Peptoid-based siderophore mimics as dinuclear Fe3+chelators

Article abstract of DOI:10.1039/d0dt00689k

A practical synthesis of preorganized tripodal enterobactin/corynebactin-type ligands (consisting of aC₃-symmetric macrocyclic peptoid core, three catecholamide coordinating units, and C₂, C₄, and C₆spacers) is

Full text of DOI:10.1039/d0dt00689k

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Peptoid-based siderophore mimics as dinuclear
3+
Fe chelators†
Cite this: DOI: 10.1039/d0dt00689k
a
b
a
Assunta D’Amato, ‡ Pritam Ghosh, ‡ Chiara Costabile,
a
a
b
Giorgio Della Sala,
Irene Izzo,
Galia Maayan * and
a
Francesco De Riccardis
*
A practical synthesis of preorganized tripodal enterobactin/corynebactin-type ligands (consisting of a C
3
-
symmetric macrocyclic peptoid core, three catecholamide coordinating units, and C , C , and C spacers)
2
4
6
3
+
is reported. The formation of complexes with Fe was investigated by spectrophotometric (UV-Vis) and
spectrometric (ESI, negative ionization mode) methods and corroborated by theoretical (DFT) calcu-
lations. Preliminary studies revealed the intricate interplay between the conformational chirality of cyclic
trimeric peptoids and metal coordination geometry of mononuclear species similar to that of natural
catechol-based siderophores. Experimental results demonstrated the unexpected formation of unique
Received 24th February 2020,
Accepted 4th April 2020
DOI: 10.1039/d0dt00689k
3
+
rsc.li/dalton
dinuclear Fe complexes.
8
bactin/corynebactin bacterial siderophores enclose quintes-
sential attributes for the synthesis of artificial iron chelators.
Introduction
Naturally occurring and synthetic siderophores are of great The presence of three convergent catecholamide (1,2-dihydrox-
interest not only due to their vital role in microorganisms, ybenzamide) units, stemming from a twelve-membered macro-
1
,2
plants and fungi, but also due to their growing clinical rele- cyclic core, promotes chirospecific trapping of ferric ions in
3
vance as antibiotic carriers and inhibitors of metalloen- natural siderophores (Fig. 1). In particular, [Fe
3
4
3−
3−
zymes, in imaging and in the treatment of iron overload (enterobactin)] , [Fe(1) ], displays a right-handed (Δ) coordi-
5
9
3−
3−
disease.
Due to its redox active nature, excess of iron in human handed (Λ) chirality of the Fe centre.
body, caused by transfusion due to anaemic disorders, Although 1 and 2 are powerful iron chelators, the hydro-
induces organ malfunction and failure. Iron chelation therapy lytic instability of ester linkages present in the macrocyclic
nation mode and [Fe(corynebactin)] , [Fe(2) ], shows a left-
3
+
9
1
1
0
(
ICT) restores the physiological concentrations of this metal core, encouraged the synthesis of novel, more stable, and bio-
4
,11
making use of high-affinity iron-chelating compounds. compatible siderophores.
Unfortunately, most of the commercially available ferric iron
sequestering agents (i.e.: deferoxamine, deferiprone, and
deferasirox) show multiple adverse side effects (neutropenia,
neurotoxicity, agranulocytosis, diarrhoea, and hepatic/cardiac
6
,7
damage) and search for new families of iron chelators is an
ebullient field of research.
1
–4
Natural products have always been the most successful
source of inspiration for the design of new drugs and entero-
a
Department of Chemistry and Biology “A. Zambelli”, University of Salerno, Via
Giovanni Paolo II, 132, Fisciano, SA, 84084, Italy. E-mail: dericca@unisa.it
b
Schulich Faculty of Chemistry, Technion – Israel Institute of Technology, Haifa,
32000, Israel. E-mail: gm92@technion.ac.il
†
Electronic supplementary information (ESI) available: NMR spectra, HPLC
chromatograms of linear and cyclic peptoids, UV titration with metal ions,
3
+
3+
ESI-MS of Fe complexes, and computational details. See DOI: 10.1039/
D0DT00689K
Fig. 1 Molecular structure and absolute configuration for the Fe tris
chelate centre (Δ or Λ) of naturally occurring enterobactin (1) and cory-
nebactin (2) metal complexes.
‡
Equal contribution.
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(
3a′). Theoretically calculated structures of the diastereomeric
3
−
3−
Λ-[Fe(3a′)] and Δ-[Fe(3a′)] models (Fig. 3a) indicated an
−
1
internal energy difference (ΔE) equal to 3.3 kcal mol in favor
of the former species (Fig. 3b), suggesting a steric mismatch
between the pR planar chirality and Δ configuration of the
metal centre.
The higher stability of the
p
R/Λ diastereoisomer was attribu-
ted to a modest dihedral angle distortion of complex Λ-[Fe
3
−
(
3a′)] (Δϕ ≈ +7°; Δψ ≈ +4°; Δω ≈ −9°) when compared to the
classic CP γ-turn values reported for cyclic trimeric peptoids
ϕ = + 95°; ψ = −95°; ω = 0°). The average deviations esti-
1
1
6
(
3
−
mated for the higher energy species Δ-[Fe(3a′)] were more
pronounced (Δϕ ≈ +17°; Δψ ≈ +17°, Δω ≈ −27.0°, see the ESI†
for atomic coordinates).
Ring size shrinkage from twelve-membered natural oligolac-
tones 1 and 2 to nine-membered cyclopeptoid hosts 3a–c was
considered uninfluential for the iron complex formation with
tricatechol-based ligands, as MECAM and TRENCAM, with
1
1d
Fig. 2 (a) Molecular structures of cyclotrimeric peptoid catecholamides
smaller 1,3,5-trisaminomethylbenzene
and β, β′, β″-
3
a–c. (b) Possible enantiomorphic forms of cyclopeptoid-based hosts
11c,e
triaminotriethylamine
ing activity.
cores, display excellent iron-chelat-
p p
showing opposite conformational chirality (( R)-form: 3a’–c’ and ( S)-
form: 3a’’–c’’).
The main problem to be addressed, in the experimental
studies, was heterogeneous nuclearity typical of the catechola-
1
1b,f
mide complexes.
With the aim to find a novel robust cyclooligomeric frame
for catecholamide chelating groups, we designed a new family
of siderophores based on
stable peptoid scaffold displaying C , C , and C spacers and
a
proteolytically/hydrolytically
1
2
2
4
6
a preorganized cyclotrimeric structure (3a–c, Fig. 2a).
The presence of a sterically strained nine-membered macro-
1
3
cycle and a relatively high thermodynamic barrier for the oli-
‡
−1
goamide ring interconversion (ΔG ≈ 19 kcal mol , for cyclic
tripeptoids)
1
3,14
indicated the formation of 3a′-c′/3a″-c″ enan-
tiomeric conformational isomers, with opposite planar chiral-
1
3,15
ity at the tertiary amide bond centres (Fig. 2b).
In the present contribution we report the synthesis of
cyclooligomeric 3a–c, as amide-based analogues of enterobac-
tin/corynebactin-type siderophores. In this study we reveal
3
+
their conformational properties as free hosts and Fe com-
plexes, and demonstrate, through accurate spectrophoto-
metric/spectrometric analyses, the formation of unique dinuc-
lear complexes.
Results and discussion
DFT studies on mononuclear complexes
The concurrent presence of amide bonds at stereogenic
1
3,15
sites
and left-(Λ) or right-handed (Δ) coordination geome-
9
3+
try at the Fe centre, leads to the formation of energetically
nonequivalent metal-cyclopeptoid complexes with R/Λ;
and S/Δ; S/Λ configurations.
