To characterize the inhibition mechanism of human DMT1 and its prokaryotic homologue EcoDMT by bis-isothiourea-containing aromatic compounds, we have synthesized seven molecules of this substance class. These include five compounds carrying two isothiourea moieties for which we have varied the aromatic scaffolds (i.e. a brominated dibenzofuran and a single phenyl ring with different substituents) to investigate the influence of their respective size and geometry on inhibition (Figure 1A, Appendix 1). For simplicity, we termed the tri-methyl and tri-ethyl substituted benzyl bis-isothiourea compounds TMBIT and TEBIT, respectively, and the dibenzofuran-based compound Br-DBFIT. Br-DBFIT, TMBIT and its derivatives were previously described as inhibitors of DMT1 (Zhang et al., 2012). To ease the identification of benzyl bis-isothiourea compounds in inhibitor complexes by X-ray crystallography, we have also synthesized the brominated derivatives Br-BIT and oBr-BIT. Additionally, we have synthesized two variants of the inhibitor oBr-BIT where we have replaced one or both isothiourea moieties by bulkier thio-2-imidazoline groups. All molecules are water-soluble and thus poorly membrane-permeable with both basic isothiourea groups being predominantly charged under physiological conditions (pKa = 8.5–9.5 as measured in a titration of TMBIT and Br-BIT, Figure 1—figure supplement 1A). We first tested the activity of all compounds on human DMT1 (hDMT1) by measuring radioactive ⁵⁵Fe²⁺ transport into HEK293 cells stably expressing the protein. When assayed at a free Fe²⁺ concentration of 1 μM, all compounds inhibit metal ion uptake in a dose-dependent manner with IC₅₀ values in the micromolar range (Figure 1B, Figure 1—figure supplement 2). The most potent compounds TEBIT and TMBIT display IC₅₀ values of 0.27 μM and 0.35 μM, respectively, latter being in close quantitative agreement with a previous measurement using a calcein-based fluorescence assay (IC₅₀ = 0.29 μM) (Figure 1B, Figure 1—figure supplement 2B,C) (Zhang et al., 2012). In comparison, the larger values of Br-BIT (4.66 μM) and oBr-BIT (2.3 μM) indicate an equivalent interaction with somewhat lower affinity and the dibenzofuran compound Br-DBFIT (1.24 μM) is in our hands less potent than previously reported (Figure 1B, Figure 1—figure supplement 2A,D and E) (Zhang et al., 2012). To rule out that the observed activity would be due to chelation of divalent metal ions, we performed isothermal titration calorimetry measurements. Upon titrating MnCl₂ to either TMBIT or Br-BIT, we did not detect any pronounced response that would indicate specific binding (Figure 1—figure supplement 1B), emphasizing that the inhibition of ⁵⁵Fe²⁺ transport is caused by the specific interactions of either compound with hDMT1. We next investigated the role of the positively charged isothiourea groups for protein interactions by comparing the potency of oBr-BIT with its variants where one or two of the moieties were replaced. In case of the replacement of a single isothiourea group, we were able to measure a four-fold reduced potency of 8.13 μM whereas a much stronger reduction (IC₅₀ = 161 μM) was obtained for a compound where both isothioureas were modified (Figure 1—figure supplement 2F,G). Together these results underline the importance of the isothiourea moieties for specific protein interactions. To further characterize the mode of inhibition, we studied the effect of different extracellular inhibitor concentrations on the kinetics of iron transport (Figure 1—figure supplement 3). In the absence of inhibitors, the transport rate at different ⁵⁵Fe²⁺ concentrations can be fitted to a Michaelis-Menten equation with K_M values of 2.6 μM to 4.4 μM and v_max values of 2.7 to 6.1 pmol min⁻¹ well⁻¹, which is in general agreement with previously reported values (Gunshin et al., 1997; Mackenzie et al., 2006; Pujol-Giménez et al., 2017). At increasing inhibitor concentrations, we observed in all tested cases a pronounced increase of the apparent K_M whereas the apparent v_max values decreased only slightly (Figure 1—figure supplement 3, Table 1). These results suggest that the compounds act by a predominant competitive mechanism. When fitting the data to a mixed enzyme inhibition model the resulting equilibrium constants are in the micromolar range with inhibitors binding with much higher affinity to the substrate-free transporter (Table 1). Taken together, our data confirm the activity of aromatic isothiourea-based compounds as competitive inhibitors of hDMT1 with potencies in the low micromolar range. As all compounds are positively charged and thus membrane-impermeable, the binding site of the inhibitor is expected to be accessible from the extracellular side.

