Interfacial Supramolecular Structures of Amphiphilic Receptors Drive Aqueous Phosphate Recognition

Article abstract of DOI:10.1021/jacs.9b02148

Phosphate remediation is important for preventing eutrophication in fresh waters and maintaining water quality. One approach for phosphate removal involves the utilization of molecular receptors. However, our understanding of anion recognition in aqueous solution and at aqueous interfaces is underdeveloped, and the rational design of surface-immobilized receptors is still largely unexplored. Herein, we evaluated the driving forces controlling phosphate binding to elementary amphiphilic receptors anchored at air-water interfaces. We designed biologically inspired receptors with neutral thiourea, positively charged guanidinium, and thiouronium units that all formed Langmuir monolayers. Phosphate binding was quantitatively examined using surface pressure-area isotherms and infrared reflection-absorption spectroscopy (IRRAS). The receptors within this homologous series differ in functional group, charge, and number of alkyl chains to help distinguish the fundamental components influencing anion recognition at aqueous interfaces. The two charged receptors bearing two alkyl chains each displayed strong phosphate affinities and 10³- and 10¹-fold anti-Hofmeister selectivity over chloride, respectively. Neutral thiourea and the single-chain guanidinium receptor did not bind phosphate, revealing the importance of electrostatic interactions and supramolecular organization. Consistently, charge screening at high ionic strength weakens binding. Spectroscopic results confirmed phosphate binding to the double alkyl chain guanidinium receptor, whereas surface pressure isotherm results alone showed a minimal change, thus emphasizing the importance of interfacial spectroscopy. We found that the binding site identity, charged interface created by the electrical double layer, and supramolecular superstructure all affect interfacial binding. These detailed insights into phosphate recognition at aqueous interfaces provide a foundation to develop efficient receptors for phosphate capture.

Full text of DOI:10.1021/jacs.9b02148

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Article
Interfacial Supramolecular Structures of Amphiphilic
Receptors Drive Aqueous Phosphate Recognition
Jennifer F. Neal, Wei Zhao, Alexander J. Grooms, Morgan A.
Smeltzer, Brittany M. Shook, Amar H Flood, and Heather C. Allen
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 26 Apr 2019
Downloaded from http://pubs.acs.org on April 26, 2019
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Interfacial Supramolecular Structures of Amphiphilic Receptors Drive Aqueous Phosphate
Recognition
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Jennifer F. Neal , Wei Zhao , Alexander J. Grooms , Morgan A. Smeltzer , Brittany M. Shook ,
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Amar H. Flood* , Heather C. Allen*
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† Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210,
United States
‡ Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States
*corresponding authors
ABSTRACT
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Phosphate remediation is important for preventing eutrophication in fresh waters and
maintaining water quality. One approach for phosphate removal involves the utilization of
molecular receptors. However, our understanding of anion recognition in aqueous solution and at
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13 aqueous interfaces is underdeveloped and the rational design of surface-immobilized receptors is
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still largely unexplored. Herein, we evaluated the driving forces controlling phosphate binding to
elementary amphiphilic receptors anchored at air-water interfaces. We designed biologically-
inspired receptors with neutral thiourea, positively charged guanidinium, and thiouronium units
that all formed Langmuir monolayers. Phosphate binding was quantitatively examined using
surface pressure-area isotherms and infrared reflection-absorption spectroscopy. The receptors
within this homologous series differ in functional group, charge, and number of alkyl chains to
20 help distinguish the fundamental components influencing anion recognition at aqueous interfaces.
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The two charged receptors bearing two alkyl chains each displayed strong phosphate affinities and
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10 and 10 fold anti-Hofmeister selectivity over chloride, respectively. Neutral thiourea and the
23 single-chain guanidinium receptor did not bind phosphate, revealing the importance of
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electrostatic interactions and supramolecular organization. Consistently, charge screening at high
ionic strength weakens binding. Spectroscopic results confirmed phosphate binding for the double
26 alkyl chain guanidinium receptor, whereas surface pressure isotherm results alone showed a
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minimal change, thus emphasizing the importance of interfacial spectroscopy. We found that the
binding site identity, the charged interface created by the electrical double-layer, and
supramolecular superstructure all affect interfacial binding. These detailed insights into phosphate
recognition at aqueous interfaces provide a foundation to develop efficient receptors for phosphate
capture.
