Macrocyclic squaramides: Anion receptors with high sulfate binding affinity and selectivity in aqueous media

Article abstract of DOI:10.1039/c6sc01011c

A number of macrocyclic squaramide-containing receptors (MSQs) have been designed and synthesised and their interaction with a range of inorganic anions was studied in solution by ¹H NMR spectroscopy and ESI-HRMS. The binding data revealed remarkable binding of sulfate in aqueous mixtures from 0.5 to 50% v/v H₂O/DMSO-d₆. The larger [3]-MSQs were found to better match the size and shape of the sulfate ion than the [2]-MSQs, providing high affinity and selectivity for sulfate while other tetrahedral divalent anions such as selenate, phosphate species and chromate have substantially lower binding affinities. In mixtures of anions mimicking the composition of either nuclear waste or plasma, the [3]-MSQs were still able to bind sulfate ions with high affinity.

Full text of DOI:10.1039/c6sc01011c

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DOI: 10.1039/C6SC01011C
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Macrocyclic Squaramides: Anion Receptors with High Sulfate
Binding Affinity and Selectivity in Aqueous Media
Lei Qina, Anna Hartleya, Peter Turnera, Robert B. P. Elmesa,b* and Katrina A. Jolliffea*
Received 00th January 20xx,
Accepted 00th January 20xx
A number of macrocyclic squaramide-containing receptors (MSQs) have been designed and synthesised and their
DOI: 10.1039/x0xx00000x
1
interaction with a range of inorganic anions was studied in solution by H NMR spectroscopy and ESI-HRMS. The binding
www.rsc.org/
data revealed remarkable binding of sulfate in aqueous mixtures from 0.5 to 50% v/v H2O/DMSO-d6. The larger [3]-MSQs
were found to better match the size and shape of the sulfate ion than the [2]-MSQs, providing high affinity and selectivity
for sulfate while other tetrahedral divalent anions such as selenate, phosphate species and chromate have substantially
lower binding affinities. In mixtures of anions mimicking the composition of either nuclear waste or plasma, the [3]-MSQs
were still able to bind sulfate ions with high affinity.
Introduction
allowing for error correction and therefore selectivity.10
Following Nature’s lead, numerous receptors have been
Inorganic sulfate plays numerous roles in environmental and developed for the recognition and/or extraction of sulfate using
biological systems, so the selective recognition of this anion has hydrogen bonding interactions from ureas, thioureas, pyrroles,
applications across a range of fields. For example, in industry the indoles, and squaramides,11-24 but very few of these show
selective extraction of sulfate from nitrate rich mixtures in acidic significant sulfate affinity in aqueous solution. To overcome this,
(pH < 4) conditions would have significant benefits for nuclear charged interactions are often incorporated into sulfate receptors
waste remediation because sulfate interferes with the vitrification to provide increased binding energies to compensate for the
process that is a crucial procedure in nuclear waste treatment.1-3 In dehydration ‘penalty’. Even so, few receptors are capable of binding
the context of biology, sulfate is essential for the formation of to sulfate in aqueous solution25-29 and the introduction of charged
proteins in joints4 and mucin5, 6 and low levels of sulfate have been interactions frequently occurs at the cost of selectivity or
found in the plasma of patients with rheumatoid arthritis4 and functionality (e.g. cationic receptors are likely to be toxic30 and so
irritable bowel disease,5, 6 so selective sulfate receptors could find unable to be used in biological applications). The development of
applications in disease diagnosis and monitoring. However, binding neutral receptors capable of binding selectively to sulfate in
of sulfate in aqueous solution is very challenging as a result of the aqueous solution is therefore of particular interest.
high hydration energy of this anion (ΔG = -1090 kJ mol-1),7 which
It is well established that to achieve strong and selective
poses additional problems in terms of receptor selectivity in water binding, receptors should be preorganised to match the size and
since other anions, e.g. chloride and nitrate, have lower hydration shape of the target ion. Macrocyclic receptors have therefore found
energies (ΔG = -347 kJ mol-1 and -306 kJ mol-1, respectively).
great utility in the selective recognition of a range of cations and
Nature has overcome this challenge by employing an idealised anions.31 We report herein the first examples of a new class of
arrangement of hydrogen bonding interactions in the sulfate macrocyclic anion receptors 1 - 4 (Figure 1) containing alternating
binding protein (SBP)8 where three of the four sulfate oxygen atoms aryl spacers and squaramides spaced by methylene units, together
are bound through two hydrogen bonding interactions, while the with the more soluble triethylene glycol monomethyl ether
fourth has
a single hydrogen bond. These combined weak derivatives 5 and 6 (Figure 1).
interactions result in a binding affinity for sulfate of 8.3 × 106 M-1
across a pH range of 5-99 and it has been hypothesised that this
combination of weak interactions leads to fast exchange kinetics,
Results and discussion
Synthesis and Characterisation of MSQs 1 - 4:
While Costa and co-workers have previously demonstrated the self-
templated formation of macrocycles containing both squaramides
and a hydrogen-bond acceptor unit,32, 33 attempts to prepare a
macrocycle containing two squaramide units linked by alkyl chains
(i.e. lacking the hydrogen bond acceptor) have been reported to be
unsuccessful, giving only traces of the desired compound. We have
now developed a simple yet versatile synthetic route to
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presence of Et3N under high dilution conditions gave 2 and 4 in 79%
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DOI: 10.1039/C6SC01011C
and 68% yield, respectively.