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After the characterization of hDMT1 inhibition, we have studied the properties of different bis-isothiourea compounds on the prokaryotic SLC11 homologue EcoDMT, which catalyzes H⁺-coupled Mn²⁺ symport and whose structure was determined in an outward-facing conformation by X-ray crystallography (Ehrnstorfer et al., 2017). Due to the insufficient solubility of the dibenzofuran-based inhibitor Br-DBFIT and TEBIT for experiments with EcoDMT, we restricted this analysis to the benzyl bis-isothiourea compounds TMBIT, Br-BIT and oBr-BIT. To characterize EcoDMT-mediated transport, we have reconstituted the purified protein into liposomes and used a fluorescence-based in-vitro assay (Figure 2—figure supplement 1A). In these proteoliposomes, EcoDMT is incorporated in inside-out and outside-out orientations at about equal ratios (Figure 2—figure supplement 1B). Concentration-dependent Mn²⁺ uptake into proteoliposomes was monitored by the time-dependent quenching of the fluorophore calcein trapped inside the vesicles (Figure 2—figure supplement 1A) (Ehrnstorfer et al., 2017). In the absence of inhibitors, Mn²⁺ transport by EcoDMT saturates at low micromolar concentrations (K_M = 4.3 μM) (Figure 2—figure supplement 1C, Table 1). The addition of either benzyl bis-isothiourea compound decreases the kinetics of uptake in a dose-dependent manner thus suggesting that all tested compounds, when applied at micromolar concentrations to the outside of proteoliposomes, inhibit the transport activity of EcoDMT by binding to a saturable site of the protein (Figure 2A, Figure 2—figure supplement 1D,E). Since higher concentrations of TMBIT and oBr-BIT (i.e. >50 µM) did interfere with the assay, we restricted our quantitative characterization to Br-BIT, where we do not observe any interference at concentrations up to 200 µM. At high micromolar concentrations of Br-BIT, the decrease of transport activity approaches a maximum and even at 200 µM Br-BIT we could not detect complete inhibition. The saturation of the inhibition at high concentration results from the full occupancy of accessible binding sites, whereas the residual transport likely originates from transporters with inside-out orientation which do not expose the presumed inhibitor binding site to the external solution. The basal activity at high inhibitor concentration thus further demonstrates the sidedness of the inhibition and the membrane-impermeability of the compound. As for the inhibition of hDMT1, the K_M values of transport increased at higher inhibitor concentrations, whereas v_max did not show pronounced changes (Figure 2B, Table 1). The K_i value of 14.2 µM, representing the equilibrium dissociation constant to the substrate-free EcoDMT, is in the same range as the K_i value of 3.6 µM obtained for hDMT1, reflecting the strong structural relationship between both proteins. Together, our results suggest that Br-BIT inhibits EcoDMT and hDMT1 by a common competitive mechanism.

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To investigate the structural basis for the inhibition of divalent metal ion transporters of the SLC11 family by benzyl bis-isothiourea-based compounds, we have characterized the interaction between the brominated analogs and EcoDMT by X-ray crystallography. In our experiments we exploited the anomalous scattering properties of the inhibitors to facilitate their localization in the complex. For that purpose, we have soaked crystals of EcoDMT with Br-BIT and oBr-BIT and collected multiple datasets at a wavelength corresponding to the anomalous absorption edge of bromine (Table 2, Table 3). Whereas we were unable to detect bromine in the anomalous maps of oBr-BIT containing crystals, the majority of datasets collected from crystals soaked with Br-BIT displayed a single strong peak in the anomalous difference density at equivalent positions, which aided the localization of the bound inhibitor (Figure 3A,B, Figure 3—figure supplement 1A, Table 3). A detailed view of the complex defined in the 2Fo-Fc density at 3.8 Å is displayed in Figure 3A. In this structure, EcoDMT adopts the same substrate-free outward-facing conformation that has previously been observed in datasets of the protein in absence of the inhibitor (Figure 3C,D) (Ehrnstorfer et al., 2017). In this conformation, a funnel-shaped aqueous pocket of the protein leads from the extracellular solution to the substrate binding site. The inhibitor is bound at the base of this pocket as defined by the anomalous difference density that constrains the position of the covalently bound Br-atom and by residual density in the 2Fo-Fc omit map that was calculated with phases from a model not containing the inhibitor (Figure 3B). The fact that the Br-Atom of Br-BIT is located in the narrow apex of the pocket, whereas it would be placed in the wider part of the cavity in oBr-BIT might explain why we were unable to detect the binding position in the anomalous difference density of the latter compound. The omit map of the EcoDMT Br-BIT complex displays density for the aromatic ring and for the isothiourea group located close to the metal ion binding site (termed proximal isothiourea group), whereas the other group (the distal isothiourea group) is not defined in the electron density reflecting its increased conformational flexibility (Figure 3B). In general, the shape of the binding pocket is complementary to the structure of the inhibitor but it is sufficiently wide in the long direction of the molecule to accommodate substitutions at the aromatic ring as found in the molecules TMBIT, TEBIT and in the larger dibenzofuran ring of Br-DBFIT (Figure 3E). The aromatic group is stacked between α-helices 6 and 10 contacted by the side chains of residues Ala 231, Leu 410, Ala 409 and Leu 414. The close-by Asn 456 located on α11 might interact with the covalently attached Br atom of Br-BIT (Figure 3F, Figure 3—figure supplement 1B). The proximal isothiourea group is located in a narrow pocket in interaction distance to the conserved Asp 51 and Asn 54 in the unwound part of α−1 and to Gln 407 on α−10, which were shown to contribute to the coordination of transported metal ions (Figure 3E,F, Figure 3—figure supplement 1B) (Bozzi et al., 2019; Ehrnstorfer et al., 2014; Ehrnstorfer et al., 2017; Pujol-Giménez et al., 2017). The distal isothiourea group is located in the wider entrance of the cavity and might thus adopt different conformations, which is consistent with its undefined position in the electron density (Figure 3B). In one conformation, this group approaches residues Ser 459 and Gln 463, both located on α11. Besides the direct ionic interactions of the proximal isothiourea group with Asp 51, the positive charge of both groups would be additionally stabilized by the negative electrostatics of the pocket that is conferred by an excess of acidic residues (Figure 3—figure supplement 1C). The observed binding position and the assumed interaction of the inhibitor with the metal ion binding site is also compatible with the observed competitive nature of the inhibition.

