Article Cite This: J. Org. Chem. 2017, 82, 13368−13375
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Synthesis, Anion Recognition, and Transmembrane Anionophoric Activity of Tripodal Diaminocholoyl Conjugates Zhi Li,† Xi-Hui Yu,† Yun Chen,† De-Qi Yuan,‡ and Wen-Hua Chen*,† †
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, P.R. China ‡ Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Minatojima 1-1-3, Chuo-ku, Kobe 650-8586, Japan S Supporting Information *
ABSTRACT: In this paper, we present the synthesis, anion recognition, and anionophoric activity of 1,3,5-tris(aminomethyl)2,4,6-triethylbenzene-based tripodal 3α-hydroxy-7α,12α-diamino-5β-cholan-24-oate conjugate 1 and the corresponding tris(2aminoethyl)amine-based analogue 2 and choloyl analogue 3. Their affinity toward anions was evaluated by means of competitive displacement assay using 5-carboxyfluorescein (5-FAM) as a fluorescent indicator. The results indicate compounds 1 and 2 exhibit strong recognition toward a wide range of biologically important anions, in particular, toward sulfate and phosphate anions. In MeOH−HEPES (4/1, pH 7), the binding constants of compounds 1 and 2 are 416- and 168-fold higher for sulfate than for chloride and 35- and 25-fold higher for phosphate than for chloride, respectively. The anion transport activity was measured by use of pH discharge assay and chloride-ion-selective electrode technique. The results indicate that compounds 1 and 2 function as effective anion-selective transporters in the order of ClO4− > I− > NO3− > Br− > Cl− > SO42− > H2PO4− and exhibit anionophoric activity via a process of major anion exchange and minor anion/cation symport. In addition, some insights into the correlation of the anion binding affinity with the transport efficiency are also briefly discussed.
1. INTRODUCTION Anions are ubiquitous in nature, and their recognition and transport across cell membranes is an essential process for the functioning of biological systems.1 Synthetic anion receptors and transporters may find wide applications, for example, in the detection of biologically important species2,3 and in the treatment of cancers and bacterial infections.1,4 Powerful anion receptors may be constructed by assembling a variety of functionalities to some sophisticated molecular scaffolds with highly preorganized structures, such as crown ethers,5 cyclodextrins,6 calix[4]arenes,7 calix[4]pyrroles,8 and cucurbit[n]urils.9 Some of preorganized receptors are also especially effective anion transporters.10−14 In these aspects, tripodal conjugation is an attractive approach for the development of powerful anion receptors. Because they feature a well-dispersed but convergent 3D array of functionalities,15 tripodal receptors exhibit specific affinity toward anions, in particular, toward tetrahedral inorganic oxoanions, such as sulfate and phosphate. Flexible tris(2aminoethyl)amine (tren) and rigid 1,3,5-tris(aminomethyl)© 2017 American Chemical Society
2,4,6-triethylbenzene represent two common molecular scaffolds that are widely used to create tripodal synthetic receptors. In 2013, Jin and co-workers have described the synthesis of tren-based tripodal squaramide conjugates that are highly selective for sulfate over the other examined anions.16 Anslyn et al. have shown that a host with three guanidiniums linked to the 2,4,6-triethylbenzene platform can selectively bind citrate over carboxylic acids, phosphates, sugars, and salts in water.17 Some tripodal conjugates have also been demonstrated to act as effective anion transporters.11−14,18−22 For example, Gale and co-workers have shown that tren-based tripodal trisurea/ thiourea receptors are able to mediate the chloride/bicarbonate anions exchange and thereby belong to a new class of bicarbonate transport agents.12 Davis et al. have shown that catechol-bearing tren-based receptors are able to mediate the transmembrane transport of chloride anions.21 More recently, they have shown that 1,3,5-tris(aminomethyl)-2,4,6-triethylReceived: September 26, 2017 Published: November 22, 2017 13368
DOI: 10.1021/acs.joc.7b02447 J. Org. Chem. 2017, 82, 13368−13375
Article
The Journal of Organic Chemistry
2. RESULTS AND DISCUSSION 2.1. Chemistry. Compounds 1−3 were synthesized according to the route depicted in Scheme 1. Thus, activation of cholic acid 4 and 3α-hydroxy-7α,12α-di[N-(tbutyloxycarbonyl)amino]-5β-cholan-24-oic acid 5 with 1hydroxybenzotriazole (HOBt) and subsequent reaction with 1,3,5-tris(aminomethyl)-2,4,6-triethylbenzene and tren afforded compound 3 and Boc-protected 6 and 7, respectively. Deprotection of the Boc groups in compounds 6 and 7 with TFA afforded compounds 1 and 2, respectively. Compound 5 was prepared according to the reported protocols.23−25 Compounds 1, 2 and 6, 7 were fully characterized by NMR (1H and 13C) and MS (LR and HR) (refer to the experimental part as well as Figures S1−S14). Compound 3 is a known compound and was characterized by means of ESI MS and 1H NMR and further by comparing the structural data with the ones