[2]-MSQs 1 and 3 were both successfully recrystallised from
concentrated solutions of DMSO to yield samples suitable for
single crystal X-ray diffraction analysis (Figure 2), which
confirmed the structure of the anticipated macrocyclic [2]-
MSQs 1 and 3. Macrocycles 1 and 3 were each found to be
hydrogen-bonded to a molecule of DMSO in the solid state
with the squaramide moiety acting as both a H-bond donor
and acceptor to a pair of DMSO molecules as previously
observed for acyclic squaramide derviatives.36, 37 As expected,
though both are centred on a crystallographic inversion
site with squaramide moieties on opposite sides of the
macrocycle cavity plane, the substitution pattern around the
aryl rings of 1 and 3 gave macrocycles with substantially
different conformations. Macrocycle 1 was found to adopt a
‘chair-like’ conformation that maintains the cis/cis
conformation around each squaramide that is optimal for
anion complexation. In contrast, 3 has a cis/trans arrangement
of the squaramide NH protons (Fig 2B) and complementary
hydrogen bond interactions between the squaramide groups
of adjacent molecules that link them in a chain like manner in
the solid state (Fig. 2D). The length and width of the
macrocycle cavity is effectively determined by the separation
between the four ‘corner’ carbon atoms that link the phenyl
groups and squaramide groups and in 1 these distances are
6.062(2) Å across the squaramide group (distance between C5
and C12; see the supplementary information) and 5.075(2) Å
across the phenyl group (distance between C5 and C12). In 3
these distances are effectively ‘reversed’ and are respectively
4.732(3) and 5.837(2) Å. Notwithstanding this difference, in
both cases the cavity dimensions are promising for anion
recognition.
Figure 1: Macrocyclic squaramides (MSQs) 1 – 6.
macrocycles comprising alternating squaramide and benzylic groups
(macrocyclic squaramides: MSQs), enabling us to quickly assemble
the desired frameworks containing either two or three squaramide
units and starting from the readily available diethyl squarate and
appropriate benzyl diamines. We envisaged that this would allow
the development of analogues that could be readily functionalised
by substitution of the aromatic rings to tailor the solubility of the
macrocycles for a variety of applications.
[2]-MSQs 1 and 3 were synthesised in a two-step process
(Scheme 1), exploiting the ability of diethylsquarate 7 to undergo
sequential amidation reactions.34 Treatment of an excess of
diethylsquarate with one equivalent of the appropriately
substituted benzyl diamines 8 and 9 gave the corresponding
disquarate derivatives 10 and 11. Subsequent reaction of 10 and 11
Figure 2: : Single crystal X-ray diffraction structures of (A) 1, (B) 3 and the solid
state intermolecular hydrogen bond interactions of (C) 1 and (D) 3 in the
with the benzyl diamines 8 and 9 in the presence of Et3N under high presence of DMSO; hydrogen atoms not shown
dilution conditions yielded macrocycles 1 and 2 in 87% and 93%
.
The cavity shape in 1 is essentially rectangular, with internal
corner angles of approximately 87.1° and 92.9°, while that of 3 is
that of a parallelogram with internal corner angles of 82.6° and
97.4°. In 1 the least squares planes of the squaramide groups are
separated by 4.832(6) Å, while in 3 the distance is 6.288(6) Å. The
centroid to centroid distance and the approximate relative lateral
offset of the squaramide groups is respectively 7.233(2) and 5.38 Å
yield, respectively. Similarly, the larger [3]-MSQs (2 and 4) were
assembled (Scheme 1), by initial reaction of two equivalents of the
known mono Boc-protected benzyl diamines 12 and 1335 with 7 in
the presence of Et3N to yield the corresponding dibenzyl squarate
derivatives 14 and 15. Following Boc deprotection in the presence
of trifluoracetic acid (TFA), reaction of the subsequently formed
diamines with the aforementioned disquarates 10 and 11 in the
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in 1, and 8.590(2) and 5.85 Å in 3. The least squares planes of the anionic guests for these macrocycles. For receptors 1 and 2, anion
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phenyl groups in 1 are separated by 3.672(5) Å and in 3 the screening experiments were performed by DadOdI:it1i0o.1n03o9f/C106SeCq0.10o1f1Ca
-
-
-
separation is 4.635(2)Å. The centroid to centroid distance and the range of common anions (F-, Cl-, Br-, NO3 , HSO4 , AcO-, H2PO4 and
2-
approximate relative lateral offset of the phenyl groups is SO4 as their tetrabutylammonium salts) to 1 and 2 in DMSO-d6.
