Anion Transport with Halogen-Bonding Cascades

The lessons learned with calixarenes and small halogen-bond donors will herein be applied to explore another classic motif for ion transport in order to produce an anion transport system that shows a better performance.

General Considerations and Design

The design of a different ion transport system taking into account the lim-itations of the previous examples, was most desirable. While the lack of sophistication and the necessity to self-assemble in the case of small halogen-bond donors had consequences on their reported activities, the opposite was observed with the calixarene system, where its preoganization would lead to weak activities.

An extremely well studied system for ion transport that offered in addition some of the characteristics considered favorable (viz. a preorganized structure with moderate multivalency) was the octiphenyl rigid rod.199,200,205–208

Decoration of the octiphenyl “arms” with perfluoro-4-iodobenzyl units was expected to lead to a rod-like system similar to the polyol reported by Matile et al. As a control, it was decided to prepare also the anion-π interactions version without the iodo substituent on the perfluorophenyl ring as in the calixarene case.

In addition, an increase in size was proposed to further explore the properties of increasing oligomer size; indeed the already described systems would work by adopting a parallel orientation inside the lipid bilayer membrane, spanning over the whole thickness of it and, therefore, being able to connect both sides by a selective relay system.^199,200

3 Results and Discussion 123

Scheme 24:Synthesis of halogen bonding cascades. a. TFA, DCM, 1 - 3 h, quant.

For n = 0: a) 156, DIPEA, DMF, µW, 120 ^◦C, 3 h, 75%; c) 146,

Synthesis

The protected tert-butyl bi-, quarter-, sexi- and octiphenyl rods182 to185 were prepared as reported in the literature^209–211 starting from commercially available materials.

Tert-butyl deprotection with tetrafluoroacetic acid (TFA) would yield the free acids (186 to189). Nucleophylic substitution by the carboxylates with the corresponding benzyl halides under basic conditions (diisopropylamine, DIPEA) and microwave irradiation (when needed) gave compounds 190 to 197in good to moderate yields (Scheme 24).

An increasing difficulty in the substitution reaction with increasing length of the oligomer was observed: the required temperature for the reaction to proceed increased from -10^◦C to 170^◦C. This phenomenon was attributed to the increasing steric hindrance preventing the access to the inner carboxylates.

Higher oligomers are most likely not accessible following the same procedure.

Interdigitation of the π-acidic phenyl units could also be involved in the increased activation barrier. π-πinteractions between the rods and the unre-acted benzyl halide units were observed by NMR, nevertheless purification with standard chromatographic methods is enough to get the desired compounds in pure form.

Anion Transport and Cooperative Effects

Compounds 190to 197 were tested in the HPTS assay and showed an in-creasing activity with inin-creasing size of the oligomer (Figure 72 and Table 16).

Compound193with anEC50of 110 ±20 nM was the most active.

Overall, the activities in the HPTS assay, as shown by theEC₅₀ values, in-crease quickly with increasing length of the oligomer, whereas Hill coefficients drop to one as a cooperative behavior is no longer needed.

Activity was observed as well with the anion-π versions, but the activities where less important and the oligomeric effect was comparably low.

One way to verify if the enhancement of the transport is “only” due to the individual contributions of the monomers is to calculate the effective concen-tration per unitEC^N₅₀, that is the individual contribution as if the effect would

3 Results and Discussion 125

0 0.2 0.4 0.6 0.8 1

0.001 0.01 0.1 1 10

Y

c / µM

0 0.2 0.4 0.6 0.8 1

0.001 0.01 0.1 1 10 100

Y

c / µM

Figure 72: Dose response curves for compounds190 (x), 191 (♦), 192 () and 193() (left) and compounds194(x),195(♦),196() and197() in the HPTS assay.

be linearly dependent. These values are reported in Table 16 and, just as for theEC50 itself, the value decreases (which represents an increase in activity) steadily.

From theEC^N₅₀values and evolution, the gain in activity per monomeric unit is clearly not linear; non-linear increases in a macroscopic parameter (most of-ten the bonding constants) are generally related to a multivalency effect.^212–214 The effect of cooperativity on the activity according to the length of the grow-ing oligomer can be characterized with Equation (7),^215,216

EC₅₀∝N^−m (7)

Where,Nis the number of monomers and m is the cooperativity coefficient.

The obtained value for the halogen bonding series is 3.37, whereas a less impressive cooperativity of 1.74 is observed for the anion-π series; the latter value is in the usual range for intrinsic multivalent contributions²¹⁵and, almost as a consequence of that fact, the former value is very high.

