A general concept for the introduction of hydroxamic acids into polymers

Article abstract of DOI:10.1039/c9sc02557j

Hydroxamic acids (HA) form stable complexes with a large variety of metal-ions, affording hydroxamates with high complexation constants. Hydroxamic acid moieties play a crucial role in the natural iron metabolism. In this work, 1,4,2-dioxazoles linked to a hydroxyl group are introduced as key compounds for the installation of hydroxamic acids at synthetic polymers in well-defined positions. A general synthetic scheme is developed that gives access to a series of novel functional key building blocks that can be universally used to obtain hydroxamic acid-based monomers and polymers, for instance as protected HA-functional initiators or for the synthesis of a variety of novel HA-based monomers, such as epoxides or methacrylates. To demonstrate the excellent stability of the dioxazole-protected hydroxamic acids, direct incorporation of the dioxazole-protected hydroxamic acids into polyethers is demonstrated via oxyanionic polymerization. Convenient subsequent deprotection is feasible under mild acidic conditions. α-Functional HA-polyethers, i.e. poly ethylene glycol, polypropylene glycol and polyglycerol based on ethylene oxide, propylene oxide and ethoxy ethyl glycidyl ether, respectively are prepared with low dispersities (<1.2) in the molecular weight range of 1000 to 8500 g mol^-1. Water-soluble hydroxamic acid functional poly(ethylene glycol) (HA-PEG) is explored for a variety of biomedical applications and surface coating. Complexation of Fe(iii) ions, coating of various metal surfaces, enabling e.g., solubilization of FeO_x nanoparticles by HA-PEGs, are presented. No impact of the polyether chain on the chelation properties was observed, while significantly lower anti-proliferative effects were observed than for deferoxamine. HA-PEGs show the same complexation behavior as their low molecular weight counterparts. Hydroxamic acid functional polymers are proposed as an oxidatively stable alternative to the highly established catechol-based systems.

Full text of DOI:10.1039/c9sc02557j

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A general concept for the introduction of
hydroxamic acids into polymers†
Cite this: DOI: 10.1039/c9sc02557j
a
Tobias Johann,a Jennifer Keth,a Matthias Brosb and Holger Frey
*
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Hydroxamic acids (HA) form stable complexes with a large variety of metal-ions, affording hydroxamates
with high complexation constants. Hydroxamic acid moieties play a crucial role in the natural iron
metabolism. In this work, 1,4,2-dioxazoles linked to a hydroxyl group are introduced as key compounds
for the installation of hydroxamic acids at synthetic polymers in well-defined positions. A general
synthetic scheme is developed that gives access to a series of novel functional key building blocks that
can be universally used to obtain hydroxamic acid-based monomers and polymers, for instance as
protected HA-functional initiators or for the synthesis of a variety of novel HA-based monomers, such as
epoxides or methacrylates. To demonstrate the excellent stability of the dioxazole-protected hydroxamic
acids, direct incorporation of the dioxazole-protected hydroxamic acids into polyethers is demonstrated
via oxyanionic polymerization. Convenient subsequent deprotection is feasible under mild acidic
conditions. a-Functional HA-polyethers, i.e. poly ethylene glycol, polypropylene glycol and polyglycerol
based on ethylene oxide, propylene oxide and ethoxy ethyl glycidyl ether, respectively are prepared with
low dispersities (<1.2) in the molecular weight range of 1000 to 8500 g molꢀ1. Water-soluble hydroxamic
acid functional poly(ethylene glycol) (HA-PEG) is explored for a variety of biomedical applications and
surface coating. Complexation of Fe(III) ions, coating of various metal surfaces, enabling e.g.,
solubilization of FeOx nanoparticles by HA-PEGs, are presented. No impact of the polyether chain on the
chelation properties was observed, while significantly lower anti-proliferative effects were observed than
for deferoxamine. HA-PEGs show the same complexation behavior as their low molecular weight
counterparts. Hydroxamic acid functional polymers are proposed as an oxidatively stable alternative to
the highly established catechol-based systems.
Received 26th May 2019
Accepted 4th June 2019
DOI: 10.1039/c9sc02557j
rsc.li/chemical-science
used for the stabilization of MnO nanoparticles in solution. Via
a tailored catechol epoxide monomer, multiple units were
Introduction
introduced along the polyether backbone enabling gelation
upon iron(III) addition.6 However, most catechols are unstable
under aqueous conditions and are known to crosslink via
oxidative coupling. This irreversible reaction leads to highly
toxic Michael systems. Even though no Michael adduct of
amines and the quinone structure was found in studies of
Deming et al., the quinone structures lead to cross-linked
materials, impeding the reversibility of the formed
complexes.4 Nevertheless, catechols are ubiquitous in nature as
one of two moieties to form so called siderophores (greek: “iron
carrier”). The second prominent class of siderophores relies on
hydroxamic acids. These N-hydroxylated amides are capable of
forming stable complexes with a large variety of metal-ions,
affording hydroxamates with complexation constants in the
range of log K ¼ 28 for tris-complexes to log K ¼ 20 for bis-
complexes.7,8 Their binding affinity to metals is generally 7
magnitudes higher compared to carboxylic acids due to the
formation of a ve membered ring.9 In nature this class of
siderophores is essential to solubilize water-insoluble iron
oxide, thus increasing the bioavailability of this key element.10
In the last two decades, biomimetic materials with chelating
and interface-adhesive moieties, such as catechols or their
derivatives, have been investigated intensely.1,2 Particularly
catechol-bearing polymers that mimic mussel-foot proteins
containing dopamine have attracted broad interest, as their
outstanding adhesive properties enable strong interaction with
almost every surface.1,3,4 Numerous applications like self-
healing hydrogels, surface coatings for anti-fouling purposes
or the formation of supramolecular networks, especially by
combination of chelating moieties with polymers, have been
demonstrated.5 In two of our previous works we focused on the
direct incorporation of catechols into polyethers via anionic
polymerization. Polymers bearing a single catechol moiety were
aInstitute of Organic Chemistry, Johannes Gutenberg University, Duesbergweg 10-14,
55128 Mainz, Germany. E-mail: hfrey@uni-mainz.de
bDepartment of Dermatology, University Medical Center of the Johannes Gutenberg
University Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany
† Electronic supplementary information (ESI) available: NMR, IR, SEC, LCST and
mass spectroscopy characterization, cell toxicity and proliferation assay data. See
DOI: 10.1039/c9sc02557j
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More than 42 different metal-hydroxamate complexes including are amenable to both radical and anionic polymerization.
(but not limited to) Fe(III), Mn(II), Zn(II), Cu(II) and Ni(II) are Polymers from epoxide and methacrylate based monomers
currently known.7,8,11 In contrast to catechols, hydroxamic acids utilizing the 1,4,2-dioxazole group to introduce multiple
are stable under physiological conditions and neither undergo hydroxamic acids along the polymer backbone are currently in
oxidation nor crosslinking. They show similar pKs-values in the progress and will reported in forthcoming works. At the
range of 7–9.12
example of the harsh conditions of the anionic ring opening
While there are only few studies on hydroxamic acids in the polymerization, we demonstrate in the second part of this work
eld of synthetic polymers, they are well established in the the introduction of precisely one hydroxamic acid end group in
therapy of iron overload diseases or as potent inhibitors for polyethers, in particular in poly(ethylene glycol), aiming at
histone deacetylases that are mostly associated with cancer a narrow molecular weight distribution for medical application,
development.13 Early efforts to combine hydroxamic acids with combined with a well-dened polymer structure, enabling
polymers were patented in 1942 by Du Pont.14 First systematic investigation of the structure-related properties of hydroxamic
studies were reported by Winston et al. in the late 1970s. In acid functional polyethers. We aim at combining the chelating
these works, poly(hydroxamic acids) were synthesized by poly- properties of hydroxamic acids with PEG as the current “gold
mer modication reaction of poly(methacrylate)-based active standard” of biocompatible, water-soluble polymers.26 Based on
esters with hydroxylamine derivatives.15,16
this work we propose hydroxamic acids as an oxidation-stable
A variety of applications was proposed for polymers con- alternative to catechols in the eld of materials science.
taining hydroxamic acids. Due to their strong complexation of
metal-ions the most prominent application is their use as ion-
Results and discussion
A. Molecular construction kit for hydroxamic acid synthons
exchange resins or for waste-water treatment.17–19 In other
works, poly(hydroxamic acid)s were used for the separation of
rare earth metals.17,19 Furthermore, application of poly(-
based on 1,4,2-dioxazole protected functional structures
hydroxamic acid)s as starting material for peptide synthesis and For the introduction of hydroxamic acids to polymers we
as resin-bound acryl transfer reagent was reported.20 We initially screened various polymer modication approaches.
