Modular tripodal receptors for the hydrosulfide (HS-) anion

Article abstract of DOI:10.1039/c7cc09405a

Hydrogen sulfide (H₂S) is an endogenously-produced gasotransmitter and is predominantly speciated as HS^- at physiological pH. Despite this importance, reversible binding of HS^- to synthetic receptors remains rare and confined to highly-engineered receptor systems. Here we demonstrate the generality of reversible HS^- binding in a family of tren-based receptors.

Full text of DOI:10.1039/c7cc09405a

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
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Modular Tripodal Receptors for the Hydrosulfide (HS–) Anion
Nathanael Lau, Lev N. Zakharov, Michael D. Pluth*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Hydrogen sulfide (H2S) is an endogenously-produced
Our group hypothesized that supramolecular receptors
gasotransmitter and is predominantly speciated as HS– at capable of binding Cl– should also be able to bind HS– due to
physiological pH. Despite this importance, reversible binding of HS– their similar ionic radii.8-9 Supporting this hypothesis, HS– has
to synthetic receptors remains rare and confined to highly- been shown to interact with Cl– ion channels and anion-
engineered receptor systems. Here we demonstrate the generality exchange proteins in biological systems.10-13 To this end, on
of reversible HS– binding in a family of tren-based receptors.
collaboration with the Johnson and Haley labs, we recently
reported that a series of bis(ethynylaniline)-based Cl– receptors
could also be used to bind HS–.14-15 These receptors were able
to reversibly bind HS– through non-covalent hydrogen bonding
interactions between the host urea N–H and aromatic C–H
moieties and HS–.3 Despite this report, we are unaware of
subsequent examples of well-characterized HS– binding to
synthetic receptors, which raised the question of the generality
of HS– binding in synthetic motifs.
Anions play vital roles in biological and environmental
processes,1 and thus considerable effort has been directed
toward the detection and recognition of specific anions.2 For
example, many supramolecular hosts have been developed to
detect different types of anions, such as monovalent halides
and multivalent phosphates and sulfates.2 Such receptors
primarily rely on non-covalent interactions to bind anionic
guests. Additionally, these receptors can often be structurally or
electronically tuned to target specific anion properties such as
shape, basicity, and hard/soft characteristics. Despite advances
in this field, only one well-characterized class of synthetic
supramolecular receptor capable of binding the hydrosulfide
anion (HS–) has been reported.3
To directly address this question and to demonstrate the
generality of HS– binding, we prepared a family of readily-
modifiable tripodal receptors capable of binding HS–. Based on
N,N',N''-(nitrilotris(ethane-2,1-diyl))tribenzamide
(baTren,
Figure 1), which has a binding affinity for Cl– in acetonitrile
(CH3CN) of ~100 M–1,16 these receptors contain N–H and
aromatic C–H moieties that were previously used in the
bis(ethynylaniline) system to bind HS–.3 One benefit of the
baTren system is its modularity; substituted versions of baTren
can be prepared in one step by reacting different commercially
available benzoyl chlorides with tris(2-aminoethyl)amine (tren)
in the presence of base.16-17
The hydrosulfide anion is the conjugate base of the
important biological signaling molecule hydrogen sulfide (H2S).4-
5
H2S is involved in the regulation of cellular processes and
responses in the cardiovascular, immune, and nervous systems
and is one of three recognized gasotransmitters alongside
carbon monoxide (CO) and nitric oxide (NO).6-7 At physiological
pH, H2S exists primarily as HS–, suggesting that HS– is an
important, and almost completely overlooked, biological
anion.4-5 Thus, the development and application of
supramolecular HS– receptors is poised to fill a gap in the field
of anionic recognition, and also provide insights into factors
influencing HS– binding.
We initially prepared baTren and measured its binding
affinity for HS– in anhydrous CD2Cl2 by titrating 1.0 – 2.0 mM
1
solutions of baTren with NBu4(SH) and monitoring by H NMR
spectroscopy (Figure 2). Pronounced shifts in the resonances
associated with the amide N–H protons and ortho-aromatic C–
H protons (Figure 2b) were observed, indicating that these
protons were involved in HS– binding as predicted. Only minor
shifts were observed in the resonances associated with the
ethylene groups of the tren backbone (Figure 2c), suggesting
that HS– did not interact with the tren backbone and ruling out
a strong hydrogen bonding interaction between the S–H proton
a.Materials Science Institute, Institute of Molecular Biology,
Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR
97403. E-mail: pluth@uoregon.edu
† Electronic Supplementary Information (ESI) available: Experimental details, NMR
spectra, crystallographic information, titration data. See DOI: 10.1039/x0xx00000x
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and the lone pair of the tertiary amine nitrogen of the tren bonding within the receptor in the absence of thVeiegwuAertisctle, Othnluines
DOI: 10.1039/C7CC09405A
backbone.
changing the ground state stability of the receptors (Figure S2).