reported in literature.28 2.2. Anion Binding Affinity. The anion binding affinity of compounds 1 and 2 was investigated by means of competitive displacement assay using 5-carboxyfluorescein (5-FAM) as a fluorescent indicator.17,29,30 First, we measured the affinity of compounds 1−3 toward 5-FAM in MeOH−HEPES (4/1, pH 7). As shown in Figures 2a and S15, upon the addition of compounds 1 and 2, the fluorescence of 5-FAM increased largely and shows a typical saturation behavior, revealing the very strong interaction of compounds 1 and 2 with 5-FAM. In contrast, no change in the fluorescence of 5-FAM was observed upon the addition of compound 3, suggesting that the interaction between compound 3 and 5-FAM is weak or the fluorescence of 5-FAM is not affected by compound 3. The binding constants of compounds 1 and 2 with 5-FAM were calculated from the nonlinear least-squares fitting of the experimental data to a 1:1 binding model and are (1.59 ± 0.08) × 105 M−1 for compound 1 and (9.23 ± 0.52) × 104 M−1 for compound 2 in MeOH−HEPES (4/1, pH 7) (Table 1). The system composed of 5-FAM and compound 1 or 2 as a platform for the detection of anions was confirmed by the fluorescence quenching induced by the competitive displacement of 5-FAM with a wide range of biologically important anions, including halogen, nitrate, perchlorate, acetate, sulfate and phosphate anions. As shown in Figure 2b and S16a, in MeOH-HEPES (4/1, pH 7), the addition of these anions led to the fluorescence quenching of 5-FAM, indicating that they are able to compete with 5-FAM for binding to compounds 1 and 2. The strongest decrease in the fluorescence intensity was observed with SO42− and H2PO4−, suggesting that compounds 1 and 2 have high selectivity for these two anions. To quantify the affinity and selectivity of compounds 1 and 2 toward the above-mentioned anions, we examined the concentration-
benzene-derived tris-N-arylthioureas act as extremely potent anion carriers with the optimized activities being among the best currently known.22 Recently, we have reported the synthesis of 3α-hydroxy7α,12α-diamino-5β-cholan-24-oate dimer A (Figure 1) and the
Figure 1. Structures of dimeric diaminocholoyl conjugate A, 1,3,5tris(aminomethyl)-2,4,6-triethylbenzene-based tripodal diaminocholoyl conjugate 1, the tren-based analogue 2, and choloyl analogue 3.
derivatives modified with alkyl chains of varying lengths on the amido linkages.23 This series of compounds exhibits efficient anion transport activity, which is a likely consequence of the enhanced interactions of these dimeric diaminocholoyl conjugates with anions by the replacement of the choloyl 7and 12-hydroxyl groups with amino groups.24,25 Inspired by this result, we reason that a tripodal 3α-hydroxy-7α,12α-diamino5β-cholan-24-oate conjugate, in particular, based on the socalled “pinwheel” 2,4,6-triethylbenzene scaffold, that is, conjugate 1 (Figure 1), would be developed as a powerful, cleft-shaped anion receptor. This sterically hindered core scaffold, in which the ideal structure has the 1,3,5-functional substituents and the 2,4,6-triethyl groups directed in opposite directions from the phenyl plane, tends to drive the three diaminocholoyl subunits to align in the same direction, so as to form a contact, highly preorganized binding site for anions,17,22,26 where the multiple electrostatic and hydrogenbonding interactions with anions may be in full operation.27 Herein, we report the synthesis, anion recognition, and transmembrane anion transport activity of conjugate 1, the corresponding tren-based analogue 2 and choloyl analogue 3 for comparison (Figure 1). Their anion binding affinity was assessed by means of indicator displacement assay, and the transmembrane anion transport activity was measured by means of pyranine assay and chloride-ion-selective electrode technique. Scheme 1. Synthesis of Compounds 1−3
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DOI: 10.1021/acs.joc.7b02447 J. Org. Chem. 2017, 82, 13368−13375
Article
The Journal of Organic Chemistry
Figure 2. (a) Plots of the relative fluorescence intensity (F/F0) of 5-FAM (0.5 μM) against the concentrations of compounds 1−3 in MeOH/ HEPES buffer (1.0 mM, pH 7.0, 4/1, v/v). λEx 480 nm/λEm 518 nm and hereafter. The solid lines for compounds 1 and 2 are the nonlinear least fitting of the experimental data to a 1:1 binding model. (b) Relative fluorescence intensity (F/F1) of 5-FAM (0.5 μM) mixed with compound 1 (12 μM) and various anions (1.2 mM), in MeOH/HEPES buffer (1.0 mM, pH 7.0, 4/1, v/v). (c) Fluorescence spectra of 5-FAM (0.5 μM) mixed with compound 1 (12 μM) and sulfate anions of increasing concentrations, in MeOH/HEPES buffer (1.0 mM, pH 7.0, 4/1, v/v). Inset: plot of the fluorescence intensity against the concentrations of sulfate anions. The solid line is the nonlinear least fitting of the experimental data to the model based on an indicator displacement assay. (d) Negative ESI MS spectrum of a mixture of compound 1 (2.0 mM) with tetramethylammonium sulfate (20 mM) in MeOH/H2O (4/1, v/v).