emphasize that in all published reports the hydroxamic acid Typically hydroxamic acids are introduced at the end of
containing polymers were synthesized via polymer modication a synthetic sequence, e.g. at a polymer structure by reaction with
(post-polymerization) of carbonyl compounds, such as esters or hydroxylamine or its protected derivatives, leading to incom-
amides with hydroxylamine. However, this approach inevitably plete functionalization with many side products. This strategy
leads to limited conversion due to the harsh reaction condi- also lacks general applicability.22,27 Hence, for the introduction
tions, which is an obstacle for many applications, particularly of hydroxamic acids into polymers, we decided to avoid post-
for medical purposes.21 Nome and coworkers showed that the polymerization processes with hydroxylamine as a common
conversion of polyamides to hydroxamic acids by reaction with option for functionalization, as they are restricted to a partic-
hydroxylamine leads to an alternating copolymer with both ular polymer system and thus limit the universal character for
carboxylic and hydroxamic acids due to neighboring effects.22 In further applications. To establish functional groups at well-
recent works Kizhakkedathu et al. reported conjugates of dened sites during chain-growth polymerization either (i)
hyperbranched polyethers with deferoxamine (DFO, Desferal), functional initiators (ii) functional monomers or (iii) functional
a biologically produced tris-hydroxamic acid used for the termination reagents can be employed. Protected hydroxyl
therapy of iron overload diseases.23 These modied polyethers functional hydroxamic acids were developed for this approach,
improved the plasma half time of deferoxamine 500-fold. This featuring both a protected hydroxamic acid to ensure stability
enhancement in pharmacokinetic properties impressively during polymerization and a hydroxyl group for further trans-
demonstrates the potential of hydroxamic acid functional pol- formation or use as initiators or monomers.
yethers. Besides the medical use of hydroxamic acids for the
The hydroxyl moiety can be conveniently transformed to
therapy of iron overload diseases, structures analogous to bio- methacrylate monomers and epoxide monomers (Scheme 2),
logical siderophores like desferrichrome containing multiple for instance glycidyl ethers. Additionally, the hydroxyl group can
hydroxamic acids have been investigated as potent Zr(IV) directly be used in anionic ring opening polymerization (AROP)
chelators for PET imaging.24 Radical polymerization of as an initiator for epoxide or lactide polymerization, thus
hydroxamic acid functional methacrylates was shown to creating a platform for both radical as well as anionic poly-
proceed with incomplete conversion due to radical transfer to merization (Scheme 1). In this work we place the focus on the
the hydroxamic acid moiety.25 Hence, the attachment of use of hydroxyl functional initiators for AROP of common
hydroxamic acids to synthetic polymer architectures via poly- epoxides (EO, PO, glycidyl ethers) to combine the hydroxamic
mer modication or direct radical polymerization represents acids with medically relevant polymers. Multifunctional poly-
a challenge, and new strategies based on HA-based building ethers from hydroxamic acid functional epoxides, as well as
blocks are required.
methacrylate based HA copolymers are currently in progress
In this work we describe a systematic approach for the and will be reported in forthcoming works.
introduction of hydroxamic acids to polymers via tailored
Selection and optimization process of the protected
hydroxyl functional 1,4,2-dioxazoles. The concept presented can hydroxamic acid initiator. Based on the abovementioned
be applied to different classes of monomers and initiators that prerequisites, we focused on (i) the selection of suitable
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for facile conversion to 1,4,2-dioxazoles due to the close prox-
imity of the hydroxyl group in ortho position to the hydroxamic
acid moiety, prohibiting the synthesis via scalable processes.28
Hence, N,4-dihydroxybenzamide (2a) was chosen as a rst
candidate for the preparation of the desired initiators. Even
though N,4-dihydroxybenzamide (2a) is available at specialized
chemical distributors, albeit its high price impedes larger scale
synthesis of the 1,4,2-dioxazole. The preparation of 2a is known
in literature, but typically multistep syntheses or costly pro-
tected hydroxylamine derivatives are employed.29
To maintain a scalable process, we focused on preparation
methods for hydroxamic acids based on esters and hydroxyl-
amine, due to the absence of protecting group chemistry,
excellent commercial availability and the preparation in one
step. Hence, para-methyl paraben was chosen as a starting
material for the synthesis of 2a via nucleophilic cleavage of the
ester bond by hydroxylamine (See ESI Fig. S5a–f† for NMR and
IR characterization data). Unfortunately, all attempts to
synthesize the corresponding hydroxamic acid led to low yields
with many side products and thus troublesome purication.
This is attributed to the deprotonation of the phenolic hydroxyl
group, leading to a delocalized negative charge within the
aromatic system. Hence, nucleophilic attack by hydroxylamine
to form the hydroxamic acid is highly obstructed. To overcome
this issue, we introduced an ethylene glycol C2 spacer, which
enabled facile synthesis of the hydroxyl functional hydroxamic
acid (“HA-OH”, 1b) in high yields (77%) (ESI Fig. S3a–f†). Aim-
ing at a reduction of the necessary synthetic effort, the aromatic
hydroxamic acid (“benzylHA-OH”, 2a) was also prepared from the
commercially available ester precursor (ESI Fig. S7a–e†). Addi-
tionally lactones were explored for the preparation of aliphatic
derivatives (4a, see ESI Scheme S1† for further details), since
naturally occurring hydroxamic acid-based siderophores are
typically aliphatic structures.10 The preparation of hydroxamic
acids from lactones is known in literature.30 The conversion of
caprolactone to N,6-dihydroxyhexanamide (4a) (ESI Fig. S9a–f†)
and valerolactone to N,5-dihydroxypentanamide (Scheme S1†
compound 5) proceeded smoothly in moderate yields, but all
attempts to convert butyrolactone to N,4-dihydroxybutanamide
led to complex mixtures requiring excessive purication efforts.
In summary, the aromatic hydroxamic acids 1b and 3a, as well
the aliphatic hydroxamic acids 4a and 5 fullled the necessary
requirements for facile and large-scale synthesis and were
subsequently used for conversion to 1,4,2-dioxazoles.
Scheme 1 General concept for the introduction of hydroxamic acids
at polymers: the hydroxamic acid is protected in the 1,4,2-dioxazole
group via transketalization. The remaining free hydroxyl group can be
used as a starting point either for monomer synthesis (methacrylates,
epoxides) or as an initiator for anionic ring opening polymerization.
After acidic treatment the 1,4,2-dioxazole group is cleaved to release
the free hydroxamic acid for metal complexation.
functional hydroxamic acids and (ii) the optimization of
protection and deprotection procedures to ensure stability of
the hydroxamic acid during the polymerization process, while
maintaining the capability of mild and facile deprotection and
release of the functional moiety.
Choice of suitable hydroxyl functional hydroxamic acids. No
compound containing
a hydroxyl group combined with
a hydroxamic acid, except for salicylhydroxamic acid and N,4-
dihydroxybenzamide (Scheme 2, compound 2a), is currently
commercially available. Salicylhydroxamic acid is not suitable
Optimization of the protection and deprotection reaction.
The anionic ring opening polymerization of epoxides employs
both strong nucleophilic as well as harsh basic conditions.
Therefore, any carbonyl compound and protic groups have to be
protected. The 1,4,2-dioxazole group was chosen as a suitable
protection group, as it can be expected to withstand the harsh
conditions of AROP. Besides its inert character against strongly
Scheme 2 Preparation of hydroxyl functional hydroxamic acids, their
corresponding protected 1,4,2-dioxazole form and further trans-
formation to monomers (1d, 1e) developed in this work. See ESI basic conditions, the 1,4,2-dioxazole group has been reported to
Scheme S1† for additional reactions with C3 and C4 aliphatic spacers.
Conditions for the preparation of hydroxamic acids (1b, 2b, 3a, 4a):
NH2OH/KOH/MeOH or water, RT; conditions for the preparation of
1,4,2-dioxazoles: 2,2-dimethoxy propane or 2,2-diethoxy propane,
be stable against other nucleophiles, such as EtMgBr, oxidation
with KMnO4 or reduction by NaBH4. In this context, the four
hydroxamic acid derivatives 1b, 2a, 3a and 4a were converted to
the corresponding 1,4,2-dioxazoles (Scheme 2). This 1,4,2-
dioxazole group can be viewed as the acetonide derivative of
CSA, DCM, RT. For detailed synthesis description see experimental
section.
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a tautomeric form of 1b and is therefore designated “hydroxa-
mic acid acetonide” (HAA). As reported in a different context by
32
Couturier et al.31 and Geen and Frobose, the trans-
¨
ketalization of hydroxamic acids is unfortunately not straight-
forward. Varying amounts of side products, depending on the
substituent adjacent to the hydroxamic acid, is formed during
the reaction. In this side reaction O-acyl acetone oxime (see
Scheme 3I for an exemplary structure) is formed, which readily
reacts with nucleophiles such as alcohols to form esters. The
amount of side product is signicantly higher for aliphatic
hydroxamic acids (8 : 1 dioxazole : side product) compared to
aromatic derivatives (25–30 : 1 dioxazole : side product).31 For
evaluation purposes, all three aromatic hydroxamic acids (1b,
2a and 3a) were converted to the corresponding 1,4,2-dioxa-
zoles. As expected, the side reaction occurred in only negligible
amounts (ESI Fig. S4a–f, S6a–e and S8a–e†).