3−
Fig. 3 (a) Schematic structures of cyclopeptoid complexes Λ-[Fe-3a’]
3
−
3−
R/Δ and Δ-[Fe-3a’] . (b) Minimum energy structures of Λ-[Fe-3a’] and Δ-
p
p
3
[
Fe-3a’] and their respective internal energies calculated at the B3LYP/
-311G* level of theory expressed in kcal mol− (see the ESI† for
p
p
1
6
To obtain the lowest energy conformation and a quantum
mechanical refinement, density functional theory (DFT) calcu-
lations at the B3LYP/6-311G* level were performed on the
Cartesian coordinates). Dihedral angle definition for residue i: ω [Cα(i−1)
;
CO(i−1); N(i); Cα(i)], φ [CO(i−1); N(i); Cα(i); CO(i)], ψ [N(i); Cα(i); CO(i); N(i+1)].
Hydrogen atoms are omitted for clarity. Atom type: C light grey, N blue,
model with the C2 spacer and pR amide bond configuration O red, and Fe yellow.
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Scheme 1 Synthesis of N-(ω-aminoalkyl)-2,3-bis(benzyloxy)benza-
mides 8a–c.
Scheme 2 Final steps of the synthetic route of tris-catecholamide
ligands 3a–c. For simplicity, enantiomorphous conformational isomers
were not reported.
Synthesis of cyclic peptoid ligands 3a–c
The synthesis of enterobactin/corynebactin peptoid analogues
temperature, of conformational isomers in slow equilibrium
on the NMR time scale.
1
7
3
a–c relied on the solid-phase “sub-monomer” approach and
2
5
required preliminary elaboration of the appropriately protected
primary amines for their insertion in the growing oligoamide
chain. The amine precursors were known N-(2-aminoethy)-2,3-
Catalytic debenzylation in the presence of H2 and Pd/C
afforded the cyclic catecholamides 3a–c in quantitative yields.
All-cis “crown” conformations of the target 2,3-dihydroxycate-
cholamides were established on the basis of the three-fold
1
8
bis(benzyloxy)benzamide (8a) and N-(4-aminobutyl)-2,3-bis
1
9
(
benzyloxy)benzamide (8b), plus new N-(6-aminohexyl)-2,3-
1
13
symmetry present in the H and C NMR spectra (600 MHz,
CD OD, see the ESI†). The inequivalence of intra-annular
bis(benzyloxy)benzamide (8c) intermediates. 8a–c were syn-
1
8
3
thesized according the general procedure
Scheme 1.
reported in
glycine proton resonances (Δδ ∼ 1.5 ppm) inferred the pres-
ence of conformational enantiomers 3a′–c′/3a″–c″ in slow equi-
librium on the NMR time scale.
2
0
The formation of 2,3-bis(benzyloxy)benzoyl chloride from
the corresponding acid 4 and subsequent amidation in the
2
1
presence of mono-Boc diamines 5a–c, gave the required fully
protected amidocarbamates 6a–c. The acid-induced N-Boc de-
Metal ion binding studies
protection and formation of the corresponding free bases pro- Metal-free peptoids 3a–c exhibited three absorption bands
2
6
duced the primary amines 8a–c useful for solid-phase each, in methanol, arising from 2,3-dihydroxybenzamide.
synthesis.
Upon addition of FeCl , the intensity of these three bands
Intermediates 8a–c readily reacted with the bromoacetic increased and new absorption bands were obtained, indicating
3
3
+
residues of the growing peptoid chains, according to the that all three peptoids bind with Fe . For 3a, the bands near λ
1
7
3+
classic solid phase procedure and gave linear oligoamides = 209, 251 and 316 nm, shifted upon binding with Fe to λmax
a–c (Scheme 2) in an excellent overall yield with decent = 215, 258 and 322 nm, respectively (Fig. 4a). Similarly, for 3b
analytical purity (see the ESI†).
the bands near λ = 207, 250 and 318 nm were shifted to
9
Linear peptoids 9a–c were successfully cyclized in the pres- 211 nm with a shoulder at 227 nm, 258 nm and 330 with a
ence of an efficient HATU condensing agent under high shoulder at 365 nm, respectively (Fig. 4b). For 3c, a similar
dilution conditions (3.0 mM). The fully protected targets 10a–c trend was observed and the bands near λ = 208, 248 and
were subsequently isolated as all-cis “crown” conformational 314 nm shifted to 209 nm with a shoulder at 227 nm, 258 and
1
3,22
isomers.
C
3
-symmetry, in accordance with previously syn- 332 nm with a shoulder at 363 nm, respectively (Fig. 4c). In
2
3,24
thesized cyclic trimeric peptoid congeners,
the revelation of one third of the expected H and C NMR 565 nm for 3a–c, respectively, was obtained, due to ligand-to-
signals of 10a–c ( H NMR analysis, 600 MHz, CDCl
ESI†). The presence of cis amide bonds was supported by the (catecholate) rather than tris(catecholate) complexes.
extensive X-ray/NMR/modelling studies on similar cyclic these UV-Vis titration results, metal-to-peptoid ratio plots were
was inferred by addition to these shifts, a new band near λ = 570, 562 and
1
13
1
3
, see the metal charge transfer (LMCT), indicating the formation of bis
2
7,28
From
1
3,22
tripeptoids.
constructed, revealing a saturation of the absorbance value at
1
3+
Variable temperature H NMR experiments on strained a 2 : 1 ratio for the Fe : peptoid.
N,N′,N″-tri-2,3-bis(benzyloxy)benzamidethyl-cyclo-triglycine 10a
demonstrated, for the diastereotopic glycine protons, no 570 nm with 17 μM Fe and varying the concentrations of 3a
2
9
The Job’s plot, obtained by monitoring the absorbance at
3
+
3
+
coalescence up to 373 K (C
2
D
2
Cl
4
solution, 600 MHz, implying in methanol (Fig. S14a, ESI†) also indicated a 2 : 1 Fe : 3a
ΔG > 16.9 kcal mol−1), confirming the presence, at room ratio, supporting the coordination of two Fe ions to each
‡
3+
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two catechol ligands (binding site 1, BS1, in red colour),
3
+
−
forming bis(catecholate)Fe complexes, [Fe(3a–c)] , with a
certain number of solvent molecules (methanol and/or water)
3
+
depending on the Fe coordination geometry. The second
3
+
equiv. of the Fe ions binds to the remaining catechol ligand
binding site 2, BS2, in blue colour) to give the hypothesized
(
dinuclear complex [Fe
molecules).
We also suggest that the mononuclear complex [Fe(3a–c)]
is formed upon the first addition of one equiv. of the Fe
ions, such that K1 ≫ K , assuming a stronger (and faster)
2
(3a–c)] (plus methanol and/or water
−
3
+
2
3
0
binding to the two catechol chelators of BS1.
2
+
Interestingly, the titration of 3a–c with Fe did not produce
any observable changes in the UV-Vis spectra, indicating that
2
+
these peptoids do not bind with Fe . The UV-Vis titration of
2
+
3
a with Fe is depicted in Fig. 4d (see the ESI† for 3b–c). The
2
+
2+
2+
Fig. 4 UV-Vis titration of: (a–c) 3a–c (17 µM in methanol) with UV-Vis titration of peptoids 3a–c with Co , Cu , Ni and
additions of 2 µL aliquots of 5 mM methanol solution of Fe in multiple Zn metal ions showed changes in the spectra, indicating that
3
+
2+
3+
steps (black: free peptoid and blue: Fe complex) (d) 3a (17 µM in
methanol) with additions of 2 µL aliquots of 5 mM methanol solution of
Fe in multiple steps (black: free peptoid and green: peptoid without
the three peptoids can coordinate to these metal ions (see the
ESI†).