In addition to increasing binding affinity, these modified
receptors should provide insight into the relative importance of
amide N–H versus aromatic C–H groups toward HS– binding. We
expected that 4CF3-baTren should increase the acidity of the
amide N–H moieties via inductive effects, whereas 3CF3-baTren
should increase the acidity of the aromatic C–H moieties
oriented towards the anion.
Figure 1. Tripodal receptors used in this study: baTren, 3CF3-baTren, 4CF3-
baTren,18 3CH3-baTren, 4CH3-baTren,19 and 2F-baTren.
Previous studies of baTren-type systems with halides
suggested that the binding stoichiometry of this family of
receptors should be limited to a simple 1:1 model.17 To
determine whether similar binding was present for HS–, we
constructed a Job plot for HS– binding to baTren (Figure 2b). This
plot supported a 1:1 binding stoichiometry as evidenced by the
plot maximum near 0.5, which is further substantiated by direct
fitting of the titration data to a 1:1 binding isotherm model.20
Building from this binding stoichiometry, we measured the 1:1
binding constants for baTren and both HS– and Cl– in CD2Cl2
using the Thordarson method (Table 1).21 The binding affinities
for HS– (149 ± 8 M–1) and Cl– (160 ± 20 M–1) were equivalent
within error, suggesting a lack of selectivity between the two
anions for this receptor. These values in CD2Cl2 were similar to
the previously reported value between baTren and Cl– in
CH3CN;16 we found baTren to be poorly soluble in CD3CN and
D2O and thus conducted our experiments in CD2Cl2.
Figure 2. (a) Representation of the HS– host-guest equilibrium with baTren, (b)
representative 1H NMR titration of 1.0 mM baTren with NBu4(SH) in anhydrous CD2Cl2,
and (c) Job plot of baTren with NBu4(SH) in anhydrous CD2Cl2.
Supporting our hypothesis, the measured binding affinity of
4CF3-baTren (3,500 ± 300 M–1) toward HS– was approximately
three times greater than that of 3CF3-baTren (1160 ± 90 M–1)
(Table 1). This result suggested that N–H groups in the baTren
scaffolds are more influential in anion binding than aromatic C–
H groups, a conclusion consistent with the fact that N–H bonds
are more polarized than those of aromatic C–H bonds.
Additionally, variable temperature 1H NMR studies on a solution
of 4CF3-baTren with 2 equivalents of NBu4(SH) in CD2Cl2
revealed that the amide N–H peak shifted downfield at –35 °C.
In contrast, the ortho-aromatic C–H peak shifted slightly upfield
and was not further resolved. Thus, decreasing the temperature
increased the anionic interaction with the N–H groups but
decreased the interaction with the C–H moieties as the
aromatic rings continued to rotate freely despite the lowered
temperature. We note, however, that the inductive effects of
the CF3 groups, as measured by Hammett parameters, are not
equivalent for the para (σp = 0.54) and meta (σm = 0.43)
position.22 Also, since substituent positions are relative, the
inductive effect of a given substitution cannot exclusively target
either the N–H or C–H groups; both will experience inductive
+
Additionally, the NBu4 counter cation did not appear to
influence anion binding in this system as evidenced by the
identical binding affinities obtained from titrations using
NEt4(SH) to those using NBu4(SH).
To determine whether the binding affinity of HS– could be
modulated within the baTren scaffold, variants of baTren
containing electron-withdrawing CF3 substituents on the aryl
rings were prepared (3CF3-baTren and 4CF3-baTren, Figure 1).
Such modifications should increase the acidity and therefore
the H-bond donating ability of the amide N–H and aromatic C–
H moieties, resulting in stronger interactions with anions. These
modifications should also modify the intramolecular hydrogen-
2 | Chem. Commun., 2017, 00, 1-3
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effects, which complicates deriving a direct correlation between NBu4(SH) could reform the HS– complex, demonstrating the
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DOI: 10.1039/C7CC09405A
substituent position and the hydrogen bonding ability of either reversibility of this binding event.
the N–H and C–H groups in this system. Nevertheless, the
substantial difference in binding affinity between the para and
meta substituted receptors supports our initial hypothesis that
HS– affinities can be modulated by electronic effects.