Surprisingly, the reaction starting from the aliphatic
hydroxamic acid 4a proceeded to form mainly caprolactone by
intramolecular ring closure of the O-acyl acetone oxime side
product (Scheme 2, see Fig. S10† for 1H NMR of 1,4,2-dioxazole,
aer purication still some caprolactone is le due to trouble-
some separation). Hence, intramolecular attack of the hydroxyl
group appears to enhance the side reaction, limiting the overall
yield (<23%) of the desired aliphatic 1,4,2-dioxazole derivative.
Attempts to prepare 4-(5,5-dimethyl-1,4,2-dioxazol-3-yl)butan-1-
ol from valerolactone via the hydroxamic acid 5 completely
failed and led to this side reaction exclusively (Scheme 4).
Considering the synthetic effort and low yields, the aliphatic
(4b) as well aromatic (2b) protected hydroxamic acid were not
further used for any polymerization. These ndings suggest that
a rigid spacer like the aromatic ring is benecial for the prep-
aration of hydroxyl functional 1,4,2-dioxazoles via
transketalization.
Scheme 4 Side reaction during transketalization of 5 leading to
valerolactone (I) via formation of O-acyl acetone oxime, subsequent
intramolecular ring closure.
scale-up to 20 g with 64% overall yield. We have to emphasize
that although the aliphatic initiator 4b was not used in this
work for further polymerization, aliphatic hydroxamic acids can
be installed in polyethers via this approach. Additionally, to
further demonstrate the wide applicability of this approach
HAA-OH (1b) was used as a starting material for the preparation
of an 1,4,2-dioxazol bearing glycidyl ether (1d) and a methacry-
late derivative (1e). The hydroxamic acid acetonide glycidyl
ether (HAAGE) was synthesized in high yields (81%) from 1b via
phase transfer catalysis in a one-step reaction (See ESI Fig. S11†
for NMR analysis). Methacrylate hydroxamic acid acetonide
(MAHAA) (1e) was obtained via Steglich esterication of 1b with
methacrylic acid (See ESI Fig. S12† for NMR analysis). Polymers
based on both monomers are currently in progress and will be
reported in a forthcoming work. To sum up, hydroxyl-functional
1,4,2-dioxazoles represent the key structure for the preparation
of monomers and polymers.
B. Polymer synthesis: hydroxamic acid functional polyethers
a-Functional polyethers were prepared from the two selected
functional initiators 1c, 3b to introduce one terminal hydroxa-
mic acid (HA) moiety. Although this limits the binding capacity
for metal ions, the use of a single complexing moiety inherently
prevents crosslinking via metal chelation, thus providing water-
soluble complexes in case of PEG. This requirement is essential
to avoid aggregation of coated nanoparticles or gelation.
Suitable hydroxamic acid initiators for the oxyanionic poly-
merization have to be both aprotic and base-stable and must
possess one hydroxyl group that can be used to form the cor-
responding alkoxide-initiator salt for the anionic ring opening
polymerization. Both aromatic protected hydroxamic acids (1c
“HAA-OH”, 3b “benzylHAA-OH”) full these requirements for
AROP and have been successfully used for the preparation of
functional polyethers (see below). For the different polymer
molecular weight series, 1c was selected due to methyl paraben
being a cheap and widely commercially available starting
material. We emphasize that this synthetic route employs only
inexpensive and commercially available chemicals, permitting
Scheme 5 Synthesis of HAA based polyethers (HAA-PEG, HAA-PPO,
HAA-PEEGE) and subsequent cleavage of the protecting group to
release the free hydroxamic acid functional polyethers (HA-PEG, HA-
PPO, HA-linPG).
Scheme 3 Side reaction during transketalization of 4a leading to
caprolactone (II) via formation of O-acyl acetone oxime (I) and
subsequent intramolecular ring closure.
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HAA-poly(ethylene glycol)s (HAA-PEGs) P1a, P2a with
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molecular weights in the range of 1000 to 8500 g molꢀ1 were
prepared via AROP of ethylene oxide (Scheme 5, Table 1) in THF/
DMSO. The preparation of polyethylene glycol based polymers
was chosen as PEG represents the gold standard polymer for
biomedical application. Also polymers based on propylene
oxide (PO) P3a, P5a and ethoxy ethyl glycidyl ether (EEGE) P4a,
P6a have been prepared to broaden the scope of materials and
to demonstrate general application of 1c. Polypropylene oxide
(PPO) was chosen as a hydrophobic polyether counterpart to
PEG. PPO exhibits a lower critical solution temperature (LCST),
enabling temperature dependent aqueous solubility below
20 ꢁC (for PPO50).26,33 One downside of linear polyethers is their
limited number of functional groups. In the case of functional
initiated polyethers like in this work, only the terminal OH
group can be addressed for further functionalization of the
polymers. To overcome this issue, poly(ethoxy ethyl glycidyl
ether) (PEEGE) polymers were prepared. EEGE is an acetal-
protected glycidol monomer. These hydrophobic PEEGE poly-
mers can easily be converted (by acidic treatment) to hydro-
philic linear polyglycerol (linPG), which exhibits one hydroxyl
group per repeating unit, enabling access to multi-functional
polyethers.33
In all cases full conversion was achieved within 24 h reaction
time. All HAA-PEG, HAA-PPO and HAA-PEEGE polymers exhibit
monomodal molecular weight distributions with narrow dis-
persities below 1.20 (Fig. 1, Table 1, ESI Fig. S1†). The degree of
polymerization was kept low (10–20) for both the PPO and
PEEGE polymers to limit this side reaction. Higher degrees of
polymerization are possible, based on these proof-of-principle
studies and further optimization. Purication of the polymers
was performed via partitioning between water and DCM to
obtain the salt-free, pure polymers in good yields (73–94%).
For all polymerizations conducted good agreement of target
molecular weights with molecular weights determined via NMR
end group analysis can be conrmed. Slight differences are
accounted to the normalization of the integrals to the initiator,
leading to uncertainties in the determination of approx. ꢂ250 g
Fig. 1 SEC traces of the prepared HAA-PEGs (DMF, PEG-calibration).
HA-PEGs after deprotection strongly interact with SEC column
material and hence cannot be analyzed via SEC. See Fig. S1† for SEC
traces of PPO and PEEGE as well benzylHAA-OH initiated polymers.
molꢀ1. The discrepancy between SEC and NMR molecular
weight can be explained by the PEG standards used for cali-
bration. Similar underestimation of molecular weights via SEC
have been reported for both PPO as well PEEGE polymers.33 In
case of PEEGE slight broadening of the molecular weight
distribution can be detected. This is attributed to the high
polymerization temperature of EEGE (80 ꢁC), increasing known
side reactions such as proton abstraction and elimination,
leading to overall broadening of the molecular weight
distribution.34
1H NMR analysis conrms (ESI Fig. S13–S18†) the successful
initiation of the polymers from either HAA-OH or benzylHAA-OH
respectively. This can further be veried by the excellent overlap
of UV and RI signal in SEC measurements (not shown). The
thermo-responsive behaviour of HAA-PPO21 was investigated in
deionized water. As expected, a cloud point at 12.9 ꢁC was
observable (ESI Fig. S2†), at which the aqueous solution turned
turbid and HAA-PPO21 precipitated. In summary, both hydroxyl-
functional protected hydroxamic acids have been proven to be
Table 1 Overview of the prepared protected hydroxamic acid functional polyethers
Polymer
Mn target [g molꢀ1
]
Mn (NMR)a [g molꢀ1
]
Mn (SEC)b [g molꢀ1
]
Đb
HAA-PEG21
HAA-PEG31
HAA-PEG41
HAA-PEG64
990
1160
1600
2040
3050
3760
5830
8520
2010
3510
1460
1430
1410
4180
1080
960
1.06
1.05
1.06
1.11
1.06
1.09
1.13
1.09
1.07
1.06
1.07
1.22
1.14
1.20
1470
1950
3010
3800
6180
8080
2200
4220
1400
1370
1700
4470
1670
1370
1850
2210
3380
4360
6010
1250
1810
1360
1310
860
HAA-PEG80
HAA-PEG127
HAA-PEG188
benzylHAA-PEG41
benzylHAA-PEG75
HAA-PPO21
benzylHAA-PPO21
HAA-PEEGE7
HAA-PEEGE26
benzylHAA-PEEGE6
1790
880
a
Determined via end group analysis in NMR. b DMF SEC, PEG standard.