2
+
2
+
The UV-Vis titration suggested the formation of dinuclear
interaction with the Fe ion).
[
Fe
2 3
(3a–c)] species. As the titration was carried out with FeCl ,
−
we assumed that the counterion, in this case Cl , might help
in the coordination of Fe by bridging. Thus, to gain further
insight and to explore the effect of the counterion on Fe
binding, peptoid 3a was titrated with Fe(ClO ) and Fe(NO )
3 3
3
+
31
peptoid rather than 1 : 1 binding. Interestingly, a rapid linear
increase in the intensity of each LMCT band was observed
3
+
3
+
4
3
until one equiv. of the Fe ions was added (Fig. 4a–c, insets).
3
+
under the same reaction conditions as with FeCl . The titra-
tion exhibited similar UV-Vis bands to those obtained in the
3
Further addition of another equiv. of Fe ions led to a slower
gradual intensity increase, reaching the saturation upon two
equiv. addition (Fig. 4a–c, insets).
presence of FeCl , including the LMCT band at 570 nm
3
(
Fig. 5). The metal-to-peptoid ratio plots also showed a 2 : 1
Rationalization of the experimental results has been recapi-
3
+
3
+
Fe to peptoid ratio (see the ESI†). It was therefore concluded
that Fe complexation was independent from the counterion.
ESI-MS spectroscopy (negative mode), performed on the
tulated in Scheme 3. The first equivalent of Fe is captured by
3
+
samples of 3a–c with 2 equiv. of FeCl
3
, further supported the 1
:
2 host : guest ratio evidencing a cluster of pseudomolecular
ion peaks with a variable number of solvent molecules bound
to the dinuclear complexes (Fig. S16–S18, ESI†). The survey of
prominent metalated mass peaks, in conjunction with the
experimental isotopic analysis (Fig. S19–S24, ESI†), suggested
a general molecular formula, [Fe (3a–c)(H O) (CH OH)6−n],
2 2 n 3
confirming the formation of dinuclear complexes rather than
a mononuclear one (typical of enterobactin/corynebactin side-
4 3 3 3
Fig. 5 UV-Vis titration of 3a with (a) Fe(ClO ) and (b) Fe(NO ) in
Scheme 3 Proposed two-step coordination of peptoids 3a–c to two
methanol. Peptoid concentration: 17 µM, with additions of 2 µL aliquots
of 5 mM methanol solution of Fe in multiple steps.
3
+
3+
equiv. of Fe with the formation of zwitterionic species [Fe
2
(3a–c)].
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1
rophores). To explore the influence of the solvent on the
3
+
coordination of Fe , 3a was titrated with several aliquots of
3
+
the Fe
ion in acetonitrile (Fig. S14b†). Saturation was
3
+
achieved after the addition of 2 equivalents of Fe , suggesting
that the final metallopeptoid complex has a metal : peptoid
ratio of 2 : 1, as observed in methanol. The UV-Vis spectrum in
acetonitrile, however, is different from the one obtained in
methanol, having two intense maxima near 305 and 372 nm
and an LMCT band near 540 nm (Fig. S14c†). Similar solvent
dependency of the LMCT band is also reported in the literature
3
+
for an Fe phenolate type complex where the LMCT band is
observed at a higher wavelength for methanol and is shifted to
3
2
2 2 4 3 2
a lower wavelength in acetonitrile. The solution obtained Fig. 6 Minimum energy structure of [Fe (3a)(H O) ]·(CH OH)·(H O).
Hydrogen atoms are omitted for clarity, except for water and methanol
molecules, and for hydrogens involved in hydrogen bonds. Relevant
hydrogen bonds are indicated by dashed lines. Distances are given in Å.
Atom type: C light grey, N blue, O red, and Fe yellow.
from the UV-Vis titration was directly analyzed by ESI-MS,
which suggests multiple complexes in solution, similar to the
spectrum obtained for the titration in methanol; all have a
2
: 1 metal : peptoid ratio with various solvent molecules as
ligands including acetonitrile (see the Experimental section
and Fig. S16†). Based on these results we propose that a 2 : 1
3
+
Fe : peptoid coordination complex is obtained both in metha-
nol and in acetonitrile.
3+
cholate)Fe , we observed the expulsion of the methanol mole-
cule from the first coordination sphere during the optimiz-
ation. The final structure [Fe
3
+
To estimate the affinity of 3a–c for Fe , we measured the
dissociation constants, K , of the complexes [Fe (3a–c)
2
2 4 3 2
(3a)(H O) ]·(CH OH)·(H O)
d
2
showed CH
structure.
3
OH bound only by hydrogen bonds to the main
(
H
2
O)
n
3
(CH
3
OH)6−n
]
via competition experiments with
≫K , the actual chelator competing with
EDTA over Fe ions was supposed to be BS1, rather than the
entire peptoid, and it was reasonable to assume that K ≈ K
In a typical experiment a mixture of peptoid and EDTA
3,34
EDTA.
As K
1
2
Dinuclear complex formation indicated the intrinsic
difficulty of the three catecholamide groups to form mono-
nuclear octahedral Λ-[Fe(3a′)]
support the formation of a bis(catecholate)Fe
3
+
d
1
.
3−
species. Our calculations
3+
chelating
3
+
(
16.61 μM of each chelator in methanol) was titrated with Fe .
The UV-Vis signal of these titrations, in the range of
00–800 nm, was monitored according to the method devel-
oped by Wedd, and Xiao et al. Based on this method, the
slope between ([BS1 site]total/[FeBS1]) − 1 and ([EDTA]total
3+
complex (rather than a tris(catecholate)Fe
complex) as
suggested by our experimental data. Distorted square pyrami-
dal geometry pentacoordination in BS1 precludes the stereo-
chemical complications discussed for mononuclear complex
4
3
3
/
3−
Λ-[Fe(3a′)] and increases the bite angle between the two cate-
[
Fe-EDTA]) − 1 represented the dissociation constant K
d
.
M
cholamide units in [Fe
130° in Λ-[Fe(3a′)] to ≈150° in dinuclear complexes).
2
2 4 3 2
(3a)(H O) ]·(CH OH)·(H O) (from
−
19
−19
The values obtained were K = 6.86 × 10
M, 4.21 × 10
3−
d
≈
and 4.01 × 10− M for [Fe
19
(3a–c)(H
O)
(CH
OH)6−n], respect-
2
2
n
3
ively (Fig. S15†). The recorded dissociation constants
were higher than those of enterobactin and commercially
f
available siderophores (the log K value for enterobactin, amo-
Conclusions
nabactin T and desferrioxamine E is 49.0, 34.5 and 32.5
1
respectively).
New ligands 3a–c containing three catechol side chains
attached to a preorganized cyclotrimeric peptoid scaffold
through C , C , and C aliphatic spacers were prepared and
2 4 6
DFT studies on dinuclear [Fe (3a)(H O) (CH OH)] complexes
2
2
5
3
The coordination geometry of the dinuclear complex [Fe (3a) spectroscopically characterized. The complexation behaviour
2
3
+
(H
2
O)
5 3
(CH OH)] emerging from the spectrophotometric and of these ligands towards Fe was first studied at a theoretical
spectrometric studies was investigated by DFT calculations level (considering the implications of conformational isomer-
3
+
(
considering octahedral Fe ions). Two molecules of water ism of the cyclic peptoid core in conjunction with the octa-
3
+
completed the geometry of bis(catecholate)Fe (BS1); three hedral coordination geometry) and then determined through
molecules of water and one of methanol were located on less UV-Vis spectrophotometry. The results showed that sidero-
sterically demanding mono(catecholate)Fe (BS2).