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INTRODUCTION
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The development of receptors capable of overcoming the high hydration energies and inherent
difficulties of aqueous phosphate recognition is a necessary challenge for a modern sustainable
35 society.1–4 Demand for phosphate fertilizers grows in conjunction with the global population.5,6
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Consequently, phosphate runoff from agricultural non-point sources threatens fresh water
supplies,7–10 and phosphate-driven eutrophication in natural waters can generate harmful algal
38 blooms which negatively impact aquatic life.7,9,11–13 Furthermore, phosphate rock is a non-
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renewable resource and all losses have negative impacts on long-term food security. Thus, there is
a continued need to understand the principles of recognition and underlying physical-organic
interactions to design synthetic phosphate receptors for use in sensing and remediation
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42 applications. Challenges of phosphate recognition in bulk water consist of the high energetic cost
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of phosphate dehydration (Ghyd = –465 kJ/mol),15 the large dielectric constant (ε) of bulk water
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(ε = 80) that screens all electrostatic interactions, the large size-to-charge ratio of the phosphate
guest,17,18 and the acid-base properties of phosphate1,17,19 (pK = 2.16, pK = 7.21, pK =
a1
a2
a3
12.32).20 We seek to exploit the unique and technologically-relevant environment present at
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aqueous interfaces, akin to those of remediation membranes, to overcome some of the inherent
disadvantages of phosphate recognition in bulk solution.
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The air-water interface is a unique microenvironment. It has a much lower dielectric constant
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50 than bulk water (ε < 40),21,22 thus amplifying electrostatic interactions to increase phosphate
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binding. In addition, Langmuir monolayers provide a confined local environment and have the
advantage of preorganization,23,24 which is expected to enhance recognition.2,25 The long
hydrophobic tails of the receptor are well ordered upon compression while the hydrophilic
component (the binding site) is anchored down into the water and available for phosphate binding.
Once anchored, they can also benefit from the lower dielectric in the interfacial microenvironment
to boost affinity.
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Pioneering work by Kunitake on interactions between monolayers of guanidinium
58 amphiphiles, bearing a single hydrophobic tail, and aqueous solutions of adenosine triphosphate
revealed that affinity is greatly enhanced at the monolayer interface and by multivalency.26–29
A
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recent study showed that the interfacial binding between a monolayer of organophosphate and
61 aqueous guanidiniums could be studied in situ. The affinity was also significantly enhanced at the
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air-water interface relative to aqueous solution (>10,000 times) in the model system composed of
the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) and the amino acid
arginine.30 Although the enhancement in binding at the air-water interface is promising, the
65 governing principles of interfacial binding are underexplored, which has hindered the rational
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design of effective surface-bound receptors.
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Figure 1. Structures of the octadecyl-based, U-shaped neutral receptor U-thio, U-shaped and
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for phosphate binding at the air-water interface.
73 Molecular and Supramolecular Design
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We explored a series of elementary and biologically-inspired amphiphilic receptors to help
elucidate the design principles of phosphate recognition. The guanidinium and thiourea motifs are
76 well-known to display binding selectivity towards phosphate,28,31–35 with only a few studies
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extended to interfacial environments for guanidinium, thiouronium, and thiourea. We expect
the monolayer-organized receptors with these binding sites will retain selectivity for phosphate
binding over chloride at interfaces.
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We tuned the structures of the homologous receptor series by modulating the binding sites and
alkyl chains, and evaluated their binding to aqueous phosphate in the subphase using in situ
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methods. Octadecyl chains (C H ) were chosen to facilitate monolayer formation, and the
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83 number of alkyl chains was varied to determine how the organization of the monolayer affects
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phosphate affinity. For the binding motif, the charged guanidinium and thiouronium moieties are
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almost isosteric while the charge-neutral thiourea group was chosen to investigate how different
chemical and physical driving forces contribute to phosphate recognition. Four elementary
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87 receptors were synthesized (Figure 1) to test phosphate binding: bis-octadecyl substituted
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guanidinium where the two chains are directed upwards to make a U-shape (U-guan ), the similar
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thiouronium (U-thiouro ), and thiourea (U-thio), as well as mono-octadecyl substituted
guanidinium with a single chain (S-guan+).
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The guanidinium functional group of U-guan and S-guan has a trigonal planar geometry
with a positive charge formally distributed across three nitrogen atoms by resonance stabilization
and several possible hydrogen bonding donor sites.36–38 Thus, the guanidinium moiety typically
94 binds to phosphate with two charge-assisted hydrogen bonds complementary to the structure of
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phosphate’s hydrogen bond acceptor sites. U-thiouro also has the advantage of directed, planar
hydrogen bonding interactions.39–41 Thiouronium binding sites are largely unexplored in
97 supramolecular chemistry, but nevertheless have been shown to have strong binding affinity to
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oxyanions42–46 arising from the charge on the large polarizable sulfur atom, providing charge-
assisted hydrogen bonding. While the thiourea binding site of U-thio has the advantage of
hydrogen bonding, it does not bear the positive charge. In addition to the chemical features of
these receptors, differences in the binding may be modulated by the number of alkyl groups.