Table 1. Binding Constants (Ka, M−1) of Compounds 1, 2, and A with 5-FAM and the Other Anions Examined in This Worka compound 1 Ka
anion 5-FAM F− Cl− Br− I− NO3− AcO− ClO4− H2PO4−c SO42−
(1.59 (4.72 (1.11 (1.58 (1.99 (1.16 (2.84 (1.86 (3.90 (4.62
± ± ± ± ± ± ± ± ± ±
0.08) 1.20) 0.07) 0.11) 0.03) 0.05) 0.39) 0.14) 0.54) 0.51)
compound 2 b
Ka
RA × × × × × × × × × ×
5
10 103 103 103 103 103 103 103 104 105
143 4.3 1.0 1.4 1.8 1.0 2.6 1.7 35 416
(9.23 (4.41 (1.03 (1.05 (1.07 (9.40 (2.46 (1.48 (2.53 (1.73
± ± ± ± ± ± ± ± ± ±
0.52) 1.20) 0.07) 0.12) 0.06) 0.80) 0.44) 0.17) 0.18) 0.34)
compound A b
× × × × × × × × × ×
10 103 103 103 103 102 103 103 104 105
90 4.3 1.0 1.0 1.0 0.9 2.4 1.4 25 168
RAb
Ka
RA 4
(5.69 (5.22 (2.58 (3.44 (2.66 (2.48 (3.05 (2.07 (7.93 (2.17
± ± ± ± ± ± ± ± ± ±
0.39) 3.85) 0.94) 1.04) 0.88) 0.42) 0.74) 0.25) 3.69) 0.47)
× × × × × × × × × ×
3
10 102 102 102 102 102 102 102 102 103
22 2.0 1.0 1.3 1.0 0.9 1.2 0.8 3.1 8.4
a Measured in MeOH/HEPES buffer (1.0 mM, pH 7.0, 4/1, v/v). Except 5-FAM, tetrabutylammonium, or tetramethylammonium salts of the other anions were used. bRA refers to the relative affinity of each compound to chloride anions. cExists as a mixture of H2PO4− (62%) and HPO42− (38%) under the conditions of MeOH/HEPES buffer (1.0 mM, pH 7.0, 4/1, v/v), according to the fact that phosphoric acid has a pKa1 of 2.12, pKa2 of 7.21, and pKa3 of 12.67.
toward F−, Cl−, Br−, I−, NO3−, AcO−, and ClO4− but much higher affinity toward SO42− and H2PO4−. For example, the binding constants of compound 1 reach 4.62 × 105 M−1 for SO42− and 3.90 × 104 M−1 for H2PO4−, 416- and 35-fold higher than that for Cl−, respectively. Similarly, strong recognition of SO42− and H2PO4− by compound 2 was also observed. The complexation of compounds 1 and 2 with SO42− (and Me4N· SO4−) was also confirmed by means of ESI MS (Figures 2d and S17). The ion peaks at m/z 1525.82 for compound 1 (Figure
dependent quenching of 5-FAM by each anion (Figures 2c and S18−S23) and calculated the corresponding binding constants of compounds 1 and 2 with each anion, by using the nonlinear least-squares fitting method (Table 1). It can be seen from Table 1 that in MeOH−HEPES (4/1, pH 7), compounds 1 and 2 exhibit high affinity toward all the anions with the binding constants being 1.11 × 103 to 4.62 × 105 M−1 and 9.40 × 102 to 1.73 × 105 M−1, respectively. Interestingly, both compounds 1 and 2 exhibit similar affinity 13370
DOI: 10.1021/acs.joc.7b02447 J. Org. Chem. 2017, 82, 13368−13375
Article
The Journal of Organic Chemistry
Figure 3. (a,b) pH discharge across EYPC-based liposomal membranes induced by (a) 0.5 mol % of compounds 1−3 and (b) compound 1 of increasing concentrations. The liposomes were loaded with 0.1 mM pyranine in 25 mM HEPES buffer (50 mM NaCl, pH 7.0) and added to 25 mM HEPES buffer (50 mM NaCl, pH 8.0). λEx 460 nm/λEm 510 nm. (c,d) Relative chloride efflux promoted by (c) 3 mol % of compounds 1 and 2 and (d) compound 1 of varying concentrations across EYPC liposomes with 500 mM NaCl in 25 mM HEPES buffer (pH 7.0) encapsulated. The liposomes were added to a solution of 500 mM NaNO3 in 25 mM HEPES buffer (pH 7.0).