3
+
phores 3a–c display a unique coordination mode forming
Dinuclear complex optimization led to pentacoordinated square pyramidal dinuclear complexes instead of the expected
iron centres, as shown by the minimum energy structure octahedral mononuclear ones, as evident from the UV-Vis titra-
3
+
reported in Fig. 6. More in detail, one of the water molecules tion and MS data. Such exceptional double chelation of Fe ,
3
+
expected to be coordinated to bis(catecholate)Fe , was, corroborated by detailed DFT calculations, emphasizes the
instead, located far away from the metal centre and stabilized importance of the backbone structure in the design of novel
by four hydrogen bonds. As for pentacoordinated mono(cate- peptoid-based siderophores.
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We are now preparing twelve-membered macrocyclic tricate- impact instrument with a direct probe of ACN : H
2
O (70 : 30)
chol ligands which may more closely mimic the morphologic having a flow rate of 0.3 mL min− was used.
1
attributes of enterobactin/corynebactin-type natural ligands
and unfold new avenues for the design of novel biostable/bio-
compatible ferric ion chelators.
1
8
19
General methods for the preparation of the amines 8a, 8b,
and 8c
Synthesis of derivatives 6a–c. A catalytic amount of dry di-
methylformamide was added, followed by oxalyl chloride (1.50
eq., 1.52 mL, 18.0 mmol) to a dry dichloromethane (48.0 mL)
2
0
solution of 2,3-bis(benzyloxy)benzoic acid
2.0 mmol), under a nitrogen atmosphere. The mixture was
stirred for 1 hour under a nitrogen atmosphere, then the
(4.00 g,
Experimental
General procedures
1
Starting materials and reagents purchased from commercial solvent was evaporated. Dry triethylamine (1.70 eq., 2.84 mL,
2
1
suppliers were generally used without purification unless 0.0200 mmol) was added to the desired N-Boc-diamine
otherwise mentioned. TLC was performed on Macherey-Nagel (11.0 mmol), dissolved in 46.0 mL of dry dichloromethane.
precoated silica gel plates (0.25 mm) and observed under a UV- The crude residue 2,3-bis(benzyloxy)benzoyl chloride was dis-
Vis lamp at 254 nm or sprayed with ninhydrin. HPLC analyses solved in a further 46.0 mL of dry dichloromethane and added
were performed using a JASCO LC-NET II/ADC equipped with dropwise to the reaction mixture. The mixture was stirred at
a JASCO Model PU-2089 Plus Pump and a JASCO MD-2010 room temperature overnight. CH Cl was removed in vacuo to
2 2
Plus UV-Vis multiple wavelength detector set at 220 nm. The yield a light yellow solid residue. The crude products were pur-
column used was a C18 reversed-phase analytical column ified using flash silica gel, conditions: 100%–95% A (A: di-
1
8
19
(
Waters, Bondapak, 10 μm, 125 Å, 3.9 mm × 300 mm) which chloromethane; B: methanol), to give known 6a, and 6b,
runs with linear gradients of ACN (0.1% TFA) in H O (0.1% and new 6c.
TFA) over 30 min, at a flow rate of 1.0 mL min . ESI-MS ana- N-(N-Boc-ethylenediamine)-2,3-bis(benzyloxy)benzamide
lysis in the positive ion mode was performed using a Finnigan (6a), light yellow amorphous solid, 3.77 g, 72%. ES-MS: m/z;
2
−
1
+
1
LCQ Deca ion trap mass spectrometer (ThermoFinnigan, San 477.3 [M + H] . H NMR (400 MHz, CDCl
Josè, CA, USA) and the mass spectra were acquired and pro- NH), 7.62 (1H, d, J 2.4 Hz, o-Ar–H), 7.38–7.17 (11H, br signals,
cessed using the Xcalibur software provided by Thermo overlapping, m-Ar–H and CH –Ph–H), 7.06 (1H, d, J 1.8 Hz,
Finnigan. The samples were dissolved in 1 : 1 CH OH/H O, p-Ar–H), 5.07 (2H, s, CH –Ph), 5.01 (2H, s, CH
3
) δ: 7.98 (1H, br s,
2
3
2
2
2
–Ph), 3.27 (2H,
t
0
.1% formic acid, and infused in the ESI source by using a m, CvO–NH–CH ), 3.08 (2H, m, CvOO Bu–NH–CH ), 1.32
2
2
−
1
syringe pump; the flow rate was 5 μL min . The capillary (9H, s, CvOO(CH
voltage was set at 4.0 V, the spray voltage at 5 kV, and the tube N-(N-Boc-butanediamine)-2,3-bis(benzyloxy)benzamide (6b),
lens offset at −40 V. The capillary temperature was 220 °C. light yellow amorphous solid, 5.10 g, 92%. ES-MS: m/z; 505.2
3 3
) .
+
1
3
Data were acquired in MS1 and MSn scanning modes. Zoom [M + H] . H NMR (400 MHz, CDCl ) δ: 7.93 (1H, br s, NH),
scan was used in these experiments. High-resolution mass 7.74 (1H, br dd, J 5.1, 3.7 Hz, o-Ar–H), 7.48–7.29 (11H, br
spectra (HRMS) were recorded on a Bruker Solarix XR Fourier signals, overlapping, m-Ar–H and CH –Ph–H), 7.15 (1H, br dd,
2
transform ion cyclotron resonance mass spectrometer J 4.2 Hz, p-Ar–H), 5.16 (2H, s, CH
FTICR-MS) equipped with a 7 T magnet, using matrix assisted 3.27 (2H, m, CvO–NH–CH ), 3.03 (2H, m, CvOO Bu–NH–
laser desorption ionization (MALDI) or ESI.† Yields refer to CH ), 1.58 (4H, br signals, overlapping, NH–CH –CH –CH ),
2 2
–Ph), 5.09 (2H, s, CH –Ph),
t
(
2
2
2
2
2
1
13
chromatographically and spectroscopically ( H- and C NMR) 1.44 (9H, s, CvOO(CH
pure materials. NMR spectra were recorded on a Bruker DRX N-(N-Boc-hexamethylenediamine)-2,3-bis(benzyloxy)benz-
00 ( H at 600.13 MHz, C at 150.90 MHz), and Bruker DRX amide (6c), light yellow amorphous solid, 5.86 g, 100%. ES-MS:
3 3
) .