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The high sequence similarity between bacterial and human orthologs (i.e. 52% similar and 29% identical residues between EcoDMT and hDMT1) facilitates the construction of a homology model of human DMT1 (Figure 3—figure supplement 2A,B), which permits a glimpse of potential interactions of the inhibitor with the human transporter. As this model does not contain any insertions or deletions in the binding region, we expect a similar-shaped outward-facing cavity binding the inhibitor in hDMT1 as observed for EcoDMT (Figure 3G, Figure 3—figure supplement 2B). The conservation is particularly high for α-helices 1 and 6 constituting the metal ion coordination site, but differences are observed for pocket-lining residues located on α-helices 10 and 11: While the corresponding residues Leu 414 in EcoDMT and Leu 479 in hDMT1 (both located on α10) seal the bottom of the binding cavity in both proteins, the hydrophobic character of Leu 410 and Ala 409 in EcoDMT, which contact one face of the aromatic ring is altered by the polar sidechains of Gln 475 and Ser 476 in hDMT1 (Figure 3F,H, Figure 3—figure supplement 2A). Close to the ion coordination site, Gln 407 of EcoDMT, which potentially interacts with the proximal isothiourea group, is substituted by an Asn 472 in hDMT1 (Figure 3F,H). Among the α-helices constituting the inhibitor binding site, α−11 is least conserved between hDMT1 and EcoDMT (Figure 3—figure supplement 2A). Whereas Asn 456 of EcoDMT, which contacts the Br atom of Br-BIT is conserved in hDMT1 (Asn 520), Ser 459 and Gln 463 of the bacterial transporter are replaced by bulky aromatic residues (Phe 523 and Tyr 527), which could decrease the volume of the binding pocket in hDMT1 and thus potentially constrain the conformation of the distal isothiourea group (Figure 3F,G,H). Nevertheless, since both residues are located in the wider part of the binding pocket, it is justified to assume a similar general binding mode of the inhibitor in bacterial and human orthologues. As for EcoDMT, we expect that the strongly negative electrostatic potential within the binding pocket of hDMT1 favors the binding of the positively charged inhibitor (Figure 3—figure supplement 2C). Taken together, our structural data thus provide a detailed view of the molecular basis of the interaction of benzyl bis-isothiourea-based inhibitors with divalent metal ion transporters of the SLC11/NRAMP family.

To further characterize the binding of Br-BIT to EcoDMT, we have studied the effect of mutations of putative contact residues identified in the structure on inhibition (Figure 4A). Although the described results emphasize the importance of interactions of the isothiourea group with the metal-coordination site, these cannot be probed with the applied transport assays as mutations of coordinating resides interfere with ion uptake. We have thus employed isothermal titration calorimetry (ITC) to directly measure the effect of a metal-binding site mutant in EcoDMT on inhibitor binding. In ITC experiments, we find two signals in the thermograms in response to the titration of the inhibitor to the WT protein. A weak endothermic contribution, which saturates at low μM concentrations (K_D = 34.5 ± 5.0 μM) can be attributed to the loading of the inhibitor binding site and an exothermic signal saturating with an affinity in the mM range to a potential non-specific interaction with the protein (Figure 4B, Figure 4—figure supplement 1A,B). To characterize the observed interaction between the positively charged isothiourea group and the negatively charged Asp 51 of the metal-binding site, we have expressed and purified the mutant D51A and measured inhibitor binding. Whereas the low-affinity signal in the thermograms appears unaltered, the high affinity component is absent, as expected if the mutant has removed an important interaction which interferes with inhibitor binding (Figure 4C, Figure 4—figure supplement 1A,B). Thus, despite the weak signal originating from the low enthalpic contribution to binding, our titration calorimetry experiments indicate a direct interaction of the isothiourea group with the metal binding site as expected for a competitive inhibitor.

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To probe the role of other residues of EcoDMT in the vicinity of the bound inhibitor, we have characterized the effect of alterations of three hydrophilic residues on α-helix 11 on the inhibition of Mn²⁺ transport. Based on our structures, we suspected Gln 463 and Ser 459 to interact with the distal isothiourea group and Asn 456 with the bromine atom on the aromatic ring of Br-BIT (Figure 4A). The three constructs, the single mutants N456A and N456L and the triple mutant N456A/S459A/Q463A transport Mn²⁺ with similar kinetics as WT (Figure 4—figure supplement 2A–C, Table 1). As for WT, Mn²⁺ transport in all three mutants is inhibited upon addition of Br-BIT, although with slightly decreased potency (K_i: WT, 14.2 μM; N456A, 29.3 μM; N456L, 28.8 μM; N456A/S459A/Q463A, 23.8 μM) (Figure 4D, Figure 4—figure supplement 2D–F, Table 1). In light of the small difference in K_i compared to WT, our data excludes a large energetic contribution of residues on α11 to inhibitor binding, consistent with the assumed mobility of the distal isothiourea group that is manifested in the lack of electron density of the group in the structures of EcoDMT Br-BIT complexes.