Interfacial binding of preorganized Langmuir monolayers was evaluated using the surface
sensitive techniques of surface pressure – area isotherms (Π-A) and infrared reflection absorption
spectroscopy (IRRAS). Although a few other in situ surface techniques (vibrational sum frequency
generation spectroscopy,47 Brewster angle30 and fluorescence48 imaging, and X-ray
reflectometry)49,50 would also be useful, Π-A isotherms and IRRAS were the preferred tools
because of their high throughput capabilities. Ex situ methods require Langmuir-Blodgett transfer
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of the monolayer to a solid substrate before further analysis by X-ray photoelectron spectroscopy,
UV-vis spectroscopy, or other spectroscopic techniques.26,31
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The use of IRRAS in this study enabled a phosphate binding response for the charged U-guan+
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and U-thiouro receptors to be recorded, verifying the effectiveness of the technique for detecting
these binding interactions. Although Π-A isotherms have been used extensively in the literature to
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demonstrate binding between ions of salts and organic compounds,
our isotherm results
often showed a null response to binding. We attribute this outcome to the two alkyl chains
impeding macroscopic changes in the molecular area of the monolayer despite phosphate binding
at the interface. Competitive binding between phosphate and chloride revealed that monolayers of
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the two charged U-guan and U-thiouro receptors are selective towards phosphate. This behavior
is the opposite from that expected from the Hofmeister series, which highlights the benefit of
binding at the interface where there is only partial dehydration. However, at a constant ratio of
phosphate to chloride, the ionic strength weakens the observed selectivity; at high ionic strength
with significant charge screening there is a weakening of electrostatic interactions. We have found
that a constant chloride concentration, <10 mM in the bulk aqueous subphase, is necessary to limit
screening of the electrostatic potential of the receptors. Our findings suggest that the nature of the
binding site (chemical), the contribution of electrostatics (physical), and the organization of the
receptor in the molecular monolayer (supramolecular superstructural) all affect phosphate binding
affinity. These results provide some of the first fundamental understanding of rational
supramolecular design and of how anion recognition is influenced by the local environment at the
receptor-water interface.
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RESULTS AND DISCUSSION
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Identification of Receptor-Phosphate Binding at the Air-Water Interface
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Spectroscopic measurements by IRRAS proved to be a valuable approach to interrogate the
associated binding interaction between the molecular monolayer and aqueous phosphate. IRRAS
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spectra in the low frequency binding region (1600 to 950 cm ) were collected across the receptor
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series to elucidate whether the S-guan , U-guan , U-thio, and U-thiouro receptors interact with
aqueous phosphate ions. The IRRAS spectra were collected at a surface pressure (Π) of 40 mN/m,
which is in the condensed phase of the monolayer for all receptors. It is apparent from these spectra
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that the charged S-guan receptor (Figure 2a) bearing only one alkyl anchor and the neutral U-
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thio receptor (Figure 2c) have negligible spectral changes with phosphate. The two U-shaped and
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charged receptors U-guan (Figure 2b) and U-thiouro (Figure 2d) both show significant changes
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upon phosphate addition.
The IRRAS spectra show clear signatures from the interfacial binding with phosphate.
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Specifically, new peaks appear in the spectra for U-guan (Figure 2b) and U-thiouro (Figure 2d)
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at approximately 1158 and 1074 cm , which we assign to the phosphate’s asymmetric (ν PO )
as
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and symmetric (ν PO ) stretching modes.
The presence of these phosphate peaks strongly
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supports phosphate binding to the receptor molecules. According to the IRRAS equation, –log
(R/R ), where R is reflectivity and R is the solution prior to depositing the monolayer, the
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phosphate modes are represented in both the numerator and denominator of the equation.
Therefore, the phosphate modes should only be present when there is binding. A series of control
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experiments further confirm that the two U-guan and U-thiouro receptors are indeed binding to
phosphate (for details, see the Supporting Information).