approximately 8.463(2) and 7.62 Å in 1, in contrast to 4.667(2) and These preliminary results indicated significant changes in the 1H
0.55 Å in 3. Additional cavity geometry details are provided in the NMR spectra of 1 and 2 in the presence of H2PO4 , AcO- and SO4
-
2-
supplementary information. Unfortunately, single crystals suitable that culminated in downfield shifts of the squaramide NH proton
2-
for X-ray crystallographic analysis could not be obtained for the signals (e.g. Δδ = 2.21 for 1 with SO42-, Δδ = 1.61 for 2 with SO4
)
larger [3]-MSQs 2 and 4. and a significant downfield shift of the aromatic proton lying within
2-
1H NMR spectroscopy provided further information about the the macrocyclic cavity in the case of SO4 addition, (CH) (e.g. Δδ =
conformations of the constrained macrocycles 1 – 4 in the solution 0.87 for 1, Δδ = 0.44 for 2), which was less pronounced in the
phase. In DMSO-d6, the meta-substituted compounds 1 and 3 presence of other anions. Conversely, less pronounced changes
exhibited broad signals in the 1H NMR spectra, suggesting that were observed in the presence of Cl-, Br-, HSO4- and NO3- suggesting
these compounds exist in multiple conformations which weaker interactions of these anions with 1 and 2. In the presence of
interconvert on a similar timescale to the NMR experiment. In the F- the signals for the NH protons could not be observed, suggesting
case of the para-substituted compounds 2 and 4, complex NMR likely deprotonation of the squaramides by this basic anion, as has
spectra were obtained with multiple signals observed for each previously been observed for simple squaramide derivatives.37
1
different type of proton, indicating that different conformers of Unfortunately, the complex H NMR spectra and low solubility of
these macrocycles exist in solution and that interconversion the para-substituted derivatives 3 and 4 did not allow the ready
between these is slow on the NMR timescale. Similarly, compounds screening of their anion binding ability. However, for both 3 and 4,
2-
2 and 4 also gave 13C NMR spectra with broadened and multiple the addition of one equivalent of either AcO- or SO4 resulted in
signals for the same type of carbon, while the signals in the 13C significant changes and simplification of the NMR spectra. Complete
NMR spectra of compounds 1 and 3 were slightly broadened.§
dissolution of suspensions of 3 and 4 in DMSO-d6 was observed
upon addition of 1.2 equiv. of sulfate and this together with the
simplified NMR spectra provides evidence that these compounds
Anion Binding Studies of MSQs 1 - 4:
With MSQs 1–4 in hand, qualitative screening experiments were bind strongly to this anion.
undertaken using 1H NMR spectroscopy to identify the optimal
Scheme 1: Synthesis of the desired macrocyclic squaramide based receptor frameworks 1 – 4. Synthesis: (i) Et3N, EtOH; (ii) 1 equiv. 8 or 9, high dilution, Et3N, EtOH,
heat. (iii) TFA/DCM (1:1); (iv) 1 equiv. 10 or 11, high dilution, Et3N, EtOH, heat.
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selectivity for SO42-, whereas 1 also bound strongly to H2PO4 and
-
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DOI: 10.1039/C6SC01011C
AcO- ions.
Analysis of the 1H NMR spectra of the complexes also provided
useful information on the binding modes of the MSQs for the
different anions. For [2]-MSQ 1, addition of either Cl- or AcO-
resulted in downfield shifts of the signals attributable to the NH
protons, but the signals attributable the aromatic protons and the
methylene bridges did not change or shift significantly, although
they did become slightly sharper. In contrast, addition of H2PO4- and
2-
SO4 to 1 resulted in signifcant downfield shifts of both the NH
proton signals and the signal attributable to the aromatic CH that
sits between the two ring substituents, with the other aromatic
proton signals exhibiting slight upfield shifts (Figure 3). In addition
the signal for the benzylic protons (CH2) broadened in the case of
-
2-
H2PO4 addition, while upon addition of SO4 this signal shifted
slightly upfield and broadened until one equivalent of sulfate was
added, at which point it sharpened and split into two distinct signals
indicating the inequivalence of the two protons on each CH2 group
and suggesting that, upon full complexation to the sulfate ion, the
macrocycle is frozen into a single conformer. The multiplicity of
these signals indicates an AB quartet, in which one of the signals is
further split by coupling to the adjacent NH proton while the other
is not, as a result of the different dihedral angles between the two
CH2 protons and the NH proton. For [3]-MSQ 2, a similar trend was
observed, with addition of Cl- and AcO- resulting in signifcant
downfield shifts only of the squaramide NH proton signal, whereas
-
addition of both H2PO4 and SO42- resulted in significant shifts for
both the NH protons (downfield) and the aromatic protons (one
signal moves downfield while the others shift upfield). In this case
the signal for the CH2 protons sharpens upon addition of the anions
and no splitting of the signal is observed, even upon addition of
excess sulfate ion.