To characterize the role of residues in the predicted inhibitor binding pocket of human DMT1, we have generated several point mutants and investigated the effect of these mutations on the interaction with different inhibitors. Due to the strong negative impact of alterations of the metal ion coordination site on transport, mutagenesis was restricted to residues lining the remainder of the binding pocket. The investigated positions encompassed residues on α−6 (Ala 291), α−10 (Gln 475, Ser 476 and Leu 479), and α−11 (Asn 520, Phe 523 and Tyr 527) (Figures 3H and 5A). In our experiments we wanted to target interactions of protein residues with the aromatic ring in the narrow part of the binding pocket by either shortening the side-chains in the mutants A291G and Q475A, or by increasing their size in the mutants A291V, Q475F, S476V and L479F. In the orthogonal direction, the binding pocket is wider and would on one side be delimited by resides located on α−11 (Figures 3G and 5A). Based on our model, we suspected the aromatic side chains of Phe 523 and Tyr 527 to be located in proximity to the distal isothiourea groups of TMBIT, TEBIT and Br-BIT or to the second phenyl-ring in the case of the dibenzofuran-based compound Br-DBFIT and Asn 520 in interaction distance with the aromatic ring harboring the proximal isothiourea group in all compounds (Figures 3H and 5D). To probe these potential interactions, we have truncated the aromatic side chains in the mutants F523A and Y527A and generated a nearly isosteric hydrophobic substitution in the mutant N520L and subsequently studied the ⁵⁵Fe²⁺ uptake properties of HEK293 cells transiently transfected with DNA coding for the respective constructs. Transport is similar to WT in case of the mutants S476V, F523A and Y527A, reduced in the mutants Q475A and N520L and undetectable in the mutants A291G, A291V, Q475F and L479F (Figure 5B, Figure 5—figure supplement 1A). Mutations that render hDMT1 inactive, most likely interfere with structural rearrangements during ion transport, as judged by the tight packing of the respective region in the inward-facing structures of SLC11 transporters (Bozzi et al., 2016b; Bozzi et al., 2019; Ehrnstorfer et al., 2014). Inhibition experiments on hDMT1 were carried out with Br-BIT used for crystallization, the more potent inhibitors TMBIT and TEBIT and the dibenzofuran-based compound Br-DBFIT to explore the influence of the aromatic scaffold and the geometric relationship between the two isothiourea groups on interactions. Similar to WT, the addition of either compound at equivalent concentrations decreases uptake in the mutants Y527A and Q475A both located towards the extracellular entrance to the binding pocket (Figures 3H and 5A,C and Figure 5—figure supplement 1B–D) thus suggesting that interactions with these residues do not strongly contribute to inhibitor binding. Conversely, the compounds had much smaller effects on the transport activity of cells expressing the mutants S476V, N520L and F523A located deeper in the binding pocket (Figures 3H and 5C,D, Figure 5—figure supplement 1B–C) thus suggesting that in these cases, the mutations affected inhibitor interactions. To further characterize the inhibitory properties of the investigated compounds, we have measured uptake at different inhibitor concentrations and found a strong reduction in potency in most cases (Figure 5E, Table 4). Whereas the effect is uniform in the mutant S476V for all investigated compounds, the mutants N520L, and F523A showed a decreased potency of inhibition for the related molecules Br-BIT, TMBIT and TEBIT but only a slight reduction for Br-DBFIT (Figure 5E, Figure 5—figure supplement 1E, Table 4) indicating that residues on α−11 might form distinct interactions with different inhibitor classes. This is consistent with the wide dimensions of the pocket in that direction that allows for a geometry-dependent placement of the aromatic ring and the attached isothiourea moiety on the distal side (Figure 5D). Taken together our results suggest an involvement of residues on α−10 and α−11 on inhibitor binding to hDMT1 although with variable specificity, consistent with the proposed general binding mode of the inhibitors, which constrain the binding of the first aromatic ring to position the proximal isothiourea group in interaction distance with the metal ion coordination site. Since equivalent mutations of α11 in EcoDMT had little impact on inhibition of Br-BIT, our results also point towards species-dependent energetic differences in inhibitor interactions on the distal side of the inhibitor binding pocket, which are reflected in the poor conservation of residues in α11 and the wide geometry of the pocket in the prokaryotic transporter. Despite the described species-dependent differences, our data is generally consistent with the notion that the characterized compounds inhibit both pro- and eukaryotic transporters by binding to equivalent regions.

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The chemical synthesis of all compounds is described in Appendix 1.

Experiments using human cell lines were conducted with HEK293 cells either stably (ATCC-CRL-1573) or transiently (ATCC-CRL3216) over-expressing DsRED-hDMT1 constructs. The cell line stably over-expressing hDMT1 has been characterized previously (Montalbetti et al., 2014). Mycoplasma contamination was negative for both cell lines as tested with the LooKOut Mycoplasm PCR Detection Kit (Sigma-MP0035). All cells were grown in DMEM media (Invitrogen) supplemented with 10% FBS, 10 mM HEPES and 1 mM Na-pyruvate at 37°C, 95% humidity and air containing 5% CO₂. For cells stably over-expressing hDMT1, the media was additionally supplemented with 500 µg ml^–1 geneticin (Life Technologies).

The sequence coding for the hDMT1 isoform 1A-IRE (+) (UniProt identifier P49281-3) was cloned into pIRES2 DsRed-Express2 bicistronic vector (Montalbetti et al., 2014). Single-point mutations A291G, A291V, Q475A, Q475F, S476V, L479F, N520L, F523A and Y527A were introduced into the hDMT1 encoding sequence as previously described (Pujol-Giménez et al., 2017). For EcoDMT, the corresponding gene (UniProtKB identifier E4KPW4) was cloned using genomic DNA isolated from a Eremococcus coleocola strain (DSM No. 15696) into the ​arabinose-inducible expression vectors pBXC3GH and pBXC3H with fragment-exchange (FX) cloning (Geertsma and Dutzler, 2011). The point mutations N456A, N456L, S459A and Q463A were introduced by site-directed mutagenesis (Li et al., 2008).