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a viable initiator for the polymerization of various epoxide
monomers, leading to water-soluble hydrophilic PEG, thermo-
responsive PPO or multi-functional PEEGE polymers bearing
a terminal hydroxamic acid unit.
Cleavage of the protecting group and release of the free
hydroxamic acid moiety. In an optimized procedure, cleavage of
the protecting group was achieved by simple treatment of the
HAA-PEGs with 0.1 molar HCl. These acidic conditions lead to
degradation of the dioxazole structure, thus liberating the free
hydroxamic functional PEG (HA-PEG, P1b) in quantitative
yields. Compared to the reported deprotection scheme of
Couturier et al. this greatly reduces the necessary effort by
omitting the usage of ion-exchange resins.31 Successful cleavage
of the acetal protecting group can both be conrmed via 1H
NMR (Fig. 2, ESI Fig. S13b†) and mass spectra (Fig. 3). No signal
of the dioxazole methyl group was detected in the product.
Furthermore, both signals of the labile NH and OH-protons are
observed at 11.06 and 8.90 ppm (1H NMR) in DMSO-d6. The
polyether backbone remained unaltered.
ESI mass spectra reveal only the desired HAA-PEG and HA-
PEG species, respectively. Comparing both polymers,
a distinct shi of 40 g molꢀ1, corresponding to cleavage of the
protecting group, can be seen. Each spectrum shows only the
distribution of the polymers with 44.05 g molꢀ1 per repeating
unit. No sub-distribution can be detected (see ESI Fig. S19 and
S20† for full spectra).
Fig. 3 ESI-mass spectra of HAA-PEG31 (bottom) and HA-PEG31 (top).
simultaneously cleaved with other protecting groups reacting
under the same acidic conditions, like acetals without inter-
ference leading to multi-functional linPG bearing one
hydroxamic acid (Scheme 6). Considering the high stability of
the 1,4,2-dioxazole group under basic conditions, orthogonal
protecting group chemistry should also be possible for selective
release of the functional moieties.
Both protecting groups employed in the synthesis of HAA-
PEEGE (P6a) are acetals. Hence, the cleavage of both protect-
ing groups can be achieved simultaneously via acidic treatment.
Due to the hydrophobicity of PEEGE the cleavage is very slow in
aqueous media. For improved reaction kinetics, the cleavage
was performed in isopropanol solution with DOWEX 10WX8
acidic ion exchange resin, leading to HA-linPG (P6b) (Scheme 6).
Chelation characterization and demonstration of the appli-
cability of hydroxamic acid functional PEG
Chelation properties. Fundamental chelation properties of the
HA-PEGs were investigated, relying on UV-Vis spectroscopy of
the highly colored iron(III) complexes. As already reported by
Winston et al.16, at low iron(III) concentration the tris(hydrox-
amato)iron(III) complex is favored. When increasing the iron(III)
to HA-PEG ([Fe]/[HA-PEG]) ratio the tris-complex is consecu-
tively cleaved to form bis- and nally mono(hydroxamato)
iron(III) complexes (Scheme 7). Simultaneously, the color
changes from the red tris-complex (lmax ¼ 500 nm) to the purple
mono-complex (lmax ¼ 550 nm) (Fig. 5, ESI Fig. S21). This
distinct shi cannot be detected in case of chelating tris-
hydroxamic acids such as deferoxamine due to their highly
favored formation of tris-complexes.16
1
Full cleavage of the protecting groups is conrmed via H
NMR spectroscopy (Fig. 4). Aer treatment with DOWEX10WX8
in isopropanol all acetal signals of PEEGE (Fig. 4 yellow) as well
the methyl signals of the 1,4,2-dioxazole group at 1.6 ppm (Fig. 4
green) have vanished. The free hydroxamic acid can be
conrmed via the NH-signal appearing at 11 ppm (Fig. 4 blue,
top). In conclusion, the 1,4,2-dioxazole group can be
This behavior can also be veried by a steep increase in
absorbance in the course of iron(III) chloride addition, until
Scheme 6 Reaction scheme for cleavage of the protecting group in
Fig. 2 1H NMR analysis of HAA-PEG (bottom) and HA-PEG (top) after HAA-PEEGE releasing both the hydroxyl groups as well as the
cleavage of the protecting group.
hydroxamic acid moiety.
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Nanoparticle and surface modication. Besides binding of low
molecular iron, the coating of macroscopic surfaces or metal
oxide nanoparticles is another prominent application of poly-
meric chelators.1 As a proof of principle for the coating of
surfaces with HA-PEGs, solubilization of iron oxide nano-
particles (FeOx-NP) in water as well as enhancement of the
hydrophilicity of different metal surfaces were demonstrated
(ESI Fig. S22 and S23†). In all cases, the hydrophilicity of the
surfaces increased due to coating with HA-PEGs. By coating
FeOx-NP with HA-PEGs with a molecular weight of 1000, 3800
and 8500 g molꢀ1 the NP diameter was systematically increased
from 20 nm for the oleate-coated NP up to 50 nm in case of HA-
PEG with a molecular weight of 8500 g molꢀ1. Control experi-
ments with non-functionalized PEG resulted only in minor
changes in size within the error range of the uncoated NP
distribution (Fig. 6, see Table S1†).
Fig. 4 Stacked 1H NMR spectra of HAA-PEEGE7 (I) before cleavage of
the protecting groups and (II, top) HA-linPG7 as the reaction product.
Aer coating, the initially water-insoluble NPs were freely
soluble in water or methanol (ESI Fig. S21†). TEM images
prepared from water show the coated, non-agglomerated NPs
(Fig. 7). As expected, no differences in size or distribution in
comparison to the NP prior to coating can be detected.
Cell toxicity and proliferation assays. First, biocompatibility
tests were performed using primary human blood cells to
explore applicability of the polymers for the transport of
Fe(III) or other metal ions in medicine. In all cases HA-PEG as
well as their protected counterpart HAA-PEG showed no
signicant impact on the metabolic cell activity within the
range of tested concentrations, thus low to no toxicity can be
expected (ESI Fig. S24 and S26†). To further investigate the
shielding effect of the PEG chain, T-Cell proliferation studies
were performed.
Scheme 7 Equilibria of the formation of mono-, bis-, or tris(PEG-
hydroxamato)iron(III) complexes.
Commercially available hydroxamic acid-based drugs like
deferoxamine mesylate (Desferal, Novartis) are known to
strongly inhibit T-cell proliferation.35 In contrast HAA-PEG, as
well as HA-PEG with a MW of 8500 g molꢀ1 did not signicantly
inuence the proliferation of T-cells, while the low molecular
weight HA 1b showed dose-dependent inhibition of the prolif-
eration (Fig. 8, ESI Fig. S25†).
Fig. 5 Maximum absorbance (blue) and lmax (red) in dependency of
the [Fe]/[HA-PEG188] ratio. The vertical lines denote a ratio of 1 : 3 (tris
complex) and 1 : 1 (mono complex) respectively.
a ratio of 1 : 3 ([Fe]/[HA-PEG]) is achieved. With further
addition of iron(III) the absorbance increases until equi-
molarity is obtained. At this ratio all hydroxamic acid moie-
ties are saturated as mono-complexes, and thus no increase in
absorbance can be induced by further addition of iron(III).
This behavior suggests that there is no inuence of the poly-
mer chain on the chelation behavior of the hydroxamic acid
end group connected to the polyether backbone. Hence, the
chelation properties of HA-PEGs are comparable to low
molecular weight hydroxamic acids and are not altered due to
the conjugation with PEG.
Fig. 6 Intensity distribution of the FeOx nanoparticles by DLS.
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monomers and polymers, for instance as protected HA-
functional initiators or for the synthesis of a variety of novel
HA-based monomers, exemplied by epoxides and methac-
rylates in this work.
Relying on these novel synthons, hydroxamic acid functional
polyethers based on ethylene oxide, propylene oxide and ethoxy
ethyl glycidyl ether, respectively, were prepared to show the
general applicability of this approach, even under the harsh
conditions of the AROP. Aer mild acidic treatment of the
resulting polyethers, the free hydroxamic acid can be recovered.
Consequently, hydroxyl-functional 1,4,2-dioxazoles are broadly
applicable for the initiation of AROP or for preparation of HA-
based monomers by modication of the hydroxyl group with
polymerizable structures.
The HA-initiated polyethers were studied both with respect
to their future use in materials science and for medical
purposes, for instance in the treatment of diseases related to
the iron metabolism. No measureable difference in terms of
chelation properties of the hydroxamic acid functional poly-
ethers was detected, compared to low molecular weight
hydroxamic acids, while anti-proliferative effects of low
molecular weight hydroxamic acid therapeutics like deferox-
amine were highly reduced. The complexation of Fe(III) ions,
the coating of various metal surfaces as well as FeOx nano-
particles conrm outstanding chelation properties. From
these proof-of-principle studies and the basic synthons
established in this work, a vast variety of typical applications
known for other polymeric chelators, such as catechol-bearing
or terpyridine-bearing polymers become viable. The combi-
nation of hydroxamic acids with polymers is a promising
alternative to catechols with their low oxidative stability.