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The presence of phosphate modes in the IRRAS spectra of U-guan and U-thiouro verify
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that these receptors bind phosphate. Conversely, the absence of phosphate peaks with S-guan and
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U-thio receptors show that they have no interaction with phosphate. Furthermore, the phosphate
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modes in the U-guan and U-thiouro spectra are blue shifted relative to aqueous phosphate. For
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the U-guan and U-thiouro receptors, changes from hydrated phosphate to less hydrated
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phosphate leads to blue shifts of approximately 20 cm and 5 cm for the ν PO and ν PO
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as
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respectively (Figures 2, S8). We propose from literature precedence56–58 that the blue shift
correlates with decreased hydrogen bonding. The logical interpretation of the blue shift, therefore,
is that the net hydrogen bonding from the receptor is weaker than from the displaced portion of
the hydration sphere. Weaker hydrogen bonding interactions stemming from the receptor are likely
compensated by the stabilizing nature of the ion-ion interactions at the air-receptor-interface. In
addition to the phosphate modes, there is further spectroscopic evidence to verify the extent of
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phosphate binding. In the U-guan spectra (Figure 2b, black), we tentatively assign the peak at
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approximately 1362 cm to the asymmetric N-C-N stretch (ν NCN) based on tetramethyl
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guanidine. This mode decreases in intensity upon phosphate addition, in a way that correlates
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with phosphate interaction. The IRRAS spectra of U-thiouro on 10 mM phosphate (Figure S9)
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with p-polarization and s-polarization shows a blue shift (~12 cm ) and narrowing of the 1600
cm−1 peak upon phosphate addition, providing additional support for binding. This mode is
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assigned to the CN stretch (ν CN) with additional contribution from the N-H bend (δ N-H).
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73
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+
+
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Figure 2. IRRAS spectra in the condensed phase (Π = 40 mN/m) for (a) S-guan , (b) U-guan ,
+
(c) U-thio, and (d) U-thiouro on water (black trace) and 10 mM phosphate (red trace) reveal that
+
+
1
1
75
76
phosphate is interacting with the U-guan and U-thiouro receptors. The symmetric and
asymmetric stretches from bound phosphate are marked.
1
1
77
78
Surface pressure-mean molecular area (Π-A) compression isotherms were also recorded to
+
+
179
test for phosphate recognition. However, Π-A isotherms of S-guan , U-guan , U-thio, and U-
+
1
1
1
1
80
81
82
83
thiouro receptors (Figure 3a-d, respectively) on water and 10 mM sodium dihydrogen phosphate
do not always show changes in the presence of phosphate. Π-A isotherm results of the U-thio and
+
S-guan receptors on 10 mM phosphate were identical to trials on water (within one standard
+
deviation), and the U-guan receptor showed only a minimal expansion. For these three receptors,
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the Π-A isotherms did not produce a significant expansion or compression of the monolayer upon
phosphate addition, giving the appearance that the receptors were not interacting with phosphate.
It is important to recognize, however, that the isotherms for the U-shaped receptors composed of
186
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92
U-guan and U-thio may not be informative to binding as it is possible that the large size of the
two alkyl chains obscure changes associated with the headgroup region. The interfacial
organization of these U-shaped receptors (Figure 4) is expected to direct the binding site (i.e.,
headgroup) and thus any bound phosphate into the region beneath the monolayer. This binding
arrangement of phosphate will not necessarily influence the alkyl chain packing. With this binding
mechanism in mind for these U-shaped receptors, it is possible that changes in the Π-A packing
density would be negligible.
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Figure 3. The Π-A isotherms of S-guan and U-thio (a,c) on water (black trace) and a 10-mM
sodium dihydrogen phosphate solution (red trace) do not produce significant changes in the surface
+
+
199
pressure with phosphate. (b) U-guan shows a minimal expansion and (d) U-thiouro exhibits a
large monolayer expansion on phosphate strongly suggesting binding. Shaded regions represent
one standard deviation above and below the mean.
2
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00
01
2
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02
03
+
However, for the single chain receptor (S-guan ), Π-A isotherms should be more sensitive
+
204
to binding because of the higher packing density. If the S-guan receptors were binding phosphate,
2
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05
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the phosphate anions could be driven in-between the receptor molecules, and a change in the mean
molecular area (MMA) of the monolayer would confirm binding. However, the isotherm for the
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S-guan receptor does not change with phosphate (Figure 3a), which suggests the S-guan
receptor is not interacting with phosphate.
2
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Figure 4. Schematic representation of the hypothesized binding event for the U-shaped receptors.
212
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Phosphate is binding beneath the monolayer instead of in-between the molecules of the monolayer.