For uptake experiments, HEK293 cells were grown in clear bottom, white-well, poly-D-lysine coated 96 well plates (Corning). Cells stably over-expressing hDMT1 were seeded 24 hr before the experiment at a density of 50,000 cells/well and cells used for transient transfection were seeded at 30.000 cells/well for 48 hr prior to the experiment and transfected 24 hr before the experiment using Lipofectamine 2000 (Life technologies) as described in the manufacturer’s protocol. Briefly, culture media was removed from the wells and the cells were washed three times with uptake buffer (140 mM NaCl, 2.5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 1.2 mM K₂HPO₄, 10 mM glucose, 5 mM HEPES, 5 mM MES, pH 7.4). After the wash, the cells were incubated for 15 min at room temperature (RT) with uptake solution containing the indicated amount of non-radioactive ferrous iron (Fe²⁺), 100 µM Ascorbic acid and 0.5 µCi/ml radiolabeled ⁵⁵Fe²⁺ (American Radiolabeled) dissolved in uptake buffer (pH 5.5). After incubation, uptake solution was removed from the wells, and the cells were washed three times in ice-cold uptake buffer (pH 7.5). Before quantification, a scintillation cocktail (Mycrosinth 20, PerkinElmer) was added to each well, and the cells were incubated during 1 hr at RT under constant agitation. Accumulated radioactivity was measured using a TopCount Microplate Scintillation Counter (PerkinElmer). Transport rates were quantified with:

To assess their inhibitory effect, cells were incubated with the indicated compounds at the specified concentrations during 5 min at RT prior to the addition of the uptake solution. To determine the kinetic parameters for the Fe²⁺ transport mediated by hDMT1 WT and point mutants, the influx rates at different iron concentrations were fitted to the Michaelis-Menten equation. For the determination of IC₅₀ values, influx rates for each inhibitor concentration were plotted and data was fitted to a 4-parameter sigmoidal curve. Plotted influx rates correspond to the mean of the indicated biological replicates, errors are s.d. Each experiment was performed in duplicates for transiently transfected cells with data obtained from at least two independent transfections or triplicates for stably overexpressed WT hDMT1.

EcoDMT WT and mutants were expressed in E. coli MC1061 as C-terminally-tagged fusion proteins containing a 3C-protease cleavage site followed by a His₁₀-tag. The tag was removed during purification unless specified otherwise. E. coli cells were grown in Terrific Broth (TB) medium supplemented with 100 µg ml^–1 ampicillin, either by fermentation or in flasks. Cells were grown at 37°C and the temperature was gradually decreased to 25°C prior to induction. Protein expression was induced by addition of 0.0045% (w/v) ​L-arabinose at an OD₆₀₀ of ~2.5 for fermenter cultures and ~0.8 for cultures in flasks. For overnight expression the temperature was decreased to 18°C and cells were subsequently harvested by centrifugation. All following protein purification steps were carried out at 4°C. The cells were lysed in buffer A (20 mM HEPES, pH 7.5, and 150 mM ​NaCl) supplemented with 1 mg ml^–1 (w/v) lysozyme and 20 µg ml^–1 DNaseI using HPL6 high-pressure cell disruptor (MAXIMATOR). The lysate was subjected to a low-spin centrifugation (10,000 g for 20 min) and subsequently the membrane vesicles were harvested by ultracentrifugation (200,000 g for 1 hr). Membrane proteins were extracted by resuspending the vesicles in buffer A containing 10% (w/v) glycerol and 1–2% (w/v) of the specified detergents and subsequently the extract was cleared by centrifugation. The detergent n-decyl-β-D-maltopyranoside (DM, Anatrace) was used to purify proteins for reconstitution or crystallization experiments and n-dodecyl-β-D-maltopyranoside (DDM, Anatrace) for isothermal titration calorimetry (ITC). The extracted proteins were purified by immobilized metal affinity chromatography (IMAC). The GFP- His₁₀ tag was removed by addition of HRV-3C protease at a protein:protease molar ratio of 5:1 for 2 hr while dialyzing the sample against 20 mM HEPES, pH 7.5, 150 mM ​NaCl, 8.7% (w/v) ​glycerol, and 0.1% (w/v) ​DM or 0.04% (w/v) DDM. A second IMAC step was used to separate the GFP-His₁₀ tag and the protease from the cleaved protein. Subsequently, the purified membrane proteins were subjected to size exclusion chromatography on a Superdex S200 column (GE Healthcare) equilibrated in 10 to 20 mM ​HEPES, pH 7.5, 150 mM ​NaCl, and either 0.25% (w/v) ​DM or 0.04% (w/v) DDM. Peak fractions were used for reconstitution into liposomes, ITC and crystallization experiments. Purified samples of WT and mutant proteins were analyzed by SDS-PAGE and mass spectrometry.