Table 1: Apparent Stability Constants (Ka) of MSQs 1 and 2a
-
MSQs
1
2
SO42-/ M-1
>104
H2PO4 / M-1
AcO-/ M-1
7530
Cl-/ M-1
28
>104
409
>104
776
144
a
Titrations were performed in DMSO-d6 with 0.5 % water at 300 K. Estimated
errors < 15%.
1
Figure 3: (A): H NMR titration of compound 1 with (TBA)2SO4 in DMSO-d6 with
0.5 % water at 300 K. (B): 1H NMR titration of compound 2 with (TBA)2SO4 in
DMSO-d6 with 0.5 % water at 300 K. (C): Comparison isotherms of squaramide
Further information about the interaction of MSQs 1 - 3 with
2-
SO4 and AcO- was ascertained from ESI mass spectrometric
NH, Aromatic protons (Ar-Ha, Hb, Hc) for 2 in the presence of increasing
2-
concentrations of SO4
.
analysis of 0.5 mM solutions of 1 - 3 in MeCN with either TBA2(SO4)
or TBAAcO. In the case of AcO- addition, mass spectra of 1 and 2
exhibited major peaks at m/z 1213.006 and 1608.336 that were
assigned to the doubly deprotonated [1 - 2H+ + 3(TBA+)]+ and triply
deprotonated receptors [2 - 3H+ + 4(TBA+)]+, respectively. Very
minor peaks were observed for the [M + OAc- + 2(TBA+)]+ ion thus
suggesting that the complex formed between AcO- and the MSQs is
not stable enough to be observed using ESI mass spectrometric
analysis. Conversely, in the case of addition of TBA2(SO4) to 1 and 2,
the major peaks observed had an m/z ratio and isotopic pattern
Given the low solubility and complex 1H NMR spectra of 3 and
4, quantitative anion binding studies were only performed for the
1
meta-substituted MSQs 1 and 2. H NMR titrations of 1 and 2 in
DMSO-d6 containing 0.5 % water were performed by addition of the
-
-
tetrabutylammonium salts of SO42-, H2PO4 , AcO- and Cl- (and HSO4
for 1) with the resulting data fit to a 1:1 binding model using a
global fitting method in Hyperquad38 (with the exception of binding
-
of 1 to HSO4 ; the data for which could not be fit to a suitable
that can be assigned to the 1:1 SO42- complexes. Major peaks at m/z
binding model) to provide the apparent stability constants (Ka),
which are summarised in Table 1. Both 1 and 2 were found to bind
2-
= 1250.954 and 1465.026 corresponded to the [M + SO4
+
2-
to SO4 with affinities too high to measure accurately (>104 M-1)
3(TBA+)]+ complexes of 1 and 2, respectively. No peaks were found
correlating to either deprotonated receptors or to ions with more
than one SO42- anion further suggesting that SO42- binds to 1 and 2
using NMR but significant differences in binding affinities for the
other anions were observed, with [3]-MSQ 2 showing excellent
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in a 1:1 fashion. These observations were further supported by calculations at the B3LYP/6-31G* level of theory. As shown in Figure
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DOI: 10.1039/C6SC01011C
NMR Job plot analysis (see SI), which also indicated a 1:1 binding 4, these calculations indicate that
1 adopts an alternative
mode between SO4 and these receptors. Conversely, the mass conformation when binding the AcO- ion. The encapsulated anion is
spectrum of a mixture of 3 displayed significant peaks at both m/z again bound by four H-bonding interactions with both available
1250.953 and m/z 1590.196 correlating to the masses of the 1:1 [M squaramide functionalities, however, in this case the aromatic CH
2-
+ SO4 + 3(TBA+)]+ and 2:1 [M + H+ + 2SO4 + 4(TBA+)]+ complexes, proton is clearly pointing away from the anion binding site, in a
2-
2-
respectively suggesting both binding stoichiometries are possible conformation reminiscent of
a
1,3-alternate calixarene
for complexes of SO42- and 3.