The explanation for a high cooperativity derives directly from the anion transporter design. Indeed, the previously investigated systems which resemble this one, but used other interactions, were found to transport ions in a hopping mechanism.¹⁹⁹ In our case, a similar mechanism is proposed where initial

Table 16:Summary of transport data for halogen-bonding cascades

1For structures see Scheme 24 ²Effective concentra-tion per monomer unit in an oligomer.

binding of an anion at the outer surface of the membrane would be followed by rotation/hopping of the anion to the next halogen-bond donor along. After repeating this process as required, the rod delivers the anion to the inner site of the membrane (Figure 73).

O O

Figure 73: Schematic representation of the proposed anion-hopping mechanism be-tween the halogen-bond donors (compound193).

In this case, a saturation was observed for theEC50 values already at the sexiphenyl level, even if the length is not ideal as compared to the membrane thickness. To dismiss the possibility of this being due to purely partitioning effects, the HPTS assay was measured at significantly higher concentrations:

1.25 mM lipid concentrations, which is 40 times the standard lipid

concen-3 Results and Discussion 127

tration used. Compounds 192and193 were studied under these conditions and in both cases an increase of the activity of three to four times, with re-spect to the lipid concentration, was observed. This demonstrates that, in general, not the whole concentration of transporter is in the membrane due to different partitioning coefficients. In addition, under these conditions, the smallest lipid molar ratios at which our transporter (e.g. 193) is still active (Figure 74) can be obtained; this is a standard description for ion transport sys-tems.^217–219WithEC50molar ratios as low as 1:1’000 (transporter/lipid), that is 0.01% (mol/mol), and activity still observed at 1:10’000 (transporter/lipid) or 0.001% (mol/mol) (Figure 74) it is only fair to assert that these halogen-bonding cascades show a good activity as anion transporters.

0

Transporter/lipid ratio / mol%

Figure 74: Time-dependent fluorescence traces for 193 with increasing trans-porter/lipid molar ratios as shown (left) and dose response curve for 193against transporter/lipid molar ratios (right).

Nevertheless, the observed saturation could not be explained in terms of partitioning. The origin of this saturation is to be found at the level of its design: indeed, for synthetic reasons, this version of oligophenyl rods has

“arms” that are clearly wider than in previous cases; this difference probably provides them with enough flexibility to reach both sides of the lipid bilayer membrane with only six monomeric units, thus showing saturation at this stage.

Ion Selectivity

The ion selectivity topologies for compounds 190 and 193 were measured following established procedures (Section 1.3.1). Not surprisingly, a very sim-ilar selectivity to the “small” molecule case was observed, namely an anti-Hofmeister selectivity with minor effect on fluoride and acetate and an en-hanced selectivity for nitrate (Figure 75).

This supports the hypothesis that the main driving force and limiting factor is binding. Interestingly, in the proposed transport mechanism the anion is expected to keep a certain hydration (under the penalty of being thermody-namically very unfavorable) which is neither in opposition nor in agreement with the proposed mechanism for the small molecules: in that case, although ideally the anion is fully and “exclusively” coordinated by halogen-bond donors, this is most likely not the actual situation and a certain hydration is probably maintained.

0 0.5 1 1.5

-550 -500 -450 -400 -350 -300 -250 Ynorm

-450 -400 -350 -300

F

Figure 75: Cation and anion selectivity topologies for compounds190() and193 ().

The most likely reasons for these similarities are to be found within the binding energies, either for the halogen-bond donors complex or for the hy-dration. Given that the binding energies are similar to each other,^220,221 as

Conditions for the HPTS assay for Figure 75: 1930µl buffer (100 mM M⁺Cl⁻(M⁺ = Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺) or Na⁺X⁻(X⁻= F⁻, OAc⁻, Cl⁻, Br⁻, NO⁻₃, I⁻, ClO⁻₄), 10 mM Hepes, pH 7.0), 25µl EYPC-LUVs⊃HPTS.

3 Results and Discussion 129

long as anion transport is induced by “enough” bindings sites, the others can be filled by water without disturbing the system too much. This was also confirmed, in collaboration with the Megrangolo and Resnati groups in the Politecnico di Milano in Milan, by the iso-structural formation of single crys-tals either with double halogen bonding coordination or with water occupying one of the coordinating sites; this is usually regarded in crystal engineering as iso-energetic.²²²

In any event, similar topologies indicate that the binding event leading to transport is similar in both systems.

Halogen-Bonding Cascades: Conclusions

In this section we have shown that it is possible to design a system that, as opposed to the previous cases, is neither limited by a self-assembly process nor exhibits inhibitory properties like overbinding. The proposed system is reminiscent of the rigid-rods studied by Matile et al. in the last decade and as such verifies the general applicability of this design.

Although the halogen-bonding cascades have not been studied as carefully as the small halogen-bond donors case, the results here presented are very neat: a big increase in activity is observed (in the order of >2’000 times as compared to163) as well as a strong cooperative effect suggesting a hopping mechanism (Figure 76).