​Crystals of EcoDMT were grown in 24-well plates in sitting drops at 4°C by mixing 1 μl of protein (at a concentration of 7–10 mg ml^–1) with 1 μl of reservoir solution consisting of 50 mM Tris-HCl pH 8.0–9.0 and 22–26% PEG 400 (v/v) and equilibrated against 500 μl of reservoir solution. Crystals grew within two weeks. For preparation of inhibitor complexes, crystals were soaked for several minutes with either Br-BIT or oBr-BIT. The two inhibitors were either added to the cryoprotection solutions at a final concentration of 5 mM or directly added as powder to the drops containing the crystals. For cryoprotection, the PEG 400 concentration was increased stepwise to 35% (v/v). All data sets were collected on frozen crystals on the X06SA or the X06DA beamline at the Swiss Light Source of the Paul Scherrer Institute on an EIGER X 16M or a PILATUS 6M detector (Dectris). Anomalous data were collected at the bromine absorption edge (0.92 Å). Data were integrated and scaled with XDS (Kabsch, 2010) and further processed with CCP4 programs (Collaborative Computational Project, 1994). Structures were refined in Phenix (Adams et al., 2002) using the EcoDMT WT structure (PDB ID 5M87) as starting model. The model was modified in COOT (Emsley and Cowtan, 2004) and constraints for the refinement of the Br-BIT ligand were generated using the CCP4 program PRODRG (Schüttelkopf and van Aalten, 2004). Five percent of the reflections not used in refinement were used to calculate R_free. The final refinement statistics is reported in Table 2. The coordinates of the EcoDMT-Br-BIT complex refined to data at 3.8 Å were deposited with the PDB under accession code 6TL2.

The electrostatic potential in the extracellular aqueous cavity harboring the inhibitor binding site was calculated by solving the linearized Poisson–Boltzmann equation in CHARMM (Brooks et al., 1983; Im et al., 1998) on a 150 Å ×150 Å × 200 Å grid (1 Å grid spacing) followed by focusing on a 100 Å x 100 Å x 120 Å grid (0.5 Å grid spacing). Partial protein charges were derived from the CHARMM36 all-hydrogen atom force field. Hydrogen positions were generated in CHARMM, histidines were protonated. The protein was assigned a dielectric constant (ϵ) of 2. Its transmembrane region was embedded in a 30 Å-thick slab (ϵ = 2) representing the hydrophobic core of the membrane and two adjacent 10 Å-thick regions (ϵ = 30) representing the headgroups. The membrane region contained a 38 Å-high and 22 Å-wide aqueous cylinder (ϵ = 80) covering the extracellular aqueous cavity and was surrounded by an aqueous environment (ϵ = 80). Calculations were carried out in the absence of monovalent mobile ions in the aqueous regions. The homology model of human DMT1 was prepared with the SWISS-MODEL homology modeling server (Biasini et al., 2014).

EcoDMT WT and mutants were reconstituted using detergent destabilized liposomes according to Geertsma et al. (2008). The liposomes were formed using the synthetic phospholipids POPE and POPG (Avanti Polar lipids) at a w/w ratio of 3:1. The lipids where resuspended in 20 mM HEPES, pH 7.5, and 100 mM KCl after washing with diethylether and drying by exsiccation. Liposomes were subjected to three freeze-thaw cycles and extruded through a 400 nm polycarbonate filter (Avestin, LiposoFast-Basic) to form unilammellar vesicles. Triton X-100 was used to destabilize the liposomes and the reconstitutions were performed at a protein to lipid ratio of 1:100 (w/w) for transport assays and a protein to lipid ratio of 1:50 (w/w) to determine the orientation of the transporters in the liposomes. After detergent removal by the successive addition of Bio-Beads SM-2 (Bio-Rad) over a period of three days, proteoliposomes were harvested by centrifugation, resuspended in buffer containing 20 mM HEPES, pH 7.5, and 100 mM KCl and stored in liquid nitrogen.

The orientation of the transporters in proteoliposomes was determined using a reconstitution of EcoDMT-His₁₀ in which the C-terminally Histidine-tag preceded by a 3C protease cleavage site has not been cleaved prior to reconstitution. Initially, proteoliposomes (containing a total of 2 mg lipids) were extruded using a 400 nm polycarbonate filter to generate unilammellar vesicles and split in two equal aliquots. Purified 3C protease was subsequently added to the outside of one aliquot of the proteoliposmes and incubated for 2 hr at room temperature. The external 3C protease was removed by washing twice with 20 volumes of 20 mM HEPES, pH 7.5, and 100 mM KCl and the liposomes were harvested by centrifugation. After removal of the protease, the liposomes were dissolved by addition of DM at a detergent to lipid ration of 1.25:1 (w/w) with half of the samples incubated with 3C protease for 2 hr on ice. All 3C cleavage steps were performed with a large excess of protease to ensure completion of the reaction. Control liposomes not treated with 3C protease at the different steps were processed the same way. A sample of purified EcoDMT-His₁₀ was used as control to follow the removal of the His₁₀-tag in a sample with unrestricted accessibility to the 3C cleavage site. The final samples were analyzed by SDS-PAGE.