Hydroxamic acid based polyethers offer promise for iron-
depletion therapies, for medical purposes related to metal
ion transport in general as well as in materials science and for
surface coating.
Fig. 7 TEM image of non-modified (oleate coated) FeOx nanoparticles
(left, blue histogram, prepared from hexane) and FeOx nanoparticles
after coating with HA-PEG188 (right, red histogram, obtained from
aqueous solution).
Based on the general concept for protected hydroxamic acids
introduced in this work, a variety of novel monomers based on
acrylates, methacrylates and epoxides has been prepared and
polymerized. These structures and their use for both material
science and medicine will be reported in due course.
Experimental section
Materials
Fig. 8 T-Cell proliferation assay. Human PBMC (4 donors) were
stimulated with aCD3 and aCD28 antibodies to induce T-cell prolif-
eration in the presence of different test compounds.
Unless otherwise stated, all reagents were used without further
purication. All chemicals and solvents were purchased from
¨
Acros, Aldrich, Fisher Scientic, Fluka, Riedel-de-Haen or Roth.
Conclusions
Deuterated solvents were purchased from Deutero GmbH. THF
was dried via distillation over sodium/benzophenone. DMSO
1,4,2-Dioxazoles with an adjacent hydroxyl group have been
demonstrated to represent key structures for the direct
introduction of hydroxamic acids into polymers. These
structures permit to install hydroxamic acids at synthetic
polymers in a well-dened manner at the a-position. A
general synthetic scheme was developed that gives access to
a large variety of novel functional key building blocks that can
be universally used to obtain hydroxamic acid-based
˚
was dried using molecular sieve 4 A. Hydrophobic FeOx nano-
particles coated with oleic acid were provided by Jan Hilgert
(Group of Prof. Tremel, University of Mainz).
Abbreviations
Ethyl acetate (EA), petrol ether (PE), dichloromethane (DCM),
tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), ethylene
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oxide (EO), camphor sulfonic acid (CSA), methanol (MeOH), slides were removed from the solution, dried under air and
correlation spectroscopy (COSY), heteronuclear single-quantum rinsed with MilliQ-water to remove non-adhered polymer. The
correlation spectroscopy (HSQC), heteronuclear multiple-bond contact angle measurement was performed using the sessile
correlation spectroscopy (HMBC).
drop method by applying one drop of MilliQ water on the
surface and determining the apparent angle between liquid
and solid phase. Every measurement was performed ve
times.
Characterization techniques
NMR analysis. 1H NMR spectra at 300 MHz and 13C spectra
Transmission electron microscopy. TEM images were
at 75 MHz were recorded on a Bruker Avance III HD 300 (5 mm recorded with a FEI Tecnai 12 microscope equipped with a LaB6
1
ꢁ
BBFO-Head with z-gradient) at 23 C. H NMR spectra at 400 cathode (120 kV acceleration voltage, nominal magnication:
MHz and 13C spectra at 100 MHz were recorded on a Bruker 68 000; nominal underfocus: 0.5–1.5 mm). For sample prepa-
Avance III HD 400 (5 mm BBFO-Smartprobe with z-gradient) ration, carbon grids for TEM applications were negatively glow
at 23 ꢁC. The spectra are referenced internally to residual discharged at 25 mA for 30 s in an Emitech K100X glow
proton signal of the deuterated solvent. In the rare case of no discharge system. A droplet of the sample (5 ml) was placed on
detectable residual proton signal in 2D measurements, the the grid and incubated for 30 s. The grid was dried with
spectra are referenced to compound signals from 1D spectra. Whatman lter paper no. 4 by side-blotting.
All signals are assigned according to IUPAC nomenclature
Metabolic activity. Potential cytotoxic effects of the various
guidelines. For better clarity, the 2D spectra are annotated agents on peripheral blood mononuclear cells (PBMC) were
using lowercase letters for proton signals and capital letters monitored by assaying their metabolic activity. For this, PBMC
for carbon signals.
were isolated from blood donations of healthy volunteers by
Size exclusion chromatography. SEC chromatography wꢀas1 density gradient centrifugation using Ficoll as recommended
performed in DMF (1 mL minꢀ1, 50 ꢁC) containing 1g L
by the manufacturer (Sigma-Aldrich, Deisenhofen, Germany).
lithium bromide as an additive, using an Agilent 1100 series PBMC (107/mL) were seeded into wells of 96 well cluster plates
˚
SEC system including a HEMA 300/100/40 A column cascade, (100 ml per well), and agents were applied to triplicates at the
a UV (254 nm) and RI detector. Calibration was carried out concentrations indicated. Samples le untreated or incubated
using poly(ethylene oxide) (PEO) standards provided by Polymer with DMSO at cytotoxic dose (10%) were included as controls.
Standard Service (PSS).
On the next day, MTT substrate was applied as recommended
Mass spectrometry. Polymer mass spectra were recorded on by the manufacturer (Promega, Madison, WI). MTT reagent is
an Agilent 6545 QTOF mass spectrometer in electron spray reduced by mitochondrial dehydrogenases to a purple for-
ionization (ESI) mode. Sample concentration was 1 mg mLꢀ1 in mazan product. Absorbance was measured at 570 nm using
methanol. Mass spectra of low molecular weight compounds a EMax Plus microplate reader (Molecular Devices, San
´
were recorded using a Finnigan MAT 95 mass spectrometer in Jose, CA).
eld desorption (FD) mode.
Proliferation assays. To assess effects of the different agents
Ultraviolet-Visible spectroscopy. UV-Vis spectra were recor- on the proliferative capacity of T cells, PBMC (106/mL) were
ded on a JASCO V-630 spectrophotometer in the range of seeded into wells of 96 well cluster plates (100 ml per well). To
230 nm to 800 nm with 1 nm data interval. Scan speed was set to induce polyclonal T cell proliferation, wells were precoated
400 nm minꢀ1. All samples were prepared in methanol.
with mouse anti-human CD3 antibody (1 mg mLꢀ1; clone
Dynamic light scattering. DLS measurements were per- HIT3a), and mouse anti-human CD28 antibody (1 mg mLꢀ1
;
formed on a Zetasizer Nano ZS (Malvern Instruments) at an clone CD28.2) was added to PBMC prior to seeding (both
angle of 173ꢁ and a wavelength of 633 nm at 25 ꢁC. Three antibodies from Biolegend, San Diego, CA). In parallel
measurements were performed per sample. All DLS measure- settings, T cells were stimulated using phytohemagglutinin
ments were performed in DCM.
(PHA) isolated from Phaseolus vulgaris (Sigma-Aldrich) at
Infrared spectroscopy. FT-IR spectra were recorded using a nal concentration of 5 mg mLꢀ1. PBMC le unstimulated
a Nicolet iS10 FT-IR spectrometer (Thermo Fisher Scientic) in served as internal controls. The different agents were applied
the range of 500 to 3500 cmꢀ1
.
to triplicates at the concentrations indicated. T cell prolifer-
3
Lower critical solution temperature measurements. Cloud ation was assayed as genomic incorporation of H-thymidine
points were determined in deionized water at a concentration of (0.5 mCi per well) added on day 3 of culture. On the next day,
2.5 mg mLꢀ1 and observed by optical transmittance of a laser cells were harvested onto glass ber lters. Retained radio-
beam (670 nm). The measurement was recorded in a Tepper activity was detected using a ß counter (1205 Betaplate, LKB
turbidimeter TP1. Heating rate was 1 K minꢀ1. For determina- Wallac, Turcu, Finnland).
tion of the LCST the temperature at 50% of initial transmittance
was used.
Synthesis procedures of hydroxamic acids and 1,4,2-
dioxazoles
Contact angle measurements. Contact angle measurements
were performed aer cleaning the appropriate metal slide in
acetone by sonication for 1 h. Then each slide was put into
Synthesis of the hydroxamic acid functional initiator (HAA-
either solution of PEG44 as a reference or HA-PEG188 (each OH) employed as an initiator for polymerization and HAA
10 mg mLꢀ1 in DCM) overnight at room temperature. The monomer derivatives (1a–1e).
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HC3,5), 4.89 (t, J ¼ 5.5 Hz, 1H, HO–CH2), 4.03 (t, J ¼ 4.9 HZ, 2H,
CH2–O–Ar), 3.72 (dt, J ¼ 5.5, 4.9 Hz, 2H, HO–CH2–).
13C NMR (75 MHz, DMSO-d6) d [ppm] ¼ 164.02 C^O, 160.95
C4, 128.62 C2,6, 124.82 C1, 123.13 C3,5, 69.70 Ar–O–C, 59.48 HO–
C.