with amino groups, leading to strong recognition of anions through multiple electrostatic and hydrogen-bonding interactions.25,27 2.3. Anion Transport Properties. 2.3.1. pH Discharge Activity. The strong and selective anion recognition properties of compounds 1 and 2 encouraged us to evaluate their ion transport activity on liposomal models. First, we assess the pH discharge activity of compounds 1 and 2 across vesicular membranes formed from egg-yolk L-α-phosphatidylcholine (EYPC).31 In these experiments, large unilamellar vesicles (100 nm diameter, extrusion) were prepared with pyranine encapsulated. Here pyranine (pKa = 7.2), a pH-sensitive dye, was used to report the pH changes within the vesicle interior. When the vesicles prepared in a pH 7.0 buffer were suspended in a pH 8.0 buffer and each compound was added, a pH increase within the internal of the vesicles induced by either H+ efflux or OH− influx was monitored by measuring the fluorescence intensity of pyranine. The maximum fluorescence intensity was obtained by adding 5 wt % aqueous Triton X-100 solution to collapse the vesicles. It can be seen from Figures 3a,b and S27 that the addition of compound 3 led to negligible increase in the fluorescence intensity, suggesting that compound 3 exhibits a very low level of activity (if there is any) under the assay conditions. In contrast, significant increase in the fluorescence intensity was observed upon the addition of compounds 1 and 2, suggesting that compounds 1 and 2 are very efficient in discharging the pH gradient from the EYPC vesicles. To quantify the transport efficiency of compounds 1 and 2, we carried out the concentration-dependent pH discharge
2d) and 1422.88 for compound 2 (Figure S17) may be assigned to the complexes of compounds 1 and 2 with MeOSO3− that is thought to be produced under the ESI assay conditions. The high affinity and selectivity of compounds 1 and 2 for SO 4 2− and H 2 PO 4 − over the other examined anions demonstrate the advantage of such tripodal scaffolds to selectively bind these two anions and may be ascribed to the unique tripodal structures. Specifically, the three diaminocholoyl subunits in compounds 1 and 2 form binding sites that are complementary to the tetrahedral SO42− and H2PO4−. To provide support for this, we used the same indicator displacement assay to measure the binding constants of compound A toward those anions in MeOH−HEPES (4/1, pH 7) (Table 1 and Figures S15e−f, S16b, and S24−S26). The results indicate that compared with compounds 1 and 2, compound A exhibits lower affinity toward all the examined anions, in particular, sulfate and phosphate. For example, the binding constant of compound A for SO42− is 212-fold lower than that of compound 1, and the selectivity for SO42− over Cl− drops dramatically from 416 for compound 1 to 8.4 for compound A. In addition, compound 1 exhibits higher binding affinity than compound 2, most probably due to the unique structure of compound 1. The steric bulk of 1,3,5-tris(aminomethyl)-2,4,6triethylbenzene drives the three diaminocholoyl subunits of compound 1 to align in a convergent manner to form a preorganized binding site for anions,17,22 whereas compound 2 is more flexible. The fact that compound 1 shows higher anion binding affinity than compound 3 is considered as a likely consequence of the replacement of 7- and 12-hydroxyl groups 13371
DOI: 10.1021/acs.joc.7b02447 J. Org. Chem. 2017, 82, 13368−13375
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The Journal of Organic Chemistry
Table 2. Hydration Energy of the Anions and Transport Efficiency of Compounds 1 and 2 in the Presence of the Anionsa,b compound 1 anion
hydration free energy (kJ/mol)
Cl− Br− I− NO3− ClO4− SO42− H2PO4− (HPO42−)
−340 −315 −275 −300 −430 −1080 −465 (−1125d)
EC50 (mol %) 0.22 ± 0.10 (7.32 ± 2.42) (2.01 ± 1.04) (4.61 ± 0.06) (5.16 ± 0.50) 0.95 ± 0.13 2.04 ± 0.56
× × × ×
10−2 10−2 10−2 10−3
compound 2 RA1c
EC50 (mol %)
RA1c
RA2c
1.0 3.0 10.9 4.8 42.6 0.2 0.1
1.17 ± 0.68 0.35 ± 0.08 (6.25 ± 1.83) × 10−2 0.24 ± 0.03 (5.91 ± 1.27) × 10−2 1.25 ± 0.27 3.69 ± 0.26
1.0 3.3 18.7 4.9 19.8 0.9 0.3
5.3 4.8 3.1 5.2 11.5 1.3 1.8
a
The data for the hydration free energy were taken from ref 44. bFor the assay conditions of anion transport, see Figures 3a,b, S27, S31, and S32. The EC50 values for F− and AcO− could not be measured because of the repeated failure in the preparation of EYPC vesicles in the presence of these two anions. cRA1 and RA2 denote the relative transport activity of each compound for a particular anion to chloride and the relative transport activity of compound 1 to compound 2 for a particular anion, respectively. dCalculated according to the method by Marcus. See ref 45.
Figure 4. (a) Chloride efflux mediated by compound 1 (3 mol %) across the EYPC liposomes with 500 mM NaCl in 25 mM HEPES (pH 7.0) encapsulated. The liposomes were added to a solution of either 500 mM NaNO3 or 250 mM Na2SO4 in 25 mM HEPES buffer (pH 7.0). The experiments that were performed in NaNO3 media were used as controls. (b) Relative chloride efflux mediated by compound 1 (3 mol %) across EYPC liposomes with a solution of 500 mM MCl (M = Li, Na, K, Rb, and Cs) in 25 mM HEPES buffer (pH 7.0) encapsulated. The liposomes were added to a solution of 500 mM NaNO3 in 25 mM HEPES buffer (pH 7.0). The experiments that were carried out in NaCl media with DMSO were used as controls.