Proteoliposomes for the Mn²⁺ transport and inhibition assays were obtained by resuspension of vesicles in buffer B containing 20 mM HEPES, pH 7.5, 100 mM KCl and 250 μM calcein (Invitrogen) and subjection to three freeze-thaw cycles followed by extrusion through a 400 nm filter. Proteoliposomes were harvested by centrifugation and washed twice with 20 volumes of buffer B without Calcein. The samples were subsequently diluted to 0.25 mg lipid ml^–1 in buffer containing 20 mM HEPES, pH 7.5 and 100 mM NaCl and varying concentrations of TMBIT, Br-BIT or oBr-BIT. Subsequently, 100 μl aliquots were placed in a black 96-well plate and after stabilization of the fluorescence signal, valinomycin (at a final concentration of 100 nM) and MnCl₂ were added to start the assay. Uptake of Mn²⁺ into liposomes was recorded by measuring the fluorescence change in a fluorimeter (Tecan Infinite M1000, λ_ex=492 nm/ λ_em=518 nm) in four-second intervals. As a positive control, Mn²⁺ ions were equilibrated by addition of the ionophore calcimycin (at a final concentration of 100 nM) (Invitrogen), which acts as a Mn²⁺/H⁺ exchanger at the end of the experiments. In presence of TMBIT or oBr-BIT at concentrations higher than 50 µM, the fluorescence signal after addition of calcimycin did not reach the same low level as observed in absence of inhibitors, which suggests an interference of the compounds with the activity of calcimycin at high concentrations. Initial transport rates (ΔF min^–1) were obtained by performing a linear regression of transport data obtained between 60 and 120 s after addition of valinomycin and MnCl₂ and fitted to a Michaelis-Menten equation. Kinetic data of WT and all mutants described in this study was measured in at least three independent experiments.

Kinetic data was fitted to a mixed enzyme inhibition model outlined below (Scheme 1) (Copeland, 2005) with GraphPad Prism (GraphPad Software, San Diego, California USA, www.graphpad.com):

Scheme 1

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This model assumes that the inhibitor (I) binds to the substrate free transporter (T) and to the transporter-substrate complex (T·M²⁺) with equilibrium constants K_i and K_ii, respectively. Both equilibrium constants can be obtained by a non-linear regression to Equation 2

For high values of α, the inhibitor preferentially binds to the substrate-free transporter and Equation 2 approaches a model for competitive inhibition. The resulting equilibrium constants obtained for hDMT1 using a radioactive ⁵⁵Fe²⁺ transport assay and for EcoDMT1 using an in vitro proteoliposome-based assay are summarized in Table 1.

Isothermal titration calorimetry experiments were performed with a MicroCal ITC200 system (GE Healthcare). The titrations of MnCl₂ to TMBIT and Br-BIT were performed at 25°C in 20 mM HEPES, pH 7.5, and 100 mM KCl. The syringe was filled with 5 mM MnCl₂ and sequential aliquots of 2 μl were added to the sample cell filled with 0.4 mM TMBIT, Br-BIT, Ethylenediaminetetraacetic acid (EDTA) or buffer. The titrations of Br-BIT to purified EcoDMT were performed at 6°C in 20 mM HEPES, pH 7.5, 150 mM and 0.04% (w/v) DDM. The syringe was filled with 1.8 mM or 2.5 mM Br-BIT and sequential aliquots of 1.5–2 μl were added to the sample cell filled with ~50 µM or ~180 µM EcoDMT WT, the mutant D51A or buffer. Data were analyzed using the Origin ITC analysis package and the MicroCal ITC program Concat and errors on the reported K_D values represent fitting errors. The data were fit using models assuming one or two sets of binding sites. In case of 2.5 mM Br-BIT in the syringe and ~180 µM EcoDMT in the cell, mainly the high affinity step saturating in the low micromolar range is titrated. Therefore, the low affinity transition can be ignored and the resulting reaction enthalpies were y-translated to zero to enable data analysis using a model assuming a single set of binding sites. For each protein, similar results were obtained for at least two experiments from independent protein preparations.

The coordinates and structure factors of the EcoDMT-Br-BIT complex have been deposited in the Protein Data Bank with the accession code 6TL2.

Brominated intermediate (one eq) and thiourea (two eq) were dissolved in ethanol. The solution was stirred at 80°C overnight and then cooled down to room temperature. Ethanol was removed except 1 ml which was kept. Hexane was added, and the precipitate was filtered and dried in vacuo (Chafeev et al., 2008).

Compound 1 was synthesized following method 1 using 10 (169 mg, 0.33 mmol,1.0 eq) and thiourea (57 mg, 0.75 mmol, 2.3 eq) in ethanol (9 ml). The precipitate was washed with diethyl ether/Ethyl acetate (50/50) and then purified by preparative HPLC (gradient from 85/15 A/D to 45/55 A/D in 18 min, t_R = 31% D) to afford 1 (120 mg, 0.33 mmol, 50%) as an off-white solid (Appendix 1—figure 1).

RP-UPLC: t_R = 3.27 min (method B). ¹H NMR (400 MHz, DMSO): δ 9.38 (s, 6H, NH + NH₂), 8.17 (2H, d, J = 8.3 Hz), 7.80 (2H, d, J = 8.3 Hz), 4.93 (4H, s, CH₂). ¹³C NMR (400 MHz, DMSO): δ 168.6, 154.2, 128.6 (CH_ar), 123.3, 123.2, 123.1 (CH_ar), 119.0, 29.9 (CH₂). HR-MS calculated for C₁₆H₁₅Br₂N₄OS₂: m/z 500.9049, m/z found 500.9053.

Compound 2 was synthesized following method 1 using 11 (200 mg, 0.65 mmol, one eq) and thiourea (109 mg, 1.4 mol, 2.2 eq) in ethanol (10 ml). The residue was purified by preparative HPLC (gradient from 95/5 A/D to 75/25 A/D in 18 min, t_R = 16% D) to afford 2 (215 mg, 0.41 mmol, 62%) as a white solid (Appendix 1—figure 2).

