Among the unnatural scaffolds actively investi-
gated for metal chelation are peptoids.17,18 Peptoids have a
N-substituted glycine backbone, a structure isomeric to that of
natural peptides, and are resistant to degradation by hydrolytic
enzymes. The chemical diversity of peptoids is large because
of the readily available primary amine submonomers,19 allow-
Iron is an essential element in living organisms and plays vital
roles in redox processes such as N2 fixation,1 oxygenation,2 res-
piration3 and photosynthesis.4 Nonetheless, free iron is very
toxic under aerobic conditions and produces reactive oxygen
species through various processes, such as the Fenton reac-
tion.5 Ferrous and ferric ions are poorly soluble in aqueous
media since they easily form hydroxide complexes.6 To circum-
vent these problems, nature utilizes various iron-binding
systems, such as transferrin, ferritin, and siderophores.
Microorganisms utilize siderophores as a crucial iron-trans-
porting system. Siderophores have a high iron binding affinity
to facilitate intracellular iron uptake from an iron-poor
environment (aqueous solution) and have a low molecular
weight to make the process efficient and economic. In particu-
lar, siderophores with tris(catecholato) iron complexes exhibit
remarkable stability constants compared with other sidero-
phores based on hydroxamates or carboxylates.7,8 For example,
the stability constant of enterobactin is the highest among
those of all existing iron-binding ligands known to date
(Fig. 1). This finely optimized iron-transport system has been
aDepartment of Chemistry, School of Physics and Chemistry, Gwangju Institute of
Science and Technology, 123 Cheomdangwagi-ro, Buk-gu, Gwangju, 61005,
Republic of Korea. E-mail: jseo@gist.ac.kr
bWestern Seoul Center, Korea Basic Science Institute, University-Industry
Cooperation Building, 150 Bukahyun-ro, Seodaemun-gu, Seoul, 120-140,
Republic of Korea
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1dt00091h
Fig. 1 Natural siderophores and catechol-containing cyclic peptoids
1–3. Iron-chelating motifs are colored red and hexameric skeletons are
colored blue.
‡These authors contributed equally to this work.
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ing peptoids to serve as highly modular scaffolds for chemical Catechol-iron complexes are known to have different binding
diversification. stoichiometries depending on the concentration of base and
In this work, we employed a cyclic hexapeptoid scaffold to Fe(III), as demonstrated by their different UV-vis spectral
display an iron-binding catechol moiety. A tris(catecholato) signatures.27,28 For catechol-iron complexes, each bis- and tris-
system attached to the macrocyclic peptoid demonstrated the complex has absorption maxima at approximately 570 nm and
importance of the orientation of the two hydroxyl groups in 480 nm respectively.29 Recently, De Riccardis and coworkers
the catechol moiety and the neighboring hydrogen bond to reported an enterobactin/bacillibactin-mimic cyclic tripeptoid
stabilize the iron complex, reminiscent of the structural and showed the formation of a dinuclear bis(catecholato) iron
characteristics of enterobactin. Our synthetic siderophore-iron (III) complex with an absorption maximum at approximately
complex exhibited an optimally arranged primary coordination 570 nm.30 The base titration of our cyclic peptoids was per-
sphere, leading to a stable iron complex.