The fact that compounds 1 and 2 have the Hill coefficients n of 1.28 and 1.37, respectively, suggests that both compounds function as unimolecular species. 2.3.2. Chloride Influx Activity. As compounds 1 and 2 show high anion-binding affinity and pH discharge activity, we are concerned about whether they have the ability to promote the transmembrane transport of anions, such as chloride anions. However, the above pH discharge experiments do not provide any clear evidence for cation- or anion-selective transport. Under the conditions of pH discharge, any of these four mechanisms, that is, H+/cation antiport, OH−/anion antiport, H+/anion symport and OH−/cation symport, would equally cause pH change in the internal of the vesicles.13,33−37 The chloride-selective transport by compounds 1 and 2 was confirmed by measuring their chloride efflux activity with chloride-ion-selective electrode.38−40 Thus, a series of EYPC vesicles loaded with NaCl was prepared and added to an external isotonic NaNO3 solution. After a DMSO solution of compound 1 or 2 of increasing concentrations was added, the efflux of chloride anions out of the liposomes was immediately monitored by the electrode for a period of 300 s. The final reading of the electrode that was used to calibrate the 100% release of chloride anions was obtained by the addition of 5 wt % aqueous Triton X-100 solution. It is evident from Figures 3c,d and S28a,b that compounds 1 and 2 are active in promoting the efflux of chloride anions out of the EYPC vesicles. This result suggests that compounds 1
experiments (Figures 3b and S27c,d) from which the initial rate constants (kin) at each concentration were calculated (Tables S1 and S2). Nonlinear curve fitting analysis of the relationship between the initial rate constants and the concentrations of each compound according to eq 1 afforded the EC50 value and the Hill coefficient n (Table 2).14,32 Here, EC50 is the effective transporter loading when 50% of the maximum rate (kmax) is reached and thereby can be used to characterize the effectiveness of a given transporter. The n value suggests that the transport-active species is composed of n molecules of each compound. k in = k 0 + k max[compound]n /([compound]n + EC50 n) (1)
Compounds 1 and 2 exhibit the EC50 values of 0.22 and 1.17 mol % transporter/lipid, respectively, which suggests that compounds 1 and 2 are effective ion transporters. Compound 1 is 5-fold more active than compound 2. This may be due to the steric bulk of 1,3,5-tris(aminomethyl)-2,4,6-triethylbenzene that leads to a better preorganized binding site for anions, as discussed above. In addition, the higher lipophilicity of compound 1 (c log P = 9.16 for nonprotonated species and −11.77 for fully protonated species) relative to compound 2 (c log P = 4.73 for nonprotonated species and −16.20 for fully protonated species)27 makes it more ready for compound 1 to partition into the hydrophobic lipid membranes. 13372
DOI: 10.1021/acs.joc.7b02447 J. Org. Chem. 2017, 82, 13368−13375
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The Journal of Organic Chemistry
could not be prepared successfully. Compounds 1 and 2 exhibit the pH discharge activity following the order of ClO4− > I− > NO3− > Br− > Cl− > SO42− > H2PO4−. The variations of the transport activity with the anions imply the involvement of anions in the transport process.39 Thus, these results suggest that compounds 1 and 2 exhibit selective transport with regard to the studied anions and these anions participate in the permeation process. Though the transport sequence does not parallel the binding affinity in the order of SO42− > H2PO4− > I− ≈ ClO4− > Br− > NO3− ≈ Cl− for compound 1 or SO42− > H2PO4− > ClO4− > I− ≈ > Br− ≈ Cl− ≈ NO3− for compound 2, or the hydration free energy of the anions in the order of I− > Br− > NO3− > Cl− > ClO4− > H2PO4− > SO42−, some insights into the correlation of transport efficiency with binding affinity and lipophilicity of the anions may be provided. First, though compounds 1 and 2 exhibit similar binding affinity toward I−, NO3−, Br−, Cl−, and ClO4−, their pH discharge activity follows the typical Hofmeister selectivity pattern, ClO4− > I− > NO3− > Br− > Cl−. The highest transport efficiency observed for the hydrophilic ClO4− may be ascribed to the tetrahedral structure of ClO4− that matches with the host molecules to form noncovalent and relatively lipophilic complexes to cross the lipid membrane. The sequence of I− > NO3− > Br− > Cl− suggests that the transport is primarily regulated by the lipophilicity of the anions themselves. Second, though ClO4−, SO42−, and H2PO4− have tetrahedral structures in common, their binding affinity increases in the order of SO42− > H2PO4− > ClO4−. Under the assay conditions of MeOH−HEPES (4/1, pH 7.0), H2PO4− (pKa = 7.21, 62%) is in equilibrium with HPO42− (38%) (see footnote c to Table 1). Thus, the charges of the anions contribute mainly to the binding. On the other hand, compounds 1 and 2 exhibit 184and 21-fold lower transport efficiency in the presence of SO42− than