1
13
13
6
4
1
+
+
00 ( H at 400.13 MHz, C at 100.03 MHz). Chemical shifts (δ) m/z; 533.6 [M + H] . HRMS (ESI-FTICR) m/z; [M + H] calcd for
+
1
are reported in ppm relative to the residual solvent peak
C
32
H
41
N
2
O
5
533.3010; found 533.3031. H NMR (600 MHz,
1
3
(
3
CHCl , δ = 7.26; CDCl , δ = 77.00; C DHCl , CD HOD, δ = CDCl ) δ: 7.90 (1H, m, NH), 7.74 (1H, br dd, J 6.2, 3.4 Hz, o-Ar–
3 3 2 4 2 3
1
3
.31; CD
3
OD, δ = 49.00) and the multiplicity of each signal is H), 7.40–7.30 (11H, br signals, overlapping, m-Ar–H and CH
–Ph), 5.08
doublet; dd, double doublet; t, triplet; sept, septet; m, multi- (2H, s, CH –Ph), 3.25 (2H, m, CvO–NH–CH ), 3.07 (2H, m,
2
–
designated with the following abbreviations: s, singlet; d, Ph–H), 7.15 (1H, br dd, p-Ar–H), 5.16 (2H, s, CH
2
2
2
t
plet; br, broad. Coupling constants (J) are reported in Hertz. CvOO Bu–NH–CH
For the titration of peptoids with metal ions, UV-Vis measure- br signals, overlapping, NH–CH
ments were executed using an Agilent Cary 60 UV-Vis spectro- Synthesis of derivatives 7a–c. A solution of trifluoroacetic
photometer comprising a double beam, Czerny–Turner mono- acid in dry dichloromethane (20% v/v, 77.0 mL) was added
chromator. Peptoids 3a–c titrated with the Fe ion were dropwise to a dry dichloromethane (77.0 mL) solution of the
directly used for ESI-MS (−ve mode) analysis using an Advion N-Boc protected N′-2,3-bis(benzyloxy)benzamides (6a–c,
expression CMS mass spectrometer under electrospray ioniza- 0.0100 mmol), brought to 0 °C in an ice bath. The reaction
2
), 1.44 (9H, s, CvOO(CH
3
)
3
, 1.42–1.22 (8H,
–CH ).
2
–CH –CH –CH
2
2
2
2
3
+
tion (ESI), with direct probe ACN : H
0
2
O (95 : 5) at a flow rate of mixture was stirred at room temperature for 3 hours, then the
.2 mL min− . For isotopic mass analysis, a Bruker Maxis completion of the reaction was assessed via TLC. The solution
1
Dalton Trans.
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Dalton Transactions
was then concentrated in vacuo and the crude oil was dissolved (ESI-FTICR) m/z; [M + H] calcd for C27
Paper
+
+
H
33
N
2
O
3
433.2486;
1
in 5.00 mL of hot ethanol, and then added dropwise to 200 mL found 433.2502. H NMR (400 MHz, CDCl ) δ: 7.87 (1H, bt, J
3
of a cold 50/50 solution of diethyl ether/petroleum ether to 6.3 Hz, NH), 7.66 (1H, dd, J 6.0, 3.6 Hz, o-Ar–H), 7.40–7.28
1
8
19
induce the precipitation of known 7a, and 7b, and a new 7c (11H, overlapping, m-Ar–H and CH
trifluoroacetate salt.
N-(Aminoethyl)-2,3-bis(benzyloxy)benzamide, TFA salt (7a), 6.3 Hz, CvO–NH–CH
white amorphous solid, 3.33 g, 68%. ES-MS: m/z; 377.3 [M + (4H, m, CH –CH –NH
H] . H NMR (400 MHz, CDCl ) δ: 8.48 (1H, br s, NH), 7.61 ping m, NH–CH –CH –CH –CH ). C NMR (100 MHz, CDCl3)
2
–Ph–H), 7.07 (1H, m, p-Ar–
H), 5.08 (2H, s, CH –Ph), 4.99 (2H, s, CH –Ph), 3.20 (2H, q, J
2
2
2
2 2
), 2.55 (2H, t, J 6.3 Hz, CH –NH ), 1.26
2
2
2 2 2
and NH–CH –CH ), 1.15 (4H, overlap-
+
1
13
3
2
2
2
2
(1H, d, J 7.5 Hz, o-Ar–H), 7.41–7.13 (12H, br signals, overlap- δ: 164.9 (CvO), 151.5 (m-C–OBn), 146.5 (o-C–OBn), 136.2 ×2
ping, m-Ar–H, p-Ar–H and CH –Ph–H), 5.17 (2H, s, CH –Ph), (C–Ph), 128.6 ×3 (C–Ph), 128.5 ×3 (C–Ph), 128.1 ×2 (C–Ph),
2
2
5
.13 (2H, s, CH –Ph), 3.33 (2H, m, CvO–NH–CH ), 2.96 (2H, 127.5 ×3 (C–Ph), 124.3 (p-C–Ar), 123.0 (o-C–Ar), 116.6 (m-C–Ar),
2
2
+
m, CH
2
–CH
2
–NH
3
).
76.2 (O–CH
2
–Ph), 71.0 (O–CH
), 32.2 (CH –CH
2
–Ph), 41.3 (CH
2 2
–NH ), 39.4
N-(Aminobutyl)-2,3-bis(benzyloxy)benzamide, TFA salt (7b), (CvO–NH–CH
2
2
2
–NH ), 29.0 (CvO–NH–CH –
2
2
white amorphous solid, 3.63 g, 70%. ES-MS: m/z; 405.0 [M + CH ), 26.5 (CvO–NH–CH –CH –CH ), 26.2 (CH –CH –CH –
2
2
2
2
2
2
2
+
1
+
H] . H NMR (400 MHz, CDCl
1H, br s, NH), 7.58 (1H, d, J 6.8 Hz, o-Ar–H), 7.46–7.12 (12H,
br signals, overlapping, m-Ar–H, p-Ar–H and CH –Ph–H), 5.13
3 3 2
) δ: 8.16 (3H, br s, NH ), 8.10 NH ).
(
2
(
2H, s, CH
CH ), 1.59 (2H, m, CH
CH –CH –CH ).
2
–Ph), 5.08 (2H, s, CH
2
–Ph), 3.17 (2H, m, CvO–NH–
+
General methods for the synthesis of the peptoid derivatives
Solid-phase synthesis of the linear precursors 9a–c. The
N-(N-Boc-hexamethylenediamine)-2,3-bis(benzyloxy)benzamide, 2-chlorotrityl chloride resin (α-dichlorobenzhydryl-polystyrene
2
2
–CH –NH ), 1.36 (4H, m, CvO–NH–
2
3
2
2
2
−
1
TFA salt (7c), white amorphous solid, 2.84 g, 52%. ES-MS: m/z; cross-linked with 1% DVB; 100–200 mesh; 1.47 mmol g
4
C
CDCl
,
+
+
33.4 [M + H] . HRMS (ESI-FTICR) m/z; [M + H] calcd for 0.300 g, 0.441 mmol) was swelled in dry CH Cl (3 mL) for
2
2
+
3
1
27 33 2
H N O
433.2486; found 433.2458. H NMR (400 MHz, 45 min and washed twice with CH
2
Cl
2
(3 mL). The first sub-
+
3
) δ: 8.04 (3H, br s, NH
3
), 7.99 (1H, m, NH), 7.57 (1H, dd, monomer was attached to the resin by adding bromoacetic
J 6.2, 3.1 Hz, o-Ar–H), 7.40–7.07 (11H, br signals, overlapping, acid (0.0980 g, 0.706 mmol) in dry CH Cl (3 mL) and DIPEA
2
2
2
m-Ar–H and CH –Ph–H), 7.07 (1H, br dd, p-Ar–H), 5.08 (2H, s, (384 μL, 2.20 mmol) on a shaker platform for 60 min at room
CH –Ph), 5.01 (2H, s, CH
2
2
–Ph), 3.12 (2H, q, CvO–NH–CH
2
+
), temperature, followed by washing with DMF (3 × 1 min),
+
2
1
.85 (2H, m, CH –NH ), 1.55 (2H, m, CH –CH –NH ), CH Cl (3 × 1 min) and again with DMF (3 × 1 min). A solution
.29–1.10 (6H, overlapping m, NH–CH
2
3
2
2
3
2
2
2
–CH
2
–CH
2
–CH
2
–CH
2
).