formed for iron binding mode characterization. In methanol
Three macrocyclic peptoids that mimic natural sidero- and in the absence of base, peptoids 1 and 2 did not show any
phores were synthesized (Fig. 1). The design of the cyclic skel- distinct peak attributed to iron complex formation (Fig. S2,†
eton (i.e., ring size) and the chelating arms (i.e., catecholate) Fig. 2A). Upon addition of six equivalents of NaOH, which can
was based on ferrichrome and enterobactin, respectively. deprotonate the six acidic protons on the three catechols, pep-
Compounds 1 and 2, both of which have dopamine moieties toids 1 and 2 appeared to form bis-complexes. These com-
as catecholate ligands, were prepared and exhibited different plexes showed a gradual transition (bis- to tris-complex) as the
hydrophilicities as a result of their extra ligands: benzyl and number of equivalents of base increased. On the other hand,
methoxyethyl groups. The amide group was incorporated into peptoid 3 formed a bis-complex in the absence of a base and
the chelating arms of compound 3 to increase the stability of in the presence of up to six equivalents of NaOH, and a bis- to
the metal complex by hydrogen bonding between the amide tris-complex transition occurred in methanol as the amount of
proton and catecholate,
enterobactin.20–22
a
strategy also utilized by added base increased (Fig. 2B). Interestingly, in acetonitrile,
the absorption maximum of the tris-complex appeared at
The sequence of the linear peptoid was elongated on a approximately 480 nm without any base and was saturated by
2-chlorotrityl chloride resin by following the peptoid submono- 6 equivalents of Et3N (Fig. 2C). This finding can be explained
mer synthesis protocol.19 A catechol-containing submonomer by the stability of the tris-complex conferred by the hydrogen
(R′-NH2) protected by either an acetonide or a benzyl group bond between the catechol and neighboring amide –NH
was used (Schemes S1 and S2†).23–25 After elongation, a clea- protons in an aprotic solvent. This structural aspect will be
vage reaction using a mildly acidic cocktail (TFE/TIS/CH2Cl2 = further discussed below. After the formation of a stable tris-
1 : 1 : 8, v/v/v) yielded a linear peptoid with high purity, which complex in acetonitrile was observed, a fluorescence quench-
then underwent head-to-tail cyclization using previously opti- ing experiment was performed to elucidate the binding stoi-
mized conditions, with modification of only the solvent to chiometry of the 3-Fe(III) complex. Emission of the dihydroxy-
CH2Cl2 for simpler evaporation.26 Kirshenbaum and coworkers
suggested that the macrocyclization reaction was most
effective when the linear peptoid was a hexamer, as in our syn-
thesis.26 The cyclic peptoids were deprotected and purified by
preparative HPLC to afford compounds 1–3 (Scheme 1). All
reactions in Scheme 1 were monitored by analytical HPLC and
ESI-MS, and the final cyclic peptoids 1–3 were obtained with
purity greater than 98% (Fig. S1†).
The Fe(III) binding mode of the peptoid was verified by UV-
vis spectral analysis and fluorescence quenching experiments.
Fig. 2 Spectral characterization of Fe(III)-binding cyclic peptoids. UV-vis
spectra of Fe(NO3)3·9H2O (100 μM) and solutions of (A) 2 (100 μM) and
(B) 3 (100 μM) in methanol titrated with NaOH(aq). (C) UV-vis spectra of
FeCl3 (100 μM) and 3 (100 μM) solutions in acetonitrile titrated with Et3N.
(D) Plot of the fluorescence titration result for 3 (100 μM) with Fe(acac)3
Scheme 1 Synthetic route for cyclic peptoids 1–3.
3460 | Dalton Trans., 2021, 50, 3459–3463
in acetonitrile (λex = 350 nm/λem = 405 nm).
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benzene chromophore at approximately 400 nm was observed nation sphere of the complex was optimized with a rhombic
upon excitation at 350 nm,31 and evidence of iron complex for- geometry. The distance between the oxygen atom of the cate-
mation was previously provided by quantitative fluorescence cholate and the Fe(III) ion was in the range of 2.02–2.11 Å. In
quenching.32 At a fixed concentration of 3, the fluorescence of accordance with a previous study on ferric enterobactin, the
the catechol chromophore was linearly quenched as the con- length of the Fe–Oortho bonds was slightly longer (2.09–2.11 Å)
centration of Fe(III) increased (Fig. 2D, inset), and the quench- than that of the Fe–Ometa bonds (2.02–2.03 Å) (Ent-Fe(III): Fe–
ing effect by the addition of Fe(III) was completed at a ratio of Oortho 2.03–2.04 Å; Fe–Ometa 1.97–2.02 Å) due to hydrogen
1 : 1 (Fig. 2D). It should be noted that the counterion of Fe(III) bonding.22 In the secondary coordination sphere, intra-
affected the titration results, as was previously observed.33,34 molecular hydrogen bonding was confirmed by the H⋯O dis-
The experiment with FeCl3 resulted in a nonlinear quenching tance of 1.80 Å (Ent-Fe(III): 1.73 Å).22 The resulting parameters
tendency (Fig. S3†), whereas the Fe(III) complex with acetyl- revealed that the 3-Fe(III) complex has a distorted octahedral
acetonate (acac) showed linear quenching, providing a clear primary sphere with intramolecular hydrogen bonding
binding ratio.
between the amide proton and catecholate oxygen in the sec-
The natural siderophore-Fe(III) complex has unique EPR ondary coordination sphere.