in the presence of ClO4−, respectively. It is a little complicated in the case of H2PO4−. Under the condition of pH discharge, H2PO4− shifts gradually to the more hydrophilic HPO42− as the pH discharge proceeds. Thus, the transport efficiency of compounds 1 and 2 in the presence of H2PO4− decreases more significantly, 395- and 62.4-fold lower than that in the presence of ClO4−, respectively. The reduction of the transport activity in the presence of SO42− and H2PO4− is thought to be due to the high hydrophilicity of the anions and the strong complexation with compounds 1 and 2. These may make it quite difficult for SO42− and H2PO4− to cross the hydrophobic lipid membranes, as discussed above, and to be released from the complexes with compounds 1 and 2.20 Further work will be done to clarify how compounds 1 and 2 exhibit transport activity in the presence of sulfate and phosphate anions.46
and 2 function as anion-selective transporters. A Hill analysis of the relationship of the chloride efflux rates with the concentrations of each compound according to eq 2 afforded the transport specificity constant k2/Kd of 0.211 mol %−1·s−1 for compound 1 and 0.071 mol %−1·s−1 for compound 2, and the Hill coefficients n of 0.74 for compound 1 and 0.94 for compound 2 (Figure S28c,d). Here, k2 and Kd stand for the intrinsic rate constant and the dissociation constant of the selfassociation process, respectively.41 Consistent with the pH discharge activity, compound 1 is more active than compound 2. In addition, compound 1 is ca. 4-fold more active than compound A (k2/Kd = 0.059 mol %−1·s−1),23 suggesting that a third diaminocholoyl subunit may be required not only for strong anion binding affinity but also for efficient transport activity. kobs = k 0 + k 2[compound]n /Kd
(2)
2.3.3. Probable Mechanism of Action. To investigate the probable mechanism of action of compounds 1 and 2, we first changed the external salt in the chloride efflux experiments from NaNO3 to Na2SO4. As shown in Figures 4a and S29a, this change slows down the chloride efflux significantly. It is known that sulfate anions are strongly hydrated and are not readily transported across a lipid bilayer.18 If a compound functions via a mechanism of Cl−/anion exchange, the chloride efflux activity would be reduced by sulfate anions. Thus, the observed reduction in the chloride efflux is evidence that compounds 1 and 2 function primarily as anion exchangers. This mode of action is further supported by the anion-dependent transport of compounds 1 and 2 (vide infra). However, it should be noted that the chloride efflux is not completely inhibited by sulfate anions (Figures 4a and S29a), which suggests that the anion exchange process is not exclusive and other mechanisms may be also involved. To gain insights into this aspect, we carried out the chloride efflux experiments with the chloride salts of group I alkali metal ions (i.e., Li+, Na+, K+, Rb+ and Cs+).42 As shown in Figures 4b and S29b, compounds 1 and 2 promote the efflux of chloride anions in the order of Na+ > Cs+ > Li+ ≈ K+ ≈ Rb+ and Na+ ≈ Cs+ > Li+ ≈ K+ ≈ Rb+, respectively. This moderate cation selectivity implies that a minor level of Cl−/ cation symport is also involved in the permeation process. To gain insight into whether compounds 1 and 2 function as a channel or mobile carrier, we measured the chloride efflux activity on EYPC-cholesterol (7/3)-derived liposomes. A reduction in the activity across a cholesterol-containing lipid bilayer is evidence that a compound may act as a mobile carrier.43 As shown in Figure S30, the chloride efflux activity of compounds 1 and 2 was found to be reduced, which is suggestive of a mobile carrier mechanism. The Hill coefficient values around 1 in both the pH discharge and chloride efflux experiments were consistent with this probable carrier mechanism. 2.3.4. Anion Selectivity. Because compounds 1 and 2 exhibit selective recognition toward various anions and are able to induce the transport of chloride anions, we are concerned about whether compounds 1 and 2 exhibit transport selectivity with regard to those anions. To clarify this, we carried out the concentration-dependent pH discharge experiments with the sodium salts of those anions and obtained the corresponding EC50 value for each anion. As shown in Table 2, Figures S31− S34 and Table S1 and S2, compounds 1 and 2 exhibit potent pH discharge activity in the presence of those anions, except fluoride and acetate in the presence of which EYPC vesicles
3. CONCLUSION In conclusion, we have synthesized 1,3,5-tris(aminomethyl)2,4,6-triethylbenzene-based tripodal diaminocholoyl conjugate and its tren-based analogue and fully confirmed their structures by NMR (1H and 13C) and MS (LR and HR). The anion recognition properties of these compounds were assessed by use of indicator displacement assay, and the results indicate that both diaminocholoyl conjugates exhibit high affinity toward a wide range of biologically important anions, in particular, sulfate and phosphate. The anion transport activity was measured by use of chloride-ion-selective electrode technique and pH discharge experiments. The results indicate that both 13373