Impact of the Supramolecular Superstructure
It is important to appreciate differences in the environment confined by the 2D air-water
interface compared to host-guest recognition chemistry commonly studied in bulk solution. Here,
the receptors are tethered to the air-water interface and when situated in the confined space of the
monolayer, increased receptor-receptor interactions can occur. With intermolecular spacing of S-
2
2
16
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218
+
2
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19
20
21
guan defined by just one alkyl chain, we believe it does not separate the guanidinium headgroups
from each other. They are thus free to interact with neighboring headgroups (receptor to receptor).
It has been shown62–68 that guanidinium moieties can form pairs in water despite being similarly
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charged, and are known as “magic” arginine complexes. They fall into a growing set of same-
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charge ions that can associate together.70–78 The guanidinium-guanidinium interactions are
consistent with our results, and it is therefore reasonable to conceive that the receptor can self-
associate and contribute to the inhibition of phosphate binding.
224
0
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+
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30
31
In the condensed state of the S-guan monolayer (i.e., compacted), we suggest that the close
proximity of the headgroups allows for self-association. By contrast, the U-shaped receptors
cannot easily form interactions with neighboring receptor headgroups on the basis of the larger
intermolecular spacing defined by the alkyl chains. All our results illustrate that the charged U-
+
+
guan and U-thiouro receptors are interacting with phosphate, yet the neutral U-thio receptor is
not. These findings demonstrate the importance of electrostatics on the binding affinity. The
+
+
232
thiouronium and guanidinium headgroups of U-thiouro and U-guan can form charge-assisted
1
2
2
33
34
hydrogen bonding to the phosphate and the combination of electrostatics and hydrogen bonding
3
0
provides greater phosphate affinity. Additionally, we propose that the low dielectric constant and
1
235
preorganization of the monolayers at the air-water interface further stabilize this interfacial
2
36
binding.
2
2
2
37
38
39
Spectroscopic results proved to be more informative than Π-A isotherms alone and give
mechanistic insight into the receptor-phosphate interaction; nevertheless, Π-A isotherms still offer
+
valuable information for the U-thiouro receptor. Returning to the Π-A isotherms (Figure 3d), U-
+
2
240
thiouro has a considerable mean molecular expansion (MME) from 39.4 (±0.6) Å on water to
2
2
2
2
41
42
99.0 (±0.7) Å on 10 mM phosphate,taken at a surface pressure of 5 mN/m. The 59.6 Å expansion
is too large to rationalize simply as coming from the binding of a single phosphate anion and, thus,
a reasonable explanation for this behavior is supramolecular reorganization50 within the
monolayer.
243
44
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46
To elucidate any reorganization, IRRAS spectra of the U-thiouro receptor were taken at
5, 10, and 40 mN/m on both water and 10 mM phosphate (Figure 5a, b). The peak at 1468 cm−1
−
1
247
is assigned to the CH scissoring (δ CH ) and peaks at 2963, 2924, 2883, and 2850 cm are the
2 2
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52
53
CH asymmetric (ν CH ), CH asymmetric (ν CH ), CH symmetric (ν CH ), and CH
3 as 3 2 as 2 3 s 3 2
symmetric (ν CH ) stretching modes, respectively. The lowering of the intensity is consistent
s
2
−
1
with lower molecular density. Yet, the scissoring mode at 1468 cm remains at the same relative
peak position at both low and high pressures. The existence of the single peak and at a constant
peak position suggests the monolayer remains in a hexagonal lattice structure throughout the
isotherm (Figure 5a).79
+
2
54
Comparing the U-thiouro receptor on phosphate at low surface coverage at 5 and 10
255
mN/m with their larger MMA to the more condensed monolayer with a smaller MMA at 40 mN/m,
2
2
56
57
we observe a diagnostic red shift in the ν CH and ν CH stretching modes (Figure 5b). To
as
2
s
2
emphasize this red-shift, the spectra recorded at different pressures (Figure 5b) are plotted on
+
258
different axis scales and the relative peak positions of U-thiouro on water are included as black
2
2
2
2
2
2
59
60
61
62
63
64
dotted lines. Upon phosphate binding, the molecular monolayer undergoes reorganization. The red
shift of the CH modes indicates greater gauche conformers in the earlier expanded regions of the
2
monolayer (i.e., lower Π, larger MMA) with more trans conformers in the later stages of the
isotherm (i.e. higher Π, smaller MMA). Furthermore, the presence of the methyls’ symmetric
stretch, ν CH at lower pressure (5 and 10 mN/m) but not at higher pressure (40 mN/m) supports
s
3
this hypothesis of reorganization.