IR-ATR n [cmꢀ1] ¼ 3279 m, 2947 w, 2750 m br, 1638 m, 1606
vs. (C]O), 1564 s, 1503 s, 1445 m, 1413 m, 1378 m, 1335 w, 1304
m, 1253 vs. (C–O), 1185 m, 1162 m, 1115 w, 1088 s, 1050 s, 1031
m, 1010 m, 919 m, 894 vs., 847 vs., 817 m.
Rf (EA : PE ¼ 2 : 1) ¼ 0.05.
Elemental analysis. Found C: 53.68, H: 6.09, N: 6.66 – calcu-
lated 54.82, H: 5.62, N: 7.10.
FD-MS (m/z) ¼ 197.2532 (100%), 198.2448 (8.1%).
(1c) 2-(4-(5,5-dimethyl-1,4,2-dioxazol-3-yl)phenoxy)ethan-1-ol.
(1c) was prepared in a modied procedure based on the work of
Couturier et al.31 19.72 g (0.10 mol, 1.00 eq.) N-hydroxy-4-(2-
hydroxyethoxy)benzamide (1b) were suspended in 2000 mL
DCM in a dry 3 L ask. To this suspension 31.25 g (0.30 mol,
3.00 eq.) 2,2-dimethoxypropane and 23.23 g (0.10 mol, 1.00 eq.)
camphor sulfonic acid were added. The suspension was vigor-
ously stirred for 15 h and then neutralized by addition of
250 mL 4 M aqueous NaOH solution. Aer stirring over night
the organic layer was separated and washed twice with 100 mL
2 M NaOH, dried using Na2SO4 and evaporated to dryness in
vacuo. The pure product was obtained by column chromatog-
raphy (SiO2, EA : PE ¼ 1 : 1 as eluent). Yield: 20.89 g (0.088 mol,
(1a) Methyl 4-(2-hydroxyethoxy)benzoate. (1a) was prepared in
a modied procedure described elsewhere.36 A 250 mL ask
equipped with a condenser and gas bubbler was charged with
76.07 g (0.500 mol, 1.00 eq.) methyl 4-hydroxybenzoate, 46.25 g
(0.525 mol, 1.05 eq.) ethylene carbonate, 7.50 g (0.050 mol, 0.10
eq.) NaI and 100 mL diglyme and heated under vigorous stirring
for 16 h at 140 ꢁC oil bath temperature. Finally, the temperature
was increased to 160 ꢁC for two hours. The solution was cooled
to room temperature and the solvent were removed until
dryness in vacuo. The residue was dissolved in 300 mL EA and
washed with 50 mL water, 50 mL saturated NaHCO3 and 50 mL
brine and dried using Na2SO4. Aer evaporation of the solvent
93.6 g (0.477 mol, 95%) of methyl 4-(2-hydroxyethoxy)benzoate
were obtained.
ꢁ
88%) as colourless liquid that solidied at ꢀ20 C.
1H NMR (300 MHz, DMSO-d6) d [ppm] ¼ 7.64 (AA0BB0, 2H,
HC3,5), 6.94 (AA0BB0, 2H, HC2,6) 4.12 (t, J ¼ 5.2 Hz, 2H, Ar–O–
CH2–), 3.98 (t, J ¼ 5.2 Hz, 2H, HO–CH2–), 1.66 (s, 6H, CH3).
13C NMR (75 MHz, DMSO-d6) d [ppm] ¼ 161.12 C1, 158.21
C]N, 128.58 C3,5, 116.50 C4, 115.34 C(CH3)2, 114.71 C2,6, 69.45
Ar–O–CH2–, 61.43 HO–CH2–, 24.97 CH3.
1H NMR (300 MHz, CDCl3) d [ppm] ¼ 7.99 (AA0BB0, 2H,
HC2,6), 6.93 (AA0BB0, 2H, HC3,5), 4.17 (t, J ¼ 4.1 Hz, 2H, CH2–O–
Ar), 3.99 (t, J ¼ 4.1 Hz, 2H, HO–CH2–), 3.88 (s, 3H, CH3).
13C NMR (75 MHz, CDCl3) d [ppm] ¼ 166.92 Ar–C]O, 162.48
C4, 131.78 C2,6, 123.13 C3,5, 114.24 C1, 69.45 Ar–O–C, 61.42 HO–
C, 52.06 CH3.
IR-ATR n [cmꢀ1] ¼ 3396 vw br (OH), 2935 vw, 1710 vw, 1606 m
(C]N), 1514 m, 1454 w, 1423 m, 1363 m, 1306 m, 1251 vs.,
1215 s, 1166 s, 1115 m, 1078 s, 1044 m, 1010 m, 978 m, 945 w,
916 m, 884 m, 836 s, 816 m, 771 m.
(1b) N-hydroxy-4-(2-hydroxyethoxy)benzamide. (1b) was
prepared in a modied procedure based on the work of Hauser
and Renfrow.37 42.9 g (0.62 mol, 2.00 eq.) H2NOH$HCl were
dissolved in 240 mL methanol and neutralized by addition of
a solution of 52.1 g (0.93 mol, 3.00 eq.) KOH in 140 mL meth-
anol. Aer cooling in an ice-bath for 5 min the solution was
ltered to remove precipitated KCl. To this solution 60.6 g
(0.31 mol, 1.00 eq.) methyl 4-(2-hydroxyethoxy)benzoate (1a)
were added in one step, and the solution was stirred until
homogenous. Aer standing for 24 h at room temperature, the
basic solution was acidied with 2 M HCl to pH 4 and all
solvents were removed until dryness in vacuo. The residue was
transferred into a soxhlet apparatus and extracted using THF
(1000 mL) overnight. Aer cooling at ꢀ20 ꢁC and vacuum
ltration 47.5 g (0.24 mol 77%) N-hydroxy-4-(2-hydroxyethoxy)
benzamide (1b) were obtained as a colourless solid.
Rf (EA : PE ¼ 1 : 1) ¼ 0.38.
FD-MS (m/z) ¼ 237.2485 (100%), 238.2470 (11.5%), 239.2456
(1.2%).
(1d) 5,5-dimethyl-3-(4-(2-(oxiran-2-ylmethoxy)ethoxy)phenyl)-
1,4,2-dioxazole. A 250 mL three-necked ask equipped with
a dropping funnel, thermometer and mechanical stirrer was
charged with 56 mL 50% (w/w) aqueous NaOH solution,
35.0 mL (0.45 mol, 5.3 eq.) epichlorhydrin and 1.18 g (3.5 mmol,
0.04 eq.) tetrabutylammonium hydrogen sulfate (TBAHS). 20.0 g
(84.3 mol, 1.0 eq.) 2-(4-(5,5-dimethyl-1,4,2-dioxazol-3-yl)
phenoxy) ethan-1-ol were added within 30 minutes while cool-
ing in an ice bath. Aer addition, the ice bath was removed and
the suspension stirred vigorously overnight. Aer complete
conversion of the alcohol was veried via TLC the reaction was
quenched by addition of 100 mL of an ice-water mixture. The
organic phase was separated, and the aqueous phase was
extracted three times each with 60 mL dichloromethane. The
combined organic phase was washed with brine until a pH of 8
was obtained, then dried using Na2SO4, ltered and all solvents
1H NMR (300 MHz, DMSO-d6) d [ppm] ¼ 11.06 (s, 1H, NH),
8.90 (s, 1H, NH–OH), 7.71 (AA0BB0, 2H, HC2,6), 6.98 (AA0BB0, 2H,
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were removed in vacuo. Aer column chromatography (SiO2,
13C NMR (75 MHz, DMSO-d6) d [ppm] ¼ 164.48 (C]O),
eluent: PE : EA ¼ 4 : 1) 20.0 g (68.2 mmol, 81%) 5,5-dimethyl-3- 160.12 C1, 128.81 C2,6, 123.40 C4, 114.98 C3,5
.
(4-(2-(oxiran-2-ylmethoxy)ethoxy)phenyl)-1,4,2-dioxazole were
obtained as a colourless liquid.
IR-ATR n [cmꢀ1] ¼ 3314 m, 3191 m, br, 2923 w, 1606 s (C]O),
1570 s, 1534 m, 1493 m, 1457 m, 1457 m, 1350 m, 1316 m,
1H NMR (400 MHz, DMSO-d6) d ¼ 7.68–7.59 (AA0BB0, 2H, 1263 s, 1128 vs. (C–O), 1177 m, 1157 vs., 1028 m, 971 m, 893 s,
HC3,5), 7.11–7.02 (AA0BB0, 2H, HC2,6), 4.21–4.14 (m, 2H, Ar–O– 848 vs., 768 s.