conformation. This observation suggests an alternative mode for
Final confirmation of the binding modes between 1 and AcO- AcO- binding and corroborates the results observed in the NMR
2-
and SO4 were identified by a combination of both X-ray measurements, accounting for the shifts of the NH protons upon
crystallographic analysis and molecular modeling (Figure 4). Crystals AcO- binding with the concomitant lack of shifts of the aromatic CH
of 1 suitable for single crystal X-ray diffraction analysis were protons that were observed for binding of SO42-. When the sulfate
obtained in the presence of 5 equiv. of (TBA)2SO4 by slow complex of 1 was modelled in a similar manner (see Fig. S31 in the
evaporation of an MeCN solution. Analysis revealed that in the supplementary material), the resulting structure was very similar to
2-
presence of SO4 the conformation adopted by 1 is markedly that observed by X-ray crystallography.
different to the S2 symmetry chair-like conformation found for the
DMSO complex of 1. Instead, 1 forms a 1:1 (receptor/anion)
complex with SO42- where the receptor adopts
a bowl-like
conformation with essentially C2 v symmetry and suggestive of a
calixarene in the cone conformation (Figure 4). The two least
squares plane defined by the squaramide carbon atoms are
inclined with respect to each other by 73.5(1)° and the two least
squares planes of the phenyl groups form an angle of 50.78(8)°.
The squaramides are inclined at 57.39(9) and 49.12(8)° with
respect to the least squares plane of the cavity, while the phenyl
group planes are inclined at 52.68(6) and 76.55(6)° with respect to
the cavity plane (see the supplementary information). Both
squaramide functionalities and two aromatic CH protons (one from
each ring) directed into the macrocyclic cavity. In this conformation,
the two protons at each benzylic position are clearly in different
environments (pseudo-axial and pseudo-equatorial), corroborating
the results observed in the 1H NMR titration experiments.
2-
The macrocycle cavity dimensions of the 1•SO4 complex are
very similar to those of 1, with ‘corner’ carbon to corner carbon
distances across the squaramide groups of 6.070 (3) and 6.041(2) Å
and corner to corner distances across the phenyl groups of 5.050(4)
4.998(3) Å. The internal angles are approximately 89.1, 90.6, 89.9
and 90.6°. The squaramide centoid to squaramide centroid and
phenyl centroid to phenyl centroid distances are respectively
7.152(3) and 7.323(2) Å. The crystals structure reveals two binding
modes (see Fig. S34 in the supplementary material), with one
having hydrogen bonds between each of the four amide protons
and three of the sulfate oxygens, while the other mode involves
just two of the sulfate oxygens. The binding mode involving three
oxygen atoms is the dominant mode (see the supplementary
information) and is shown in Figure 4 (N······O distances from
2. 745(2) to 2. 881(2) Å; details are provided in the
Figure 4: (A) The dominant binding mode of the complex formed between 1 and
2-
SO42- viewed from the side and (B) from below clearly showing the interaction of
SO4 with the squaramide NH protons inside the macrocyclic cavity. The
structure was obtained by single crystal X-ray diffraction. (C) Energy minimised
molecular model of the complex formed between 1 and AcO- viewed from the
side and (D) from above.
supplementary information).
The anionic macrocycle-sulfate
complex is ‘nestled’ between the pendant residues of a pair of
tetrabutylammonium cations (see Fig. S35).
While crystals suitable for X-ray crystallography could not be
obtained for the complex of 1 with AcO-, molecular modelling was
performed using Spartan 14 (Wavefunction Inc) to provide an
indication of the binding mode of 1 with AcO-. The structure of 1
was energy minimized using molecular mechanics then an AcO- ion
was placed into the centre of macrocyclic cavity of 1 and the
resulting complex was optimised by density functional theory (DFT)
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Subsequent reaction of compound 7 with a further portion of the
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benzyl diamine 17 in ethanol and trimethyDlaOmI:i1n0e.10(E3t93/NC)6SaCff0o1r0d1e1Cd
the desired macrocycle TEG-[2]MSQ 5 in 32% yield (Scheme 2). The
lower yield for this reaction as compared to that obtained for the
analogous synthesis of 1 is likely a result of decomposition of the
unstable 17 under the reaction conditions. Recrystallization of
compound 5 from a concentrated solution in DMSO/H2O (approx.
8:2 v/v) afforded crystals suitable for single crystal X-ray diffraction
analysis (Figure 6), providing confirmation of the structure of the
anticipated macrocyclic structure 5. The crystal structure reveals
that 5 adopts a similar S2 symmetry ‘chair-like’ conformation to
that of 1 in the absence of an anionic guest. In contrast to
however, DMSO has not been ‘captured’ and incorporated into
the solid state. Instead hydrogen bonds between the
1
squaramide groups of one adjacent molecule link them together
such that their aligned macrocycles form an approximately
rectangular channel (Fig. 6C and Figs. S37 and S38 in the
supplementary material) in the solid state. The cavity
dimensions are very similar to those of 1 and 1+SO42-, with the
corner carbon to corner carbon distance across the squaramide
group being 6.125(4) Å and that across the phenyl group being
5.031(4) Å. The internal corner angles are approximately 90.1,
and 89.9°. The least squares planes of the squaramide and
phenyl groups are respectively inclined at angles of 77.9(1) and
53.23(7)° with respect to that of the macrocycle cavity. Related
by inversion symmetry, the two planes defined by the
squaramide groups are parallel to each other and separated by
distance of 4.891(4) Å; this constitutes the smallest dimension of
the channel formed by the alignment of the macrocycles. The
squaramides of a macrocycle have an approximate relative
lateral offset of approximately 1.70 Å and a centroid to centroid
distance of 5.179(3) Å. The phenyl centroid to centroid distance
is 8.308(4)Å and they have a relative offset of approximately
6.58 Å. Additional details are provided in the supplementary
information, which includes a tabulated comparison of aspects
of the cavity geometry of 1, 3, 1+SO42- and 5.