of the chosen amine (2.20 mmol, 1.63 M in dry DMF) was
Synthesis of the free amines N-(aminoethyl)-2,3-bis(benzy- added to the bromoacetylated resin. The mixture was left on a
loxy)benzamide (8a), N-(aminobutyl)-2,3-bis(benzyloxy)benz- shaker platform for 60 min at room temperature, then the
amide (8b), and N-(aminohexyl)-2,3-bis(benzyloxy)benzamide resin was washed with DMF (3 × 1 min), CH Cl (3 × 1 min)
8c). The TFA salt derivatives (7a–c, 0.00600 mmol) were stirred and again with DMF (3 × 1 min). Subsequent bromoacetylation
2
2
(
for 30 minutes at room temperature with 25 mL of NaOH 1.25 reactions were accomplished by reacting the aminated oligo-
M. The mixture was extracted three times with 50.0 mL of di- mer with a solution of bromoacetic acid (0.613 g, 4.41 mmol)
chloromethane, then dried over anhydrous MgSO
4
and concen- and DIC (751 μL, 4.85 mmol) in dry DMF (3 mL) for 60 min at
trated in vacuo to give known 8a, and 8b, and new 8c as room temperature; the completion of the reaction was
free amines. assessed with the chloranil test. The filtered resin was washed
N-(2-Aminoethyl)-2,3-bis(benzyloxy)benzamide (8a), light with DMF (3 × 1 min), CH Cl (3 × 1 min), and again DMF (3 ×
yellow oil, 2.10 g, 93%. ES-MS: m/z; 377.2 [M + H] . H NMR 1 min) and then treated again with the appropriate amine
400 MHz, CDCl ) δ: 8.10 (1H, br s, NH), 7.71 (1H, t, J 4.7 Hz, under the same conditions reported above. This cycle of reac-
1
8
19
2
2
+
1
(
3
o-Ar–H), 7.46–7.44 (11H, br signals, overlapping, m-Ar–H and tions was iterated until the target linear trimer was obtained.
CH –Ph–H), 7.12 (1H, d, J 4.7 Hz, p-Ar–H), 5.12 (2H, s, CH – The cleavage process was performed by treating the resin, pre-
2
2
Ph), 5.08 (2H, s, CH
2
–Ph), 3.30 (2H, m, CvO–NH–CH
–CH –NH ), 0.85 (2H, br s, NH ).
N-(Aminobutyl)-2,3-bis(benzyloxy)benzamide (8b), light shaker platform at room temperature for 30 min each time.
2
), 2.65 viously washed with CH
2
Cl
2
(6 × 1 min), three times with a
(20% v/v, 3.00 mL each time) on a
(
2H, t, J 6.1 Hz, CH
2
2
2
2
solution of HFIP in CH
2
Cl
2
+
1
yellow oil, 2.40 g, 99%. ES-MS: m/z; 405.1 [M + H] . H NMR The resin was then filtered away and the combined filtrates
400 MHz, CDCl ) δ: 7.96 (1H, br s, NH), 7.74 (1H, dd, J 5.8, 3.7 were concentrated in vacuo. 1 mg of the final products was dis-
Hz, o-Ar–H), 7.48–7.35 (11H, br signals, overlapping, m-Ar–H solved in 60 μL of acetonitrile (0.1% TFA) and 60 μL of HPLC
and CH –Ph–H), 7.15 (1H, d, J 3.7 Hz, p-Ar–H), 5.16 (2H, s, grade water (0.1% TFA) and analyzed by RP-HPLC; purity
CH –Ph), 5.08 (2H, s, CH –Ph), 3.28 (2H, m, CvO–NH–CH ), >80%; conditions: 5 → 100% A in 30 min (A, 0.1% TFA in
.60 (2H, t, J 6.3 Hz, CH –CH –NH ), 1.35 (4H, m, CvO–NH– acetonitrile, B, 0.1% TFA in water); flow: 1.0 mL min
(
3
2
2
2
2
−
1
2
,
2
2
2
CH
2
–CH
2
–CH
2
).
220 nm. The linear oligomers (isolated as amorphous solids)
N-(Aminohexyl)-2,3-bis(benzyloxy)benzamide (8c), light were subjected to HRMS and, subsequently, to the cyclization
+
yellow oil, 2.54 g, 100%. ES-MS: m/z; 433.6 [M + H] . HRMS reactions without further purification.
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Dalton Trans.

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Paper
9a): white amorphous solid, 0.558 g, 100%; t
Dalton Transactions
(
R
: 15.0 min. (3H, m, CvO–NH–CHH), 3.54 (3H, d, J 15.5 Hz, CvO–CHH–
+
ES-MS m/z; HRMS (MALDI-FTICR) m/z; [M + H] calcd for N), 3.27–3.20 (9H, overlapping m, CvO–NH–CHH and N–CH2–
+
C
75
H
(
75
N
6
O
13 1267.5387; found 1267.5375.
CH
2
–CH
2
–CH
2
–NH), 1.47 (6H, m, N–CH
2
–CH
2
–CH
–NH). C NMR (150 MHz,
ES-MS m/z; HRMS (MALDI-FTICR) m/z; [M + H] calcd for CDCl ) δ: 167.2 ×3 (CvO–N), 165.3 ×3 (CvO–NH), 151.7 ×3 (m-
2 2
–CH –NH),
1
3
9b): white amorphous solid, 0.578 g, 97%; t
R
: 20.0 min. 1.27 (6H, m, N–CH –CH –CH –CH
2 2 2 2
+
3
+
C
81
H
87
N
6
O
13 1351.6326; found 1351.6390.
2
C–OBn), 146.8 ×3 (o-C–OBn), 136.4 ×6 (O–CH –C), 128.7 ×3 (C–
(
9c): white amorphous solid, 0.531 g, 84%; t
R
: 19.3 min. Ph), 128.2 ×12 (C–Ph), 127.7 ×3 (C–Ph), 127.2 ×12 (C–Ph), 124.4
+
ES-MS m/z; HRMS (MALDI-FTICR) m/z; [M + H] calcd for ×3 (m-C–Ar), 123.2 ×3 (o-C–Ar), 117.0 ×3 (p-C–Ar), 76.4 ×3 (O–
+
C
87
H
99
N
6
O
13 1435.7265; found 1435.7241.
Cyclization procedure: synthesis of the cyclic peptoids 10a– (CvO–NH–CH
c. The solutions of the linear peptoids (0.300 mmol), pre- CH –CH –NH), 23.8 ×3 (N–CH –CH –CH –CH –NH).
CH
2
–Ph), 71.3 ×3 (O–CH
2
–Ph), 48.9 ×3 (CvO–CH
2
–N), 46.5 ×3
2
), 38.9 ×3 (CvO–N–CH
2
), 26.6 ×3 (N–CH
2
–CH –
2
2
2
2
2
2
2
viously co-evaporated three times with toluene, were prepared
under nitrogen in dry DMF (15.0 mL). The mixture was added HRMS (MALDI-FTICR) m/z; [M + H] calcd for C87
dropwise to a stirred solution of HATU (0.456 g, 1.20 mmol) 1417.7159; found 1417.7185. H NMR (600 MHz, CDCl ) δ:
R
(10c): white amorphous solid, 0.0510 g, 12%; t : 22.1 min.