DOI: 10.1021/acs.joc.7b02447 J. Org. Chem. 2017, 82, 13368−13375
Article
The Journal of Organic Chemistry
(20 mg, 95%): 1H NMR (400 MHz, CD3OD) δ 4.46 (s, 6H), 3.45− 3.39 (m, 3H), 3.15 (br, 3H), 3.01 (br, 3H), 2.79−2.77 (m, 6H), 2.31− 0.97 (m, 99H), 0.80 (s, 9H); 13C NMR (100 MHz, CD3OD) δ 174.7, 143.8, 131.6, 71.1, 54.1, 45.9, 41.7, 41.6, 39.3, 39.2, 37.6, 35.3, 34.9, 34.6, 33.7, 32.4, 31.6, 29.8, 27.4, 26.6, 25.8, 23.1, 22.5, 21.6, 16.2, 15.2, 12.6; ESI-MS m/z 1415.6 ([M + H]+) and HR-ESI-MS for C87H148O6N9 ([M + H]+) calcd 1415.1547; found 1415.1558. Compound 2. Procedures as described for 1; from compound 7 (93 mg, 0.05 mmol). Yield: 33 mg (51%); 1H NMR (400 MHz, CD3OD) δ 3.45−3.37 (m, 3H), 3.25 (t, J = 6.0 Hz, 6H), 3.15 (br, 3H), 2.99 (br, 3H), 2.61 (t, J = 6.0 Hz, 6H), 2.35−1.04 (m, 81H), 0.96 (s, 9H), 0.80 (s, 9H); 13C NMR (100 MHz, CD3OD) δ 175.2, 71.2, 54.1, 53.7, 45.9, 41.8, 41.6, 39.4, 39.2, 37.4, 35.4, 34.9, 34.6, 33.8, 32.9, 31.7, 29.8, 27.4, 26.6, 25.8, 23.1, 21.7, 16.3, 12.7; ESI-MS: m/z 1312.72 ([M + H]+) and HR-ESI-MS for C78H139O6N10 ([M + H]+) calcd 1312.0874; found 1312.0881. Compound 3. DCC (380 mg, 1.84 mmol) was added to a solution of cholic acid 4 (188 mg, 0.46 mmol) and HOBt (186 mg, 1.38 mmol) in anhydrous THF (3 mL). The reaction was monitored with TLC (CH2Cl2/CH3OH = 20/1, v/v) and conducted at room temperature for 3.5 h. Then, to the mixture were added 1,3,5-tris(aminomethyl)2,4,6-triethylbenzene hydrochloride (50 mg, 0.14 mmol) and Et3N (1 mL). The reaction was monitored with TLC (CH2Cl2/CH3OH = 25/ 1, v/v). After the resulting solution was stirred at room temperature for 20 h, CHCl3 (150 mL) was added and the mixture was washed with saturated NaHCO3 (150 mL × 3). The organic phase was dried over anhydrous Na2SO4. The solvents were removed under reduced pressures. Purification was achieved by chromatography on a silica gel column, eluted with a mixture of CH2Cl2 and CH3OH (6/1, v/v) to afford compound 3 (90 mg, 42%): 1H NMR (CD3OD, 400 MHz) δ 4.45 (s, 6H), 3.96 (br, 3H), 3.81 (br, 3H), 3.43−3.37 (m, 3H), 2.80− 2.75 (m, 6H), 2.35−1.02 (m, 90H), 0.94 (s, 9H), 0.73 (s, 9H) and negative ESI-MS m/z 1420.7 ([M − H]−). The 1H NMR data were in agreement with the ones reported in literature.28 4.3. Measurement of the Anion Binding Affinity.17,29,30 4.3.1. Spectrofluorimetric Titration of 5-FAM with Compounds 1−3 and A. To a solution of 5-FAM (0.5 μM) in MeOH/HEPES buffer (1.0 mM, pH 7.0, 4/1, v/v) were added aliquots of a solution of each compound and 5-FAM (0.5 μM) in MeOH/HEPES buffer (1.0 mM, pH 7.0, 4/1, v/v). After the equilibria were achieved, the corresponding fluorescence spectra were recorded (λex 480 nm). The association constants (Ka) of each compound with 5-FAM were calculated from nonlinear least-squares fitting, using the equation based on a 1:1 binding model, I = I0 + ((I∞ − I0)/2[F]0) × {([C]0 + [F]0 + 1/Ka) − (([C]0 + [F]0 + 1/Ka)2 − 4[C]0[F]0)1/2}, where [C]0 and [F]0 are the initial analytical concentrations of compound 1 (or 2 or A) and 5-FAM, respectively; I, I0, and I∞ stand for the fluorescent intensities (at 518 nm) of the sample, 5-FAM alone, and the intensity when 5-FAM is totally complexed with compound 1 (or 2 or A), respectively. 4.3.2. Competitive Displacement of 5-FAM by Anions. To a solution of 5-FAM (0.5 μM) and compound 1 (or 2, 12 μM; or A, 185 μM) in MeOH/HEPES buffer (1.0 mM, pH 7.0, 4/1, v/v) were added aliquots of each anion solution containing 5-FAM (0.5 μM) and compound 1 (or 2, 12 μM; or A, 185 μM) in MeOH/HEPES buffer (1.0 mM, pH 7.0, 4/1, v/v). After the equilibria were achieved, the corresponding fluorescence spectra were measured (λex 480 nm). The association constants (Ka) of each compound with each anion were calculated from the nonlinear least-squares curve fitting using the equation, [A]0 = ((I∞ − I)/((I − I0) × Kd × Ka) + 1) × ([C]0 − Kd × (I − I0)/(I∞ − I) − [F]0 × (I − I0)/(I∞ − I0)), where [A]0, [C]0, and [F]0 are the initial analytical concentrations of each anion, each compound, and 5-FAM, respectively. Kd is the dissociation constant of 5-FAM with each compound. 4.4. Measurement of the Anion Transport Activity. The experimental protocols previously described by us and others37−40 were adopted to prepare the liposomes and examine the chloride efflux and pH discharge activity of compounds 1−3.
tripodal diaminocholoyl conjugates function as anion-selective transporters most probably via an anion exchange process with a minor cation/anion transport and exhibit anionophoric activity following the order of ClO4− > I− > NO3− > Br− > Cl− > SO42− > H2PO4−. Notably, both conjugates exhibit pH discharge activity even in corporation with membraneimpermeable sulfate and phosphate. Further work needs to be done to confirm how they transport sulfate and phosphate across lipid membranes so that they may find potential applications in the discovery of chemotherapeutic agents for sulfate or phosphate-involved diseases.