Figure 5. Energy minimised molecular model of the complex formed between 2
and sulfate.
In the absence of X-ray quality crystals, the complex between the
[3]-MSQ 2 and sulfate was also modelled to give an indication of the
binding mode (Figure 5). The modelled structure shows that 2 can
coordinate to sulfate through 6 N–H hydrogen bonds, with all four
of the sulfate oxygen atoms involved (in contrast to the complex
observed for 1) where two of the squaramide units each have two
hydrogen bonds to
a single oxygen atom, while the third
squaramide unit forms one hydrogen bond from each of its NH
protons to two of the oxygen atoms. The three aromatic rings all
have a single proton projecting towards the sulfate ion, explaining
the downfield shift observed for the signal attributable to these
protons in the 1H NMR experiments, and suggesting that in addition
to the 6 N–H hydrogen bonding interactions, an additional 3 C–H
bonding interactions39, 40 from the receptor to the anion may be
contributing to the high binding affinity observed for this complex.
Initial attempts to prepare 6 using a similar synthetic route to
that employed to prepare 2 were unsuccessful, presumably as a
result of the instability of diamine 17. We therefore employed a
slightly modified synthetic route to obtain 23. This began with the
reduction of diazide 16 using only one equivalent of
triphenylphosphine (Ph3P) in THF/water to afford the monoamine
19 in 71% yield. The reaction of compound 19 with diethyl squarate
7 and triethylamine in ethanol afforded diazide substituted
squaramide 20 in 77% yield. A single pot Staudinger reduction of 20
and subsequent reaction with compound 18, synthesised
previously, in the presence of triethylamine and ethanol afforded
the desired TEG-[3]MSQ 6 in 35% over the two steps. Gratifyingly,
the incorporation of the TEG esters provided significantly increased
solubility for both 5 and 6 as compared to the parent macrocycles 1
and 2. 5 and 6 were found to be soluble in mixtures of up to 33%
water in DMSO, enabling anion binding experiments to be
performed in this more competitive solvent mixture.
Synthesis and Characterisation of MSQs 5 and 6:
MSQs 1 and 2 exhibited high affinity and, in the case of 2, excellent
selectivity for sulfate in DMSO (0.5% H2O) but investigation of their
binding affinity in solvents other than DMSO was hampered by the
low solubility of these compounds. In order to overcome this
limitiation we designed and synthesised the triethylene glycol
monomethyl ether - macrocyclic squaramide based receptors “TEG-
MSQs” 5-6 (Figure 1). We anticipated that the incorporation of the
TEG esters would significantly improve the solubility of the MSQs in
DMSO as well as providing increased water solubility.
In order to prepare 5 and 6 via our previously established synthetic
route, we required the TEG-functionalised diamine 17. The
reduction of the known bis(azidomethyl)benzene derivative 1641
(Scheme 2) was performed via Staudinger reaction conditions using
triphenylphosphine (Ph3P) in THF/water to provide the benzyl
diamine 17 in 71% yield. Compound 17 was observed to be prone to
rapid decomposition at room temperature. However, rapid
purification of 17 followed by immediate reaction with diethyl
squarate 7 in ethanol afforded compound 18 in 80% yield.
6 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6SC01011C
Journal Name
ARTICLE
O
R
O
R
O
R
O
R
O
O
O
O
O
O
O
O
NH
NH
HN
NH
HN
(i)
(ii)
(iii)
HN
O
O
N3
N3
NH2
NH2
16
17
18
O
R
5
(iv)
O
O
O
Hb
O
N
H
N
H
O
O
R
R
O
O
Ha
O
R
N
H
N
H
R
R
NH
HN
(v)
(vi), (vii)
O
O
NH
HN
N3
N3
NH2
N3
O
O
19
20
O
O
R =
O
O
O
R
6
Scheme 2: Synthesis of the desired macrocyclic squaramide based receptor frameworks 5 and 6. Synthesis: (i) Ph3P (2 equiv.), THF/H2O, RT, 8 hrs, 71%; (ii) 7 (2
equiv.), EtOH, Et3N, overnight, RT, 80%; (iii) 17 (1 equiv.) EtOH, Et3N, overnight, RT, 32%; (iv) Ph3P (1 equiv.), THF/H2O, RT, 8 hrs, 52%; (v) 7 (0.5 equiv.) EtOH, Et3N,
overnight, RT, 77%; (vi) Ph3P, THF/H2O, RT, 8 hrs; (vii) 18 (1 equiv.) EtOH, Et3N, overnight, RT, 35%.