absorption (geff = 4.3), which is used as a signature to identify
The iron binding affinity of 3 was estimated through a com-
nonheme high-spin iron(III).35–37 The presence of multiple petitive Fe(III) binding assay with ethylenediamine-tetraacetic
unpaired electrons, such as in a high-spin d5 system, leads to acid (EDTA) (Fig. 4). Known iron-binding ligands, including
splitting of the electronic state in the absence of a magnetic deferiprone (DFP), 3,4-dihydroxybenzoic acid (3,4-DHBA),
field, called zero-field splitting (ZFS). As the electronic state deferoxamine (DFO), and enterobactin (Ent), were used to
splits, the states become highly mixed, and intra-Kramers tran- compare the binding affinity. The iron complex formation con-
sitions become allowed. The result of an intra-Kramers tran- stants in aqueous buffer were as follows: EDTA (β = 1025),39
sition from the | 3/2 > Kramers doublet appeared as a strong DFP (β = 1015),40,41 3,4-DHBA (β = 105),42 DFO (β = 1031),43 and
absorption at 1550 G, with a geff value of 4.3 (Fig. 3A). This Ent (β = 1049).7 Although the 3-Fe(III) complex was soluble in
iron in the complex is also called rhombic iron, which pos- water, the peptoid ligand itself was not soluble in aqueous
sesses rhombic (distorted octahedral) symmetry. Further media. Thus, the formation constant of peptoid 3 could not be
insight was subsequently obtained through EPR spectrum directly measured, and the competition assay was performed
simulation with EasySpin toolbox 5.2.27,38 which provided the in an acetonitrile/water = 9 : 1 (v/v) mixture instead. As shown
ZFS tensor value as a rhombicity (E/D) of 0.31 (E/Daxial = 0 and in Fig. 4, the natural siderophore Ent showed remarkable
E/Drhombic = 1/3). This decisive information clarified that iron stability of the iron complex. In contrast, a dramatic loss of Fe
exists as a high-spin ion in the hexadentate nonheme environ- (III) complexed with DFP and 3,4-DHBA was observed after the
ment with rhombic symmetry, similar to that in the natural addition of 1.5 equivalents (150 μM) of EDTA. Competitive iron
enterobactin-iron(III) complex.
binding affinity was observed for another natural siderophore,
Based on the EPR analysis, DFT calculations were per- DFO, and peptoid 3, which showed iron binding affinity com-
formed to visualize the structure of the 3-Fe(III) complex with parable to that of DFO. Regarding long-term stability, the 3-Fe
high-spin (6S5/2; sextet) iron and a net charge of −3 (Fig. 3B). (III) complex appeared to be more stable than the DFO-Fe(III)
The iron atom was treated by the effective core potential basis complex (Fig. S4†). Cyclic voltammetry measurements
set LANL2DZ to reduce computational effort. The geometry of suggested the stability of the 3-Fe(III) complex with a defined
the 3-Fe(III) complex was optimized in the condensed phase structure under basic conditions over −1.5–1.5 V vs. Ag/Ag+
using a self-consistent reaction field. The primary coordi- (Fig. S5†). Without the addition of a base, the 3-Fe(III) complex
Fig. 3 (A) EPR spectrum experimentally measured for the 3-Fe(III)
complex in acetonitrile at 5 K (solid black line) and obtained by simu-
lation (dashed red line). (B) DFT-calculated structure of the 3-Fe(III)
complex (B3LYP/6-31g+(d,p); LANL2DZ for Fe) under acetonitrile sol-
vation conditions. Hydrogen atoms except for the amide protons and
methoxyethyl side chains are omitted for clarity.
Fig. 4 Competitive Fe(III) binding assay with EDTA for the evaluation of
the iron binding affinity. The absorbance of the compounds without
EDTA was set as 100%, and all samples were prepared at a concentration
of 100 μM in an acetonitrile/water = 9 : 1 (v/v) solution.
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became unstable in the redox environment, likely due to the
occurrence of a cross-linking reaction44 upon application of
the reductive potential (Fig. S6†).
In summary, we report the synthesis of cyclic peptoid-Fe(III)
complexes and their characterization by UV-vis spectroscopy,
fluorescence spectroscopy, EPR spectroscopy, and DFT calcu-
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Conflicts of interest
There are no conflicts of interest to declare.
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Acknowledgements
This work was financially supported by the National
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Institute (KBSI) in Gwangju for instrumentation support (1H
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