+
+
97 6 12
H N O
1
3
and DIPEA (324 μL, 1.86 mmol) in dry DMF (85.0 mL) by using 7.90 (3H, br t, NH), 7.72 (3H, dd, J 5.4, 4.2 Hz, o-Ar–H),
2
a syringe pump in 6 h, at room temperature in an anhydrous 7.47–7.46 (6H, overlapping, o-CH –Ph–H), 7.41–7.33 (21H, over-
atmosphere. After 12 h the resulting mixture was concentrated lapping, m-Ar–H and m-CH –Ph–H), 7.14–7.13 (9H, overlapping
2
in vacuo, diluted with CH
with a solution of HCl (1.00 M, 30.0 mL). The organic phase (6H, s, CH
was washed with water (60.0 mL), dried over anhydrous (3H, m, CvO–NH–CHH), 3.56 (3H, d, J 15.3 Hz, CvO–CHH–
2
Cl
2
2 2
(60.0 mL), and washed twice signals, p-Ar–H and p-CH –Ph–H), 5.15 (6H, s, CH –Ph), 5.07
2
–Ph), 4.66 (3H, d, J 15.3 Hz, CvO–CHH–N), 3.80
MgSO
4
, filtered and concentrated in vacuo. The crude cyclic N), 3.26–3.19 (9H, overlapping m, CvO–NH–CHH and N–CH
–(CH –CH –CH –NH), 1.47 (6H, m, N–CH –CH –(CH
2
2
–
–
peptoids were purified using flash silica gel; conditions: 1 : 1 CH
2
2
)
2
2
2
2
2
2
)
petroleum ether : ethyl acetate, then 100% ethyl acetate, and, CH –CH –NH), 1.33–1.20 (18H, overlapping m, N–CH –CH –
2
2
2
2
1
3
finally, 90 : 10 ethyl acetate : methanol. The cyclic peptoids (CH
2
)
2
2 2 3
–CH –CH –NH). C NMR (150 MHz, CDCl ) δ: 167.0 ×3
were dissolved in 50% acetonitrile in HPLC grade water and (CvO–N), 165.0 ×3 (CvO–NH), 151.7 ×3 (m-C–OBn), 146.8 ×3
analysed by RP-HPLC; purity >90% conditions: 5%–100% A in (o-C–OBn), 136.5 ×6 (O–CH –C), 128.7 ×15 (C–Ph), 127.7 ×3 (C–
2
3
0 min (A, 0.1% TFA in acetonitrile, B, 0.1% TFA in water); Ph), 127.2 ×12 (C–Ph), 124.4 ×3 (m-C–Ar), 123.4 ×3 (o-C–Ar),
−
1
1
2 2
flow: 1 mL min , 220 nm, subsequently characterized via H- 117.0 ×3 (p-C–Ar), 76.4 ×3 (O–CH –Ph), 71.4 ×3 (O–CH –Ph),
and C-NMR spectroscopy and HRMS (MALDI-FTICR).
1
3
49.0 ×3 (CvO–CH –N), 47.1 ×3 (CvO–NH–CH ), 39.4 ×3
2
2
(
10a): white amorphous solid, 0.0900 g, 24%; t : 20.2 min. (CvO–N–CH
R
2
), 29.1 ×3 (N–CH
2
–CH
–CH
2
–(CH
2
)
2
–CH
2
–CH
2
–NH),
+
HRMS (MALDI-FTICR) m/z; ([M
+
H] 49) calcd for 26.6 ×3 (N–CH
2
–CH
2
–(CH
2
)
2
–CH
2
2 2
–NH), 26.4 ×3 (N–CH –
+
+
C H N O
1249.5281; found 1249.5296; ([M + Na] 51) CH –(CH ) –CH –CH –NH), 26.3 ×3 (N–CH –CH –(CH ) –
7
5
73
6
12
2
2 2
2
2
2
2
2 2
+
1
calcd for C75
H
72
N
6
NaO12 1271.5100; found 1249.5113.
) δ: 7.85 (3H, br t, NH), 7.60 (3H, dd, J
.5, 2.9 Hz, o-Ar–H), 7.46–7.45 (6H, overlapping, o-CH –Ph–H), a solution of cyclic peptoids 10a–c (0.0300 mmol), in ethyl
H
CH
2
–CH
2
–NH).
NMR (600 MHz, CDCl
3
Hydrogenation procedure: synthesis of the ligands 3a–c. To
6
7
7
5
2
.40–7.30 (21H, overlapping, m-Ar–H and m-CH
.11–7.10 (9H, overlapping signals, p-Ar–H and p-CH
.13 (6H, s, CH –Ph), 5.07 (6H, s, CH –Ph), 4.17 (3H, d, J 15.6 cycles of vacuum-hydrogen were performed, and the reaction
2
2
–Ph–H), acetate (0.430 mL), Pd 10% wt on carbon was added (half of
–Ph–H), the weight with respect to the cyclic peptoid substrate). Three
2
2
Hz, CvO–CHH–N), 3.88 (3H, m, CvO–NH–CHH), 3.62 (3H, m, was stirred for 5 hours. After 5 hours, 0.860 mL of ethanol was
CvO–NH–CHH), 3.47 (3H, d, J 15.6 Hz, CvO–CHH–N), added, and three more vacuum-hydrogen cycles were per-
13
3
.27–3.23 (6H, overlapping m, N–CH –CH –NH). C NMR formed. The reaction mixture was stirred for an additional
2 2
(
150 MHz, CDCl
3
) δ: 167.7 ×3 (CvO–N), 165.8 ×3 (CvO–NH), 19 hours, then the completion of the reaction was assessed via
1
1
×
51.8 ×3 (m-C–OBn), 146.7 ×3 (o-C–OBn), 136.6 ×3 (O–CH –C), TLC. The reaction mixture was filtered with ethanol using a
2
36.4 ×3 (O–CH –C), 128.9 ×3 (C–Ph), 128.6 ×12 (C–Ph), 128.2 Celite pad and extensively dried in vacuo to obtain the final
2
3 (C–Ph), 127.6 ×12 (C–Ph), 124.3 ×3 (m-C–Ar), 122.8 ×3 (o-C– products. The cyclic peptoids were dissolved in 50% aceto-
Ar), 116.9 ×3 (p-C–Ar), 76.2 ×3 (O–CH
2
–Ph), 71.2 ×3 (O–CH
2
–
nitrile in HPLC grade water and analysed by RP-HPLC; purity
Ph), 48.7 ×3 (CvO–CH –N), 46.1 ×3 (CvO–NH–CH ), 36.7 ×3 >90% conditions: 5%–100% A in 30 min (A, 0.1% TFA in aceto-
2
2
−
1
(
CvO–N–CH
(
2
).
nitrile, B, 0.1% TFA in water); flow: 1 mL min , 220 nm, sub-
1
13
10b): white amorphous solid, 0.0960 g, 24%; t : 18.4 min. sequently characterized via H- and C-NMR spectroscopy and
HRMS (MALDI-FTICR) m/z; [M + H] calcd for C H N O
333.6220; found 1333.6246. H NMR (600 MHz, CDCl
.99 (3H, br t, NH), 7.69 (3H, dd, J 7.6, 1.8 Hz, o-Ar–H), HRMS (MALDI-FTICR) m/z; [M
R
+
+
HRMS (MALDI-FTICR).
8
1
85
6
12
1
1
7
7
3
) δ:
(3a): white amorphous solid, 0.0210 g, 100%; t
R
: 9.9 min.
calcd for
+
+
Na]
+
1
.46–7.45 (6H, overlapping, o-CH –Ph–H) 7.40–7.33 (21H, over- C H N NaO
12
731.2283; found 731.2293.