4. EXPERIMENTAL SECTION 4.1. General. NMR (1H and 13C) spectra were measured on a Bruker Avance AV 400 spectrometer, and the deuterium solvents were used as internal standards. Waters UPLC/Quattro Premier XE and Bruker maXis 4G ESI-Q-TOF mass spectrometers were used to record the LR and HR ESI mass spectra, respectively. Liposomes (100 nm) were formed by extrusion on an Avanti’s Mini-Extruder (Avanti Polar Lipids, Inc., Alabaster, Alabama, USA). A Mettler-Toledo PerfectIon chloride-ion-selective electrode on a Mettler-Toledo Seven Compact S220 ionometer was used to monitor the chloride efflux. A PE LS55 spectrofluorimeter was used to record the fluorescence spectra. Pyranine and EYPC were obtained from Sigma Chemical Co. (St. Louis, USA). Compound 5 was prepared starting from cholic acid 4 according to the protocols described previously.23,24 All the other chemicals and reagents were purchased from commercial companies and used directly. 4.2. Chemistry. Compound 6. DCC (204 mg, 0.99 mmol) was added to a solution of compound 5 (200 mg, 0.33 mmol) and HOBt (134 mg, 0.99 mmol) in anhydrous THF (4 mL). The reaction was monitored with TLC (CH2Cl2/CH3OH = 20/1, v/v) and performed at room temperature for 2.5 h. Then, 1,3,5-tris(aminomethyl)-2,4,6triethylbenzene hydrochloride (36 mg, 0.1 mmol) and Et3N (1.0 mL) were added to the above mixture. The reaction was monitored with TLC (CH2Cl2/CH3OH = 20/1, v/v). After the mixture was stirred at room temperature for 20 h, CHCl3 (150 mL) was added, and the resulting solution was washed with saturated NaHCO3 (150 mL × 3). The organic solution was dried over anhydrous Na2SO4. After the solvents were removed under reduced pressures, the obtained residue was purified by chromatography on a silica gel column, eluted with a mixture of CH2Cl2 and CH3OH (20/1, v/v) to afford compound 6 (35 mg, 17%): 1H NMR (400 MHz, CD3OD) δ 4.44 (s, 6H), 3.96− 3.93 (m, 3H), 3.57 (br, 3H), 3.48−3.42 (m, 3H), 2.77−2.75 (m, 6H), 2.28−0.93 (m, 153H), 0.83 (s, 9H); 13C NMR (100 MHz, CD3OD) δ 174.7, 156.6, 143.8, 131.6, 78.4, 70.9, 53.2, 48.6, 44.6, 43.7, 41.4, 38.1, 37.6, 36.5, 35.0, 34.7, 34.0, 32.7, 31.8, 31.3, 29.4, 28.4, 27.5, 26.9, 26.7, 22.7, 22.5, 21.6, 17.0, 15.1, 12.4; ESI-MS m/z 2053.5 ([M + K]+) and HR-ESI-MS for C117H197O18N9 ([M + 2H]2+) calcd 1008.2383; found 1008.2397. Compound 7. Procedures as described for compound 6; from compound 5 (300 mg, 0.50 mmol) and tren (23 μL, 0.15 mmol). Yield: 88 mg (30%); 1H NMR (400 MHz, CD3OD) δ 3.96−3.94 (m, 3H), 3.56 (br, 3H), 3.47−3.41 (m, 3H), 3.25 (t, J = 6.0 Hz, 6H), 2.64 (t, J = 6.0 Hz, 6H), 2.31−1.02 (m, 135H), 0.95 (s, 9H), 0.83 (s, 9H); 13 C NMR (100 MHz, CD3OD) δ 175.2, 156.5, 78.3, 70.9, 53.6, 53.2, 48.5, 47.4, 44.6, 43.6, 41.4, 38.1, 37.3, 36.6, 35.0, 34.7, 34.0, 33.0, 31.8, 31.3, 29.4, 28.4, 27.5, 26.9, 26.7, 22.7, 21.7, 17.1, 12.5; ESI-MS m/z 1934.61 ([M + Na]+) and HR-ESI-MS for C108H187O18N10 ([M + H]+) calcd 1912.4019; found 1912.4032. Compound 1. TFA (0.20 mL, 2.25 mmol) was added to a solution of compound 6 (30 mg, 0.02 mmol) in CH2Cl2 (1.5 mL). The reaction was monitored with TLC (CH2Cl2/CH3OH = 20/1, v/v). After 6 h, the reaction solution was evaporated under reduced pressure. The solution of the obtained residue in MeOH (0.3 mL) was added dropwise to ammonia solution (25%, 10 mL). The formed precipitates were collected through filtration and redissolved in MeOH. Concentration under reduced pressure gave compound 1 13374
DOI: 10.1021/acs.joc.7b02447 J. Org. Chem. 2017, 82, 13368−13375
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The Journal of Organic Chemistry
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S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.7b02447. Data for the structural characterization, anion binding affinity, and transmembrane anion transport activity of each compound (PDF)
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Wen-Hua Chen: 0000-0001-5008-5485 Notes
The authors declare no competing financial interest.
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REFERENCES
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DOI: 10.1021/acs.joc.7b02447 J. Org. Chem. 2017, 82, 13368−13375