CH2–CH2–), 3.85–3.72 (m, 3H, Ar–O–CH2–CH2, epoxide-CH2–O),
3.35–3.27 (m, 1H, epoxide-CH2–O), 3.15–3.09 (m, CH), 2.73 (dd, J
¼ 5.1, 4.2 Hz, 1H, CH2 in epoxide), 2.56 (dd, J ¼ 5.1, 2.7 Hz, 1H,
CH2 in epoxide), 1.61 (s, 6H, acetonide CH3).
Rf (EA : PE ¼ 2 : 1) ¼ 0.09.
ꢁ
Mp: 146 C.
(2b) 4-(5,5-dimethyl-1,4,2-dioxazol-3-yl)phenol. (2b) was
prepared in a modied procedure based on the work of
(1e) 2-(4-(5,5-dimethyl-1,4,2-dioxazol-3-yl)phenoxy)ethyl meth- Couturier et al..31 A dried 100 mL Schlenk ask was charged
acrylate. In a 50 mL Schlenk ask 6.06 g (0.026 mol, 1.10 eq.) 2- with 337 mg (2.2 mmol, 1 eq.) N,4-dihydroxybenzamide (2b) and
(4-(5,5-dimethyl-1,4,2-dioxazol-3-yl)phenoxy)ethan-1-ol, 2.00 g dried with 4 mL benzene under high vacuum. The solid residue
(0.023 mol, 1.00 eq.) methacrylic acid and 0.23 g (1.9 mmol, 0.08 was suspended in 50 mL of dry DCM (dried over P2O5, followed
eq.) 4-dimethylaminopyridine (DMAP) were dissolved in 15 mL by distillation) and 872 mg (6.6 mmol, 3 eq.) 2,2-diethox-
dichloromethane. Under ice cooling it was added 3.23 g ypropane were added. To this suspension 511 mg (2.2 mmol, 1
(0.026 mol, 1.10 eq.) N,N0-diisopropylcarbodiimide (DIC) within eq.) camphor sulfonic acid was added at once under argon
5 minutes. Aer stirring for 15 minutes under ice cooling, the atmosphere. Aer 105 minutes of stirring at room temperature
mixture was stirred for 5 h at rt. The mixture was ltered the reaction was quenched by the addition of 10 mL saturated
through a frit, and the ltrate was washed twice with 100 mL of NaHCO3 solution. The mixture was stirred over night at room
a saturated sodium hydrogen carbonate solution. The organic temperature and then the organic phase separated. The
phase was dried over MgSO4 and the solvent was removed under aqueous phase was extracted three times with 50 mL ether, and
reduced pressure. The solid was puried by column chroma- the combined organic phase dried using Na2SO4. Aer evapo-
tography in a solvent mixture of PE : EA (6 : 1). Methacrylate ration of all solvents in vacuo the residue was dissolved in THF
hydroxamic acid acetonide (MAHAA, 1e) was obtained in yields and diethyl ether (to a nal ratio of 1 : 1) was added. 4-(5,5-
of 5.35 g (0.018 mol, 76%).
dimethyl-1,4,2-dioxazol-3-yl)phenol (2b) crystallized aer days
1H NMR (300 MHz, CDCl3): d [ppm] ¼ 7.73 (AA0BB0, 2H, as colourless needles. Yield: 160 mg (0.8 mmol, 38%).
HC3,5), 6.93 (AA0BB0, 2H, HC2,6), 6.15 (s, 1H, HC]C), 5.60 (s, 1H,
1H NMR (300 MHz, DMSO-d6) d [ppm] ¼ 7.65 (AA0BB0, 2H,
HC]C), 4.51 (m, 2H, MA–O–CH2–), 4.26 (m, 2H, Ar–O–CH2), HC3,5), 6.89 (AA0BB0, 2H, HC2,6), 1.68 (s, 6H, CH3).
1.96 (s, 3H, CH3–C]C), 1.67 (s, 6H, acetonide CH3).
13C NMR (75 MHz, DMSO-d6) d [ppm] ¼ 159.27 C1, 158.75
Synthesis of phenolic hydroxamic acid functional initiator (C]N), 128.90 C3,5, 115.94 C2,6, 115.58 CCH3, 115.24 C4, 24.90
not employed for polymerization (2a–2b).
CH3.
Rf (EA) ¼ 0.92.
Synthesis of benzylic hydroxamic acid functional initiator
benzylHAA-OH) not employed for polymerization (3a–3b)
(3a) N-hydroxy-4-(hydroxymethyl)benzamide. The synthesis
(
was carried out in a modied procedure based on the work of
Hauser and Renfrow analogous to the synthesis of (1b).37
Starting from 14.95 g (0.09 mol, 1 eq.) methyl 4-(hydroxymethyl)
(2a) N,4-dihydroxybenzamide. (2a) was prepared in a modied
procedure based on the work of Jeanrenaud et al.38 A 250 mL
round bottom ask was charged with 3.80 g (25 mmol, 1 eq.)
methyl paraben and 3.47 g (50 mmol, 2 eq.) H2NOH$HCl in
87.5 mL water. To this suspension 3.5 g (88 mmol, 3.5 eq.)
NaOH as a 2 M solution was added. Aer 4 hours of stirring at
room temperature the solution was acidied using 110 mL 1 M
HCl and cooled overnight at 4 ꢁC. Unreacted reagents were
ltered off and the residue reduced in vacuo to few mL residual
volume. The residual aqueous phase was extracted four times
with 50 mL EA each. The combined organic phase was dried
using Na2SO4 and freed from any solvents in vacuo. The crude
product was puried using column chromatography (SiO2,
PE : EA ¼ 1 : 1 as starting eluent, increasing elution strength
gradually up to EA : MeOH 19 : 1). Yield 820 mg (5.4 mmol,
21%) of N,4-dihydroxybenzamide (2a) as a colourless solid.
1H NMR (300 MHz, DMSO-d6) d [ppm] ¼ 10.97 (s, 1H, NH),
9.96 (s, 1H, Ar–OH), 8.84 (s, 1H, NH–OH), 7.62 (AA0BB0, 2H,
benzoate 11.28
g
(0.068 mol, 75%) of N-hydroxy-4-
(hydroxymethyl)benzamide (3a) were obtained as a colourless
solid.
1H NMR (400 MHz, DMSO-d6) d [ppm] ¼ 11.08 (s, 1H, NH),
8.98 (s, 1H, NH–OH), 7.77–7.64 (AA0BB0, 2H, HC2,4), 7.46–7.30
(AA0BB0, 2H, HC3,5), 5.28 (t, J ¼ 5.7 Hz, 1H, HO–CH2), 4.53 (d, J ¼
5.7 Hz, 2H, CH2).
13C NMR (100 MHz, DMSO-d6) d [ppm] ¼ 164.54 C]O,
145.83 C4, 131.10 C1, 126.70 C2,4, 126.10 C3,5, 62.4S4 CH2.
(3b) (4-(5,5-dimethyl-1,4,2-dioxazol-3-yl)phenyl)methanol. The
synthesis was carried out in a modied procedure based on
the work of Couturier et al. analogous to the synthesis of 1c.31
Starting from 5.03
g (0.03 mol, 1 eq.) N-hydroxy-4-
(hydroxymethyl)benzamide 3.43 g (0.016 mol, 55%) (4-(5,5-
dimethyl-1,4,2-dioxazol-3-yl)phenyl)methanol (3b) was ob-
tained as a colourless liquid which solidied aer one week
HC2,6), 6.78 (AA0BB0, 2H, HC3,5
)
ꢁ
at ꢀ20 C.
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1H NMR (400 MHz, DMSO-d6) d [ppm] ¼ 7.72–7.59 (AA0BB0,
2H, HC3,5), 7.50–7.39 (AA0BB0, 2H, HC2,4), 5.36 (t, J ¼ 5.7 Hz, 1H,
CH2OH), 4.56 (d, J ¼ 5.7 Hz, 2H, CH2), 1.63 (s, 6H, CH3).
13C NMR (100 MHz, DMSO-d6) d [ppm] ¼ 157.49 C]N,
146.58 C1, 126.68 C2,4, 126.11 C3,5, 115.36 C(CH3)2, 62.36 CH2,
24.41 CH3.
Polymerization procedures
Synthesis of HAA-polyether (P1a–P6a) and deprotection to
obtain the free hydroxamic acid functional polyether (P1b–
P6b).
Rf (EA : PE ¼ 1 : 4) ¼ 0.35.
Synthesis of aliphatic hydroxamic acid functional initiator
not employed for polymerization (4a–4b).
(4a) N,6-dihydroxyhexanamide. The synthesis was carried out
in an modied procedure of Hauser and Renfrow.37 A 250 mL
round bottom ask was charged with 17.12 g (150 mmol, 1.00
eq.) freshly distilled 3-caprolactone. In a separated ask 11.47 g
(165 mmol, 1.1 eq.) H2NOH$HCl is dissolved in 65 mL methanol
and neutralized with a solution of 12.62 g (225 mmol, 1.5 eq.)