sulfate is not only a result of the [3]MSQs having an additional
Binding Studies of TEG-MSQs 5 and 6
squaramide binding site compared to 5, together with additional C–
H hydrogen bonding interactions to the anion, but primarily
because the cavity size of the [3]MSQ is well-matched to the size of
the sulfate ion resulting in the anion sitting inside the cavity of the
TEG-[3]MSQ 6 in a manner that protects it from solvent molecules,
as indicated by the molecular model depicted in Figure 7. In
contrast, the crystal structure of the [MSQ 1·SO4]2- complex (Figure
4, A) shows that, in this smaller receptor, the sulfate is perched on
top of the macrocyclic cavity and still exposed to solvent molecules.
Given that MSQs 1 – 4 were found to bind strongly to SO42- (and
-
H2PO4 in the case of 1) we focused on determining the ability of 5
and 6 to distinguish between tetrahedral and divalent anions, since
even the natural sulfate-binding protein (SBP) is unable to provide
high levels of discrimination between its target sulfate ion (SO42- Ka
= 8.33×106 M-1) and ions such as SeO42- (Ka = 0.2×106 M-1) or CrO4
2-
(Ka = 3.33×106 M-1) at neutral pH.9 Hence, quantitative NMR binding
studies of TEG-MSQs 5 - 6 for a range of anions were conducted in
1:2 (v/v) H2O/DMSO-d6 using WATERGATE (see ESI for details) to
suppress the H2O signal. Binding affinities calaculated by fitting to a
1:1 binding model are given in Table 2. Compared with the binding
observed for MSQs 1 and 2 to sulfate (Ka > 104 M-1) in DMSO-d6, in
the more competitive solvent mixture [1:2 (v/v) H2O/DMSO-d6],
TEG-[2]MSQ 5 showed a dramatic decrease in affinity for sulfate (Ka
= 1819 M-1). However, TEG-[3]MSQ 6 still exhibited affinity for SO4
2-
that was too high to quantify (Ka >104 M-1) in these very competitive
conditions. We speculate that the high binding affinity of 6 for
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Journal Name
60) is significantly higher than that of the SBP (Ka(sulfate)/ Ka(chromate)
=
View Article Online
DOI: 10.1039/C6SC01011C
2.5). In order to better assess the ability of both 5 and 6 to
discriminate between the doubly charged SO42- and HPO42- ions we
also performed titrations with these ions in 2:1 DMSO-d6 / aq.Tris
buffer (15 mM) at pH 9.1. Under these conditions, for [3]-MSQ 6 Ka
values of 2045 M-1 and 13 M-1 were obtained for sulfate and
hydrogenphosphate, respectively giving a selectivity of Ka(sulfate)
/
Ka(hydrogenphosphate) = 157.§§§
Table 2: Association Constants (Ka) of receptors 5 and 6 for anionsa
5_1:2 v/v
H2O/DMSO H2O/DMSO
6_1:2 v/v
Anion
SO42-/ M-1
1820
57
166
17
96
--b
> 104
16
-
H2PO4 / M-1
SeO42-/ M-1
Cr2O72-/ M-1
CrO42-/M-1
1900
--b
165
--b
-
HCO3 /M-1
NO3 /M-1
--b
--b
-
AcO-/ M-1
--b
--b
BF4 / M-1
--b
--b
-
ClO4 / M-1
--b
--b
-
p-Toluenesulfonate/ M-1
--b
--b
ReO4 / M-1
--b
--b
-
a Titrations were performed in 1:2 (v/v) H2O/DMSO-d6 with at 300 K. b Binding too
weak to obtain an accurate binding constant. Estimated errors < 15%.
Figure 7: Calculated Space Filling Model of [TEG-MSQ 6·SO4]2- complex
.
Following the observation that 6 shows significantly higher
affinity for sulfate than for other anions, we performed binding
studies of 6 in aqueous mixtures mimicking the anion compositions
in both nuclear waste (Figure 8) and plasma (see ESI). Notably, in
these more complex mixtures, the solubility of 6 increased, such
that experiments in 1:1 DMSO-d6:H2O mixtures were possible. This
is attributed to binding of 6 to the anionic species already present in
solution. In a 1:1 v/v mixture of DMSO-d6 : aqueous plasma
Figure 6: X-ray crystalllography determined structure of 5 (A) side view; (B)
Hydrogen bonds link adjacent molecules to form an approximately rectangular
channel parallel to the a axis and centred on (0,1/2,0); (C) View down the a axis
of the unit cell showing the pseudo-rectangular channels arising from hydrogen
bonds between adjacent squaramides. Disorder at the end of the pendant TEG
chains is not shown.