Table 1. HS– binding parameters for the baTren based receptors. All values were
obtained by fitting 1H NMR spectroscopic data to 1:1 binding isotherm models, using the
Thordarson method, in triplicate.21
Host (Guest)
baTren
Ka (M–1)
149 ± 8
∆G (kcal/mol)
–2.96
baTren (Cl–)
3CF3-baTren
4CF3-baTren
3CH3-baTren
4CH3-baTren
160 ± 20
1160 ± 90
3500 ± 300
130 ± 20
140 ± 30
–2.99
–4.18
–4.83
–2.88
–2.92
To further probe the effect of electronic modulation on HS–
binding, we prepared para and meta CH3 substituted versions
of baTren (3CH3-baTren and 4CH3-baTren, Figure 1). These
receptors, although sterically similar to the CF3 substituted
receptors, should have lower binding affinity for HS– than even
unsubstituted baTren due to their electron-donating CH3
substituents. As expected, the binding affinities for 3CH3-baTren
and 4CH3-baTren were indeed lower than those of the CF3
substituted receptors (Table 1), but surprisingly these values
were approximately equivalent to that of unsubstituted baTren.
This observation suggested that the modest electron-donating
characteristics of the CH3 groups does not significantly impact
the overall binding affinity. Additionally, the similar binding
affinity between 3CH3-baTren and 4CH3-baTren implied that the
meta CH3 substituent does not sterically encumber anionic
binding.
Rational receptor design can also be used to prevent anion
binding in the baTren system, as demonstrated by 2F-baTren
(Figure 1). By incorporating fluorine atoms in the ortho-
positions of the receptor, we aimed to promote strong
intramolecular H-bonding at the expense of intermolecular
interactions. Stable six-membered rings can be formed if
hydrogen bonds are formed between the amide N–H groups
and the F atoms of the same arm, which we hypothesized would
prevent HS– binding. Indeed, the N···F distances of 2.713, 2.740,
2.967 Å are indicative of strong intramolecular H-bonds (Figure
S2c).23 The addition 10 equivalents of either NBu4(SH) or
NBu4(Cl) to a solution of 2F-baTren in CD2Cl2 resulted in virtually
no shifts in any 1H NMR resonances, suggesting that 2F-baTren
is not able to bind anions with appreciable affinity. These
experiments again illustrate how important receptor
modifications are to the binding affinity of this system.
Figure 3. (a) Scheme for the 3CF3-baTren anion binding reversibility experiment. 1H NMR
spectrum of (b) 0.5 mM 3CF3-baTren in 7% DMSO-d6/CD2Cl2, (c) after treatment with 2
equivalents NBu4(SH), and (d) after treatment of 6 equivalents of Zn(OAc)2.
Conclusions
In conclusion, we have expanded the library of supramolecular
HS– receptors by repurposing the known tripodal Cl– receptor
baTren towards HS– binding. Importantly, this study
demonstrates the generality of using simple synthetic receptors
1
for reversible HS– binding. H NMR titrations suggested that
these receptors utilize their amide N–H and aromatic C–H
groups to non-covalently bind HS– and that the binding affinity
could be tuned by electronic modulation. Additionally, the
observation that the HS– binding affinity of 4CF3-baTren was
substantially higher than that of 3CF3-baTren suggested that in
these simple systems, the amide N–H moieties may be more
influential to HS– binding than the aromatic C–H groups.
Moreover, the ability of these simple receptors, which only use
amide N–H and aromatic C–H bonds to bind HS–, points to the
generality of HS– binding in molecular receptors.
This work was supported by the National Science
Foundation (CHE-1454747) and the Dreyfus Foundation (MDP).
The NMR facilities at the University of Oregon are supported by
the NSF (CHE-1427987).
Finally, to examine and confirm the reversibility of HS–
binding in this system, we treated a solution of 3CF3-baTren in
7% DMSO-d6/CD2Cl2 (Figure 3b) with 2 equivalents NBu4(SH) to
form the HS– bound complex (Figure 3c). This mixed solvent
system was required for the dissolution of Zn(OAc)2, and the
addition of 6 equivalents of Zn(OAc)2 restored the spectrum to
that of free 3CF3-baTren (Figure 3d). Further additions of
Conflicts of interest
There are no conflicts to declare.
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3-fold symmetric receptors with N-H and C-H bond donors
enable reversible HS– binding.
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