2
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68
Figure 5. IRRAS spectra showing the U-thiouro monolayer reorganizes upon phosphate binding.
−
1
+
(a) The CH scissoring mode (1468 cm ) of U-thiouro with 10-mM phosphate at 10 mN/m (red)
2
269
70
and 40 mN/m (dark red), and without phosphate (black), (b) and the alkyl stretching modes
−1
2
(between 3000 to 2800 cm ) with 10 mM phosphate at 5 mN/m (light red), 10 mN/m (red), and
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40 mN/m (dark red). Alkyl peak positions of U-thiouro on water are shown as black dotted lines
+
for reference. (c) Schematic illustration of the U-thiouro headgroup reorganizing and impacting
273
274
275
alkyl chain conformations (see monolayer expansion in Figure 3d).
0
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Phase changes and reorganization induced through interfacial interactions are common in
biological systems that occur when phospholipids bind to calcium,80–82 proteins,83,84 and
2
2
2
76
77
78
cholesterol.85–87 Interestingly, this change from a highly disordered monolayer to a more ordered
+
one upon phosphate binding is unique to the U-thiouro receptor, as we do not observe this change
279
with the other receptors. We believe this could come as a result of the bulky methyl group residing
on the positively charged sulfur atom of the thiouronium headgroup. Upon binding, this methyl
may need to reorganize to accommodate the large phosphate guest (Figure 5c).
2
2
80
81
2
2
82
83
Phosphate Selectivity
+
+
It is apparent from the IRRAS results that the U-guan and U-thiouro receptors are
promising for phosphate recognition. Therefore, it is important to determine whether these
receptors have selectivity towards phosphate over competing anions. We focus here on chloride
284
2
2
2
2
2
2
85
86
87
88
89
90
–
1 88
because of its prevalence in natural water (0-100 mg L ). To evaluate phosphate selectivity, we
recorded IRRAS spectra (Figure 6) of these receptors when the subphase was composed of a
8
9
mixture of phosphate and different relative equivalents of chloride. The IRRAS spectra of the U-
+
guan receptor were recorded with the two anions present at a 1:1 molar ratio at 10 and 1 mM.
Under these conditions (Figure 6a) the phosphate modes are present, suggesting that the monolayer
+
291
292
of the U-guan receptor is selective for phosphate over chloride at this ratio.
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93
+
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95
Figure 6. Competitive binding between phosphate and chloride for the U-guan receptor at 40
mN/m investigated using IRRAS spectroscopy. The IRRAS spectrum on water is shown as a gray
+
–
296
dotted line for clarity. (a) U-guan receptor shows good selectivity at 1:1 equivalence ([H PO ] :
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–
2
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97
98
[Cl ] = [P] : [Cl]). (b) Selectivity at 1:10 molar ratio ([P] : [Cl]) depends on the total concentration
−
1
+
of chloride. (c) Absolute integration of the ν (PO ) peak (1120 to 1020 cm ) of the U-guan
s
2
+
299
receptor with the integration of U-guan on water subtracted from each integration. (d) Schematic
0
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00
01
representation of the phosphate binding with U-guan at aqueous interface.
3
3
3
02
03
04
The ratio of the two anions was altered to evaluate the extent of the selective recognition
of phosphate over chloride. Interestingly, by increasing the relative amount of chloride (10 mM
NaH PO versus 100 mM NaCl), the phosphate modes are seen to diminish (Figure 6b). We can
2
4
+
305
infer from this observation that the U-guan receptor has lost selectivity towards phosphate under
these conditions. At 100 mM, charge screening can retard the electrostatic interactions90–92
3
3
06
07
between U-guan and phosphate in a manner consistent with the electrical double layer theory.93
+
+
308
To evaluate this further, we tested the selectivity of U-guan towards phosphate at a variety of
3
3
09
10
chloride concentrations (0.1-100 mM) while maintaining the 1:10 ratio of phosphate to chloride
(Figure 6b, c). Across these concentrations, the Debye length shrinks from ~30 to 1 nm. Phosphate
modes are present at lower chloride concentrations (0.1-10 mM Cl ) suggesting the U-guan+
receptor is selective for phosphate, by outcompeting chloride (Figure 6b).