[3]TEG-MSQ 6 also reveals remarkable selectivity for sulfate
amongst other tetrahedral anions (Table 2), with binding affinity for
sulfate at least three orders of magnitude stronger than that
-
electrolytes (20mM Tris buffer, pH 7.4, 1.5 mM H2PO4 /HPO42-, 106
-
2-
observed for phosphate species (a mix of H2PO4 and HPO4 at
-
mM Cl- , 28 mM H2CO3/HCO3 ) TEG-MSQR 6 was still able to bind to
-
neutral pH) and perchlorate (ClO4 ). Furthermore, TEG-[3]MSQ 6
sulfate ions (1 equiv. SO42- = 2.5 mM, final SO42- concentration = 30
mM) with a distinct shift of the aromatic proton observed in the
NMR titration experiment (the squaramide protons were too broad
to observe under these conditions), giving an apparent Ka = 438 M-1
(±15%) when fit to a 1:1 binding model. Furthermore, a titration of
binds to sulfate with significantly higher affinity than it does to
selenate or chromate ions, respectively. While selectivity of 6 for
sulfate over selenate (Ka(sulfate)/ Ka(selenate) = > 5)§§ is likely to be lower
than that of the SBP (Ka(sulfate)/ Ka(selenate) = 41), the ability of 6 to
discriminate between sulfate and chromate (Ka(sulfate)/ Ka(chromate) = >
8 | J. Name., 2012, 00, 1-3
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2-
2-
was added dropwise at 1.7 ml/hr via syringe pumpV.ieTwhAerticmleixOtnulirnee
DOI: 10.1039/C6SC01011C
was stirred for 72 hrs. The resulting precipitate was isolated by
6 with sulfate (1 equiv. SO4 = 2.5 mM, final SO4 concentration =
30 mM) performed in mimicked nuclear waste solution (1:1 DMSO-
d6 : H2O, pH = 3.2, 2.5 mM phosphates, 10 mM TBACl, 250 mM filtration and washed with EtOH then Et2O to yield the product as a
2-
TBANO3) gave an apparent Ka for SO4 = 1204 M-1 (±15%), with
beige/yellow solid.
distinct downfield shifts of the squaramide NH protons as well as
shifts for the aromatic protons. These results directly showcase the
ability of MSQs to bind sulfate in aqueous media and in the
presence of a large range of interferants, and demonstrate the
potential applications of MSQs in sulfate separation from nuclear
wastes and in plasma sulfate assays.
Conclusions
In summary, we have successfully synthesized a new family of
squaramide based macrocycles via a facile synthetic process, and
shown that these can be readily functionalised to modulate their
solubility. Evaluation of the anion-binding affinity and selectivity of
[2]- and [3]-MSQs revealed remarkable anion binding in a range of
conditions, from 0.5ꢀ% v/v H2O/DMSO-d6 to 50ꢀ% v/v H2O/DMSO-d6
where the meta-substituted derivatives 1, 2, 5, 6 were found to be
2−
extremely potent and selective ligands for SO4 in highly
competitive conditions, with
6 showing a better ability to
discriminate between sulfate and chromate ions than the sulfate
binding protein. Experiments under simulated competitive
conditions reinforce the opportunity for applications of these
sulfate-selective receptors in both nuclear waste remediation and
plasma sulfate detection. We are currently investigating these and
related systems in more detail, and will report on these studies in
due course.
Acknowledgements
We thank the ARC (DP140100227) and the University of
Sydney for funding. Dr Michael Murphy is thanked for
assistance with aspects of the crystallography.
Notes and references
1
§ Variable temperature H NMR experiments were attempted but
these did not provide further information on the rate of
interconversion of the conformers, partly as a result of the low
solubility of these compounds in DMSO.
Figure 8: (A): 1H NMR titration of compound 6 with (TBA)2SO4 in 1:1 (v/v) H2O-
DMSO-d6 mixture (pH 3.2) with 2.5 mM phosphates, 10 mM TBACl, 250 mM
§§ Because Ka (Sulfate) for 6 is > 104 M-1, the minimum value was
chosen to calculate the selectivity ratio.
TBANO3 at 300 K. (B): Comparison isotherms of squaramide NH, Aromatic
2-
protons (Ar-Ha, Hb) in the presence of increasing concentrations of SO4
.
Estimated error in Ka <15%.
§§§ In 2:1 DMSO-d6 / aq.Tris buffer (15 mM) at pH 9.1, [2]-MSQ
gave Ka = 235 M-1 for SO42- and Ka < 10 M-1 for HPO42-, respectively.
Experimental
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