OD) δ: 7.12 (3H, dd, J 8.2, 1.1 Hz, o-Ar–H), 6.90
–Ph), 5.08 (3H, dd, J 8.2, 1.1 Hz, m-Ar–H), 6.68 (3H, t, J 8.1 Hz, p-Ar–H),
6H, s, CH –Ph), 4.63 (3H, d, J 15.5 Hz, CvO–CHH–N), 3.76 5.13 (3H, d, J 15.8 Hz, CvO–CHH–N), 3.86 (3H, m, CvO–NH–
H
NMR
2
33 36
6
lapping, m-Ar–H and m-CH
2
–Ph–H), 7.13–7.08 (9H, overlapping (600 MHz, CD
–Ph–H), 5.14 (6H, s, CH
3
signals, p-Ar–H and p-CH
2
2
(
2
Dalton Trans.
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3
+
CHH), 3.69 (3H, d, J 15.8 Hz, CvO–CHH–N), 3.61–3.57 (9H, valent of the Fe ion each time. For MS analysis of the titrated
1
3
overlapping m, CvO–NH–CHH and N–CH –CH –NH).
C
acetonitrile solution see Fig. S16.†
2
2
NMR (150 MHz, CD
3
OD) δ: 171.8 ×3 (CvO–N), 170.7 ×3
Dissociation constant calculations
(
(
CvO–NH), 150.4 ×3 (o-C–OH), 147.3 ×3 (m-C–OH), 119.7 ×3
p-C–Ar), 119.6 ×3 (m-C–Ar), 118.7 ×3 (o-C–Ar), 116.6 ×3 (CvO– The dissociation constants for Fe with peptoids 3a–c were
3
+
C–Ar), 51.0 ×3 (CvO–CH
CvO–N–CH ).
2
–N), 48.3 ×3 (CvO–NH–CH
2
), 38.0 ×3 measured by using UV-Vis spectroscopy following a compe-
(
2
tition method. The stock solutions of peptoids 3a–c, EDTA and
3
+
(3b): white amorphous solid, 0.0240 g, 100%; t : 11.5 min. Fe were prepared at 5 mM concentration in methanol. For
R
+
+
HRMS (MALDI-FTICR) m/z; [M + H] calcd for C39
H
49
N
6
O
12
EDTA, the solvent was water and the pH value was maintained
1
33,34
7
93.3403; found 793.3389. H NMR (600 MHz, CD
3
OD) δ: 7.21 at 7. In a competition experiment,
peptoids (3a–c individu-
(
3H, dd, J 8.1, 1.1 Hz, o-Ar–H), 6.91 (3H, dd, J 8.1, 1.1 Hz, ally) and EDTA were taken in a 1 : 1 ratio at 16.61 μM and gradu-
3
+
m-Ar–H), 6.70 (3H, t, J 8.0 Hz, p-Ar–H), 5.11 (3H, d, J 15.6 Hz, ally titrated with Fe up to two equivalents. The UV-Vis spectra
CvO–CHH–N), 3.65 (3H, m, CvO–NH–CHH), 3.62 (3H, d, J were monitored in the 400–800 nm range. Following the
1
5.6 Hz, CvO–CHH–N), 3.45–3.35 (9H, overlapping m, CvO– method reported by Wedd and Xiao et al., the slope between
NH–CHH and N–CH
overlapping m, N–CH
2
–CH
2
–CH
2
–CH
2
–NH), 1.67–1.53 (12H, ([BS1 site]total/[FeBS1]) − 1 and ([EDTA]total/[Fe-EDTA]) − 1 was
1
3
2
–CH
2
–CH
2
–CH
2
–NH).
C
a(Fe-EDTA) (EDTA)
NMR calculated. The slope equals to Kd(Fe-Peptoid)K α
(150 MHz, CD OD) δ: 171.6 ×3 (CvO–N), 170.1 ×3 (CvO–NH), where Kd(Fe-Peptoid) is the dissociation constant of Fe-peptoid,
3
1
50.3 ×3 (o-C–OH), 147.3 ×3 (m-C–OH), 119.6 ×6 (p-C–Ar and
Ka(Fe-EDTA) is the association constant of Fe-EDTA complexation
m-C–Ar), 118.6 ×3 (o-C–Ar), 116.8 ×3 (CvO–C–Ar), 50.8 ×3 and α(EDTA) is the pH correction factor for EDTA (see the ESI†).
(
CvO–CH –N), 40.0 ×3 (CvO–NH–CH ), 27.6 ×3 (CvO–N–
2
2
CH
2
), 25.1 ×6 (N–CH
2
–CH
2
–CH
2
–CH
2
–NH).
(
3c): white amorphous solid, 0.0260 g, 100%; t
R
: 14.0 min. Conflicts of interest
+
+
HRMS (MALDI-FTICR) m/z; [M + H] calcd for C H N O
77.4342; found 877.4353. H NMR (600 MHz, CD
4
5
61
6
12
1
There are no conflicts to declare.
8
3
OD) δ: 7.21
(
(
3H, d, J 7.8 Hz, o-Ar–H), 6.92 (3H, d, J 7.8 Hz, m-Ar–H), 6.70
3H, t, J 7.8 Hz, p-Ar–H), 5.08 (3H, d, J 15.6 Hz, CvO–CHH–N),
Acknowledgements
3
.65 (3H, m, CvO–NH–CHH), 3.58 (3H, d, J 15.6 Hz, CvO–
CHH–N), 3.40–3.36 (9H, overlapping m, CvO–NH–CHH and
N–CH –CH –(CH ) –CH –CH –NH), 1.63–1.56 (12H, overlap-
PG is thankful to Dr Zhiguang Xiao from The Florey Institute
of Neuroscience and Mental Health, Australia for many helpful
discussions and suggestions. PG gratefully acknowledges the
Lady Davis Fellowship Trust, Technion-Israel Institute of
Technology for a postdoctoral fellowship. Financial support
from the University of Salerno (FARB) is acknowledged. We
thank Dr Patrizia Iannece, University of Salerno, for HR-MS,
and Dr Patrizia Oliva, University of Salerno, for NMR spectra.
2
2
2 2
2
2
ping m, N–CH
CH –CH –CH
CH –CH –(CH ) –CH –CH –NH). C NMR (150 MHz, CD OD)
2
–CH
2
–(CH
2
)
2
–CH
2 2 2 2
–CH –NH and N–CH –CH –
(
2
)
2
2
2
–NH), 1.42–1.29 (12H, overlapping m, N–
1
3
2
2
2 2
2
2
3
δ: 171.4 ×3 (CvO–N), 170.1 ×3 (CvO–NH), 150.3 ×3 (o-C–OH),
47.3 ×3 (m-C–OH), 119.6 ×6 (p-C–Ar and m-C–Ar), 118.7 ×3 (o-
C–Ar), 116.9 ×3 (CvO–C–Ar), 50.8 ×3 (CvO–CH –N), 40.3 ×3
1
2
(
(
CvO–NH–CH
2
), 30.2 ×3 (CvO–N–CH
2
), 27.6 ×3 (N–CH
–CH –(CH
2
–CH
–CH
2
–
–
CH –CH –CH
2
)
2
2
2
–NH), 27.5 ×3 (N–CH
2
2
2
)
2
2
CH –NH), 27.4 ×6 (N–CH –CH –(CH ) –CH –CH –NH).
2
2
2
2 2
2
2
Notes and references
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2
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UV-Vis titrations
The titration experiments of peptoids 3a–c with metal ions
2
+
2+
2+
2+
2+
3+
(
Co , Cu , Ni , Zn , Fe and Fe ) were carried out in
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Fe in acetonitrile. In particular, 5 μL of peptoid 3a (5 mM
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