–
311
3
12
−
1
3
3
3
13
14
15
A plot of the absolute integration (1120 to 1020 cm ) of the phosphate peak ν (PO) as a
s 2
function of chloride concentration (Figure 6c) shows a clear trend with the phosphate integration
reaching a maximal plateau when the chloride concentration is at or below 1 mM. We further
+
316
characterized the phosphate selectivity of the U-guan receptor using a chloride concentration of
88
3
3
17
18
1 mM to mimic the concentration of chloride present in natural waters and to limit the negative
effects of charge screening. Based on the presence of the phosphate modes in the IRRAS spectra
+
319
(Figure 7), the U-guan receptor shows selectivity towards phosphate up to a factor of 1000. It is
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clear (Figure 7) that the U-guan receptor demonstrates high selectivity towards phosphate over
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1
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–
– 32,94–97
chloride. This selectivity displays anti-Hofmeister behavior, i.e., H PO >> Cl .
Typically,
2
4
322
Hofmeister binding selectivity suggests that chloride would have stronger binding than phosphate
on account of its smaller hydration energy (–381 kJ/mol). Interestingly, the selective phosphate
binding also reverses expectations from Collins’ rule which is known as: “anions and cations form
stable ion pair interactions only when their hydration enthalpies match.”98,99 The hydration
energies are ranked as: phosphate (–465 kJ/mol), sodium (–409 kJ/mol), and chloride (–381
kJ/mol), while guanidinium is much more weakly hydrated.100 Consequently, the pairing of the
most hydrated phosphate with least hydrated guanidinium is not consistent with Collins’ rule.
0
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Figure 7. Maintaining a constant chloride background concentration of 1 mM NaCl (a) IRRAS
spectra reveal the phosphate modes up to 1:1000 ratios of phosphate to chloride ([P] : [Cl]) and
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(b) the integration of the ν (PO ) further supports the high selectivity of the U-guan receptor.
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Figure 8. Competitive binding of U-thiouro shows preference for phosphate over chloride at 1:1
ratio but there is less preference towards phosphate observed at 1:10 phosphate to chloride. The
IRRAS spectrum on water is shown as a gray dotted line for clarity
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We also evaluated the selectivity of the U-thiouro receptor towards phosphate (Figure 8).
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The selectivity experiments for U-thiouro at 1:1 phosphate to chloride (1 mM) show the presence
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of the phosphate modes and the selectivity of the U-thiouro receptor. However, upon decreasing
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the phosphate concentration to test a 1:10 ratio, the U-thiouro receptor shows a sharp decrease
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in phosphate peak intensity with a loss in selectivity. We infer from this observation that the
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guanidinium receptor U-guan is more selective than the thiouronium U-thiouro receptor for
phosphate (Figures 7, 8). The exact origin is the topic of current work in our labs. First, the
calculated electrostatic potential of the hydrogen atoms on the binding motifs (N-H) are similar
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for U-guan and U-thiouro (Figure S10). However, the guanidinium has two more potential
hydrogen bonding donors than the thiouronium. Second, the bulky methyl residing on the sulfur
atom of the thiouronium is believed to undergo re-organization upon phosphate binding (Figure
5c), which would introduce a free energy penalty that needs to be paid upon binding phosphate.
The organizational differences are the most probable cause for this difference.
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Ultimately, the selectivity experiments support our findings that electrostatics play a
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dominant role in the binding affinity. U-guan and U-thiouro bind stronger to phosphate than U-
–
thio. We also see that when electrostatics are screened by Cl , only the hydrogen bonding of U-
+
guan remains and is not strong enough to bind the phosphate (Figure 9a). The finding is consistent
with the negligible binding seen with U-thio. Our results also suggest that both the charged
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receptors U-guan and U-thiouro possess selectivity towards phosphate over chloride. This
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finding is consistent with our hypothesis that the headgroups of U-guan and U-thiouro promote
phosphate selectivity by charge-assisted hydrogen bonding (Figure 9b, c).
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Figure 9. Schematic illustration of (a) the charge screening at high ionic strength observed for U-
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guan , (b) U-thiouro outcompeting chloride to bind phosphate with 10−fold selectivity, and (c)
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U-guan displaying even higher selectivity for phosphate at 10 fold.
At the aqueous interface, the driving forces we have identified to influence recognition
come from the receptor, double-layer electrostatics, and the receptor’s organization in the
monolayer. Charges are essential for synergizing with the hydrogen bonding present in the U-
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guan and U-thiouro receptors to drive phosphate binding and selectivity. We demonstrated that
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screening of the double-layer charge distribution will shut down binding. We also showed that
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charges alone are not enough to enable phosphate binding. When using the S-guan receptor,
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binding was not observed. Thus, we also found that the supramolecular superstructure of the
monolayer influenced binding and that the binding site must be accessible to the aqueous interface.