KOH in 32 mL methanol under shaking and cooling in a water
bath. Aer 15 minutes the solution was ltered from precipi-
tated KCl and added to the round bottom ask containing the
caprolactone and stirred for 90 minutes. Aer the reaction had
proceeded, the solution was acidied using conc. HCl to pH 4
and reduced under vacuum until most of the product solidied.
The residue was recrystallized twice from THF (approx. 200 mL).
11.24 g (76 mmol, 51%) N,6-dihydroxyhexanamide (4a) were
recovered aer crystallization for 72 h at ꢀ20 ꢁC as a colourless
solid.
(P1a/P2a) synthesis of HAA-PEG and benzylHAA-PEG. Prepara-
tion in example of HAA-OH as initiator. In a typical procedure,
2-(4-(5,5-dimethyl-1,4,2-dioxazol-3-yl)phenoxy)ethan-1-ol (1 eq.)
(1d) was deprotonated by 90 mol% cesium hydroxide mono-
hydrate in 10 mL benzene in a 100 mL Schlenk ask. Aer
stirring for 60 minutes at 50 ꢁC under vacuum the solvents were
removed with high vacuum (10ꢀ3 mbar) and subsequently the
ask was heated to 60 ꢁC overnight under high vacuum. The
formed alkoxide salt was dissolved in 15 mL dry THF and 2 mL
dry DMSO. EO was rst distilled in the cold into a graduated
ampoule and subsequently into the reaction ask containing
the dissolved initiator. The mixture was heated to 50 ꢁC and
stirred for 2 days. Aerwards, 1 mL methanol was added to
quench the reaction. Except for high boiling DMSO all residual
solvents were removed in vacuo. The residue was diluted with
40 mL DCM and washed three times with 10 mL water. The
organic phase was separated and dried using Na2SO4. The
volume was reduced in vacuo to approx. 5 mL and the polymer
precipitated in cold diethyl ether (45 mL, ꢀ20 ꢁC, recovery
through centrifugation at 4500 RPM for 10 minutes). Yields: 73–
94%.
1H NMR (300 MHz, DMSO-d6) d [ppm] ¼ 10.37 (s, 0.9H, NH),
9.72 s (s, 0.1H, NH), 9.03 (s, 0.1H, NH–OH), 8.68 (s, 0.9H, NH–
OH), 4.37 (s, 1H, CH2–OH), 3.35 (t, J ¼ 6.4 Hz, 2H, C6H2), 1.93 (t,
J ¼ 7.5 Hz, 2H, C2H2), 1.52–1.42 (m, 2H, C3H2), 1.42–1.32 (m, 2H,
C5H2), 1.30–1.16 (m, 2H, C4H2).
13C NMR (75 MHz, DMSO-d6) d [ppm] ¼ 169.25 C1, 60.67 C6,
1H NMR (400 MHz, DMSO-d6) d [ppm] ¼ 7.63 (AA0BB0, 2H,
ArC3,5H), 7.06 (AA0BB0, 2H, ArC2,6H), 4.56 (t, J ¼ 5.5 Hz, 1H,
terminal OH), 4.19–4.11 (m, 2H, Ar–O–CH2–), 3.79–3.71 (m, 2H,
Ar–O–CH2–CH2–O–), 3.71–3.28 (m, polyether backbone), 1.61 (s,
6H, CH3).
32.42 C2, 32.30 C5, 25.23 C4, 25.16 C3.
IR-ATR n [cmꢀ1] ¼ 3311 m, 3205 m, 3029 m, 2932 m, 2858 m
br, 1616 vs. (C]O), 1544 m, 1449 m, 1466 m, 1362 m, 1274 m,
1116 m, 1078 m, 1055 vs., 1012 vs., 977 vs., 785 m.
Rf (EA : MeOH ¼ 9 : 1) ¼ 0.42.
ꢁ
(P1b/P2b) removal of the acetal protecting group of HAA-PEG
(2a) to form HA-PEG.
Mp: 84 C.
(4b)
5-(5,5-dimethyl-1,4,2-dioxazol-3-yl)pentan-1-ol.
The
synthesis of 4b was carried out analogous to the preparation of 1
on a scale of 4.27 g (29 mmol) N,6-dihydroxyhexanamide (4a).
Purication was performed via column chromatography (SiO2,
EA : PE ¼ 1 : 1 as eluent). Yield: 1.25 g (7 mmol, 23%) 5-(5,5-
dimethyl-1,4,2-dioxazol-3-yl)pentan-1-ol (4b) as a colourless
liquid.
1H NMR (300 MHz, CDCl3) d [ppm] ¼ 3.64 (t, J ¼ 6.3 Hz, 2H,
C1H2), 2.31 (t, J ¼ 7.3 Hz, C5H2), 1.76 (s, 1H, OH), 1.69–1.56 (m,
4H, C2H2, C4H2), 1.55 (s, 6H, CH3), 1.50–1.39 (m, 2H, C3H2).
13C NMR (75 MHz, CDCl3) d [ppm] ¼ 160.39 C]N, 114.54
C(CH3)2, 62.65 C1, 32.26 C2, 25.22 C3, 25.18 C4, 24.91 CH3, 23.95
C5.
Preparation in example of HAA-PEG (P1a). In a typical
procedure, 500 mg HAA-PEG (P1a) were dissolved in 20 mL
0.1 M HCl and shaken for 24 h. The polymer was recovered by
adding 40 mL DCM and partitioning between the organic and
Rf (EA : PE ¼ 1 : 1) ¼ 0.46.
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aqueous phase. The organic phase was separated and dried over with a strong neodymium magnet at the bottom of a ask and
Na2SO4 and reduced in vacuo to a volume of approx. 5 mL. Aer subsequent decantation of the supernatant. For DLS measure-
ꢁ
precipitation in ice cold diethyl ether (45 mL, ꢀ20 C, recovery ments the nanoparticles were redissolved in DCM and sterile
using centrifugation at 4500 RPM for 10 minutes), HA-PEG ltered. For TEM measurement water was used for redissolving
(P1b) was obtained in near quantitative (>98%) yields.
1H NMR (400 MHz, DMSO-d6) d [ppm] ¼ 11.06 (s, 1H, NH),
8.90 (s, 1H, NH–OH), 7.72 (AA0BB0, 2H, ArC2,6H), 6.99 (AA0BB0,
2H, ArC3,5H), 4.57 (t, J ¼ 5.5 Hz, 1H, terminal OH), 4.19–4.08 (m,
2H, Ar–O–CH2–), 3.79–3.72 (m, 2H, Ar–O–CH2–CH2–O–), 3.73–
3.37 (m, polyether backbone).
instead.
Conflicts of interest
There are no conicts to declare.
ben-
(P3a, P4a, P5a, P6a) synthesis of HAA-PPO, HAA-PEEGE,
zylHAA-PPO, benzylHAA-PEEGE. Preparation example of HAA-PPO Acknowledgements
with HAA-OH as an initiator. In a typical procedure 2-(4-(5,5-
T. J. acknowledges a fellowship by the Max Planck Graduate
Center. The authors thank Monika Schmelzer for SEC
measurements, Tatjana Danzer for TEM measurement, Thi
Dinh for experimental assistance and Jan Hilgert for providing
FeOx nanoparticles.
dimethyl-1,4,2-dioxazol-3-yl)phenoxy)ethan-1-ol (1 eq.) (1d) was
deprotonated by 90 mol% cesium hydroxide monohydrate in
10 mL in a 25 mL Schlenk ask. Aer stirring for 60 minutes at
50 ꢁC under vacuum the solvents were removed with high
vacuum (10ꢀ3 mbar) and subsequently the ask heated to 60 ꢁC
overnight under high vacuum. 1 mL dry propylene oxide (dried
via cryo-distillation over CaH2) were added to the formed
alkoxide using a syringe and the polymerization was performed
at 40 ꢁC under vacuum overnight. Subsequently, 1 mL methanol
was added to quench the reaction and the polymer was dis-
solved in 40 mL DCM and washed three times with 10 mL. The
organic phase was separated, dried using Na2SO4 and all
solvents were evaporated in vacuo. Typical yield: 78-95% as
highly viscous amorphous polymer.
Preparation of PEEGE polymers was carried out analogously.
1H NMR (300 MHz, CDCl3) d [ppm] ¼ 7.70 (AA0BB0, 2H,
ArC3,5H), 6.94 (AA0BB0, 2H, ArC2,6H), 4.19–4.11 (m, 2H, Ar–O–
CH2–), 4.16 (m, 2H, Ar–O–CH2–CH2–O–), 3.85 (m, 2H, Ar–O–
CH2–CH2–O–), 3.69–3.28 (m, polyether backbone), 1.66 (s, 6H,
C(CH3)2), 1.34-1.08 (m, propylene oxide CH3).
¨
Notes and references
´
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