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That is, the U-shaped receptor U-guan was found to bind phosphate likely by directing the
guanidinium binding site down towards the water subphase to enable binding. In support of this
3
2
idea, Buhlmann and Umezawa previously suggested that allowing phosphate to retain some of
its hydration shell will enable recognition to proceed in a way defined by the binding site instead
of by the steep cost of complete dehydration. We therefore believe that the supramolecular
superstructure within the monolayer is critical to controlling the location of the binding site relative
to the aqueous interface. In this case, the alkyl groups in the U-shaped receptors provide adequate
separation to accommodate the guest and hinder any receptor-receptor interactions in the confined
space of the monolayer. Of all these driving forces, the selection of the binding unit and its
organization relative to the aqueous binding region are subject to molecular synthetic design and
need to be controlled to enhance phosphate recognition at aqueous interfaces.
377
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Table 1. Summary of binding interactions observed by Π-A isotherms and IRRAS.
Method
Π-A
U-thio
U-thiouro+
U-guan+
Minor response
Binding
S-guan+
Large
Expansion
No response
No response
No response
No response
IRRAS
Binding
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By mimicking the hydrophobic binding sites on the surface of proteins and the resulting
low dielectric constant present at these locations,39,101,102 we exploit the interface to boost affinity
389
in our synthetic receptor system. The role of the lower dielectric constant at the interface is
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believed to enhance electrostatic interactions. It is, therefore, not surprising that the U-thiouro+
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and U-guan are stronger receptors for phosphate recognition than U-thio on account of the
importance of this physical characteristic of the interface. Throughout this work, we have shown
that chemical, physical, and supramolecular superstructure features serve as important design
criteria and that each plays an influential role in phosphate recognition in these monolayer systems.
These principles can be applied to the design of sensor and sequestration materials targeted for
396
phosphate in which functional interfaces between aqueous solutions and solids are prevalent.32,103–
106
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CONCLUSION
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We have used a homologous series of amphiphilic receptors differing in charge (positive
or neutral), and supramolecular organization (single or double U-shaped chains) to better
401
understand the design features that impact phosphate recognition (affinity and selectivity) at
+
monolayer-aqueous interfaces. We find the guanidinium U-guan and thiouronium U-thiouro+
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receptors bind phosphate through charge-assisted hydrogen bonding in the aqueous region created
beneath the larger U-shaped anchoring groups. The uncharged U-thio and the single-chain but
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charged S-guan receptors do not bind phosphate. The S-guan receptor does not interact with
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phosphate as a result of compaction of the monolayer possibly from headgroup-headgroup
+
interactions facilitated by smaller anchoring groups. For the U-thiouro receptor, phosphate
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09
binding within the monolayer is evident from changes in the ratio of gauche to trans conformers
+
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during compression of the Π-A isotherm. Only the U-thiouro receptor underwent this structural
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reorganization upon binding, giving insights into the behavior of the additional bulky methyl
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group. Competition studies show that the U-guan receptor is selective to phosphate over chloride
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by a factor of 1000, but careful consideration must be given to the ionic strength of the solution
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since charge screening of the electrical double layer (≥ 10 mM NaCl) can weaken binding and alter
+
the interpretation of the results. The U-thiouro receptor was also selective towards phosphate
over chloride but only by a factor of 10. Our results provide mechanistic insights into phosphate
binding at aqueous interfaces, advancing a deeper understanding of anion recognition and
promoting the remarkable advantages of the interfacial environment over traditional homogenous
solution.
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SUPPORTING INFORMATION
The Supporting Information is available free of charge on the website at DOI:
Experimental materials, instrumentation details, synthetic procedures and characterization
+
+
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422
of the U-thio, S-guan , U-guan , and U-thiouro receptors, Raman spectroscopy calibration
curve of the filtered phosphate solution, Π-A and IRRAS of the receptors using a filtered phosphate
solution, IRRAS control experiments with eicosane, perfluorotetradecanoic acid, and tripalmitin,
and calculated electrostatic potential maps.
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AUTHOR INFORMATION
Corresponding authors
*E-mail: aflood@indiana.edu. Phone: +1-812-856-3642. Fax: +1-812-855-8300.
*Email: allen@chemistry.ohio-state.edu. Phone: +1-614-292-4707. Fax: +1-614-292-1685.
Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGEMENTS
This research was supported under grant CHE 1609672 from the National Science Foundation.
We thank Taylor A. Neal and Stephen M. Baumler for valuable feedback on the manuscript.
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