RP-UPLC: t_R = 1.31 min (method A). ¹H NMR (400 MHz, DMSO): δ 9.41 (6H, s, NH), 7.03 (1H, s, CH_ar), 4.50 (4H, s, CH₂), 2.40 (3H, s, CH₃), 2.33 (6H, s, CH₃). ¹³C NMR (101 MHz, DMSO): δ 169.9, 158.4, 138.1, 137.8, 130.7 (CH_ar), 127.6, 30.6 (CH₂), 19.3 (CH₃), 15.1 (CH₃). HR-MS calculated for C₁₃H₂₁N₄S₂: m/z 297.1202, m/z found 287.1205.

Compound 3 was synthesized following method 1 using 12 (42 mg, 0.1 mmol, one eq) and thiourea (16 mg, 0.21 mmol, 2.2 eq) in ethanol (4 ml). The residue was purified by preparative HPLC (gradient from 95/5 A/D to 75/25 A/D in 18 min, t_R = 24% D) to afford 3 (52 mg, 0.09 mmol, 94%) as a white solid (Appendix 1—figure 3).

RP-UPLC: t_R = 1.52 min (method A). ¹H NMR (300 MHz, DMSO): δ 9.26 (6H, s, NH + NH₂), 7.08 (1H, s, H_ar), 4.45 (4H, s, CH₂), 2.71 (6H, m, CH₂CH₃), 1.21 (9H, t, J = 7.5 Hz, CH₃). ¹³C NMR (101 MHz, DMSO): δ 169.5, 144.6, 143.8, 127.8 (CH_ar), 126.1, 29.5 (CH₂), 25.3 (CH₂), 22.3 (CH₂), 16.5 (CH₃), 15.4 (CH₃). HR-MS calculated for C₁₆H₂₇N₄S₂: m/z 339.1672, m/z found 339.1670.

Compound 4 was synthesized following method 1 using 1-Bromo-3,5-bis(bromomethyl)benzene (200 mg, 0.58 mmol, one eq) and thiourea (98 mg, 1.3 mmol, 2.2 eq) in ethanol (10 ml). The precipitate was washed with EtOAc and dried in vacuo to afford 4 (262 mg, 0.53 mmol, 90%) as a white solid (Appendix 1—figure 4).

RP-UPLC: t_R = 1.25 min (method A). ¹H NMR (400 MHz, DMSO): δ 9.13 (s, 6H), 7.63 (d, J = 1.2 Hz, 2H), 7.46 (s, 1H), 4.50 (s, 4H). ¹³C NMR (101 MHz, DMSO): δ 168.5, 138.5, 131.2 (CH_ar), 128.5 (CH_ar), 121.8, 33.2 (CH₂). HR-MS calculated for C₁₀H₁₄BrN₄S₂: m/z 332.9838, m/z found 332.9836.

Compound 5 was synthesized following method 1 using 2-bromo-1,3-bis(bromomethyl)-benzene (158 mg, 0.46 mmol, one eq) and thiourea (79 mg, 1.05 mmol, 2.3 eq) in ethanol (8 ml). The residue was purified by preparative HPLC (gradient from 100/0 A/D to 85/15 A/D in 18 min, t_R = 4% D) to afford 5 (70 mg, 0.13 mmol, 27%) as a white solid (Appendix 1—figure 5).

RP-UPLC: t_R = 1.18 min (method A). ¹H NMR (400 MHz, DMSO): δ 9.36 (6H, s, NH + NH₂), 7.59 (2H, d, J = 7.6 Hz, H_ar), 7.47 (1H, t, J = 7.2 Hz, H_ar), 4.60 (4H, s, CH₂). ¹³C NMR (101 MHz, DMSO): δ 168.9, 135.5, 131.3 (CH_ar), 128.5 (CH_ar), 126.5, 35.8 (CH₂). HR-MS calculated for C₁₀H₁₄BrN₄S₂: m/z 332.9838, m/z found 332.9842.

2-bromo-1,3-bis(bromomethyl)-benzene (150 mg, 0.44 mmol, one eq) was dissolved in ethanol (8 mL) before the addition of thiourea (34 mg, 0.45 mmol, one eq). The solution was stirred at 40°C overnight and then cooled down to room temperature. Ethanol was removed and the residue was purified by column chromatography on silica gel (DCM/MeOH 90/10). The product was dissolved in ethanol (8 ml) and imidazolidine-2-thione (47 mg, 0.44 mmol, one eq) was added. The solution was stirred at 40°C overnight. The solution was cooled down to room temperature and ethanol was removed except 1 ml which was kept. Hexane was added and the precipitate was filtered and dried in vacuo. The product was purified by preparative HPLC (gradient from 100% A to 90/10 A/D in 28 min, t_R = 7% D) to afford 6 (73 mg, 0.13 mmol, 28%) as a white solid (Appendix 1—figure 6).

RP-UPLC: t_R = 1.21 min (method A). ¹H NMR (300 MHz, MeOD): δ 7.58 (2H, d, J = 7.6 Hz, H_ar), 7.47 (1H, t, J = 7.5 Hz, H_ar), 4.67 (2H, s, CH₂), 4.63 (2H, s, CH₂), 4.00 (4H, s, CH₂). ¹³C NMR (101 MHz, MeOD): δ 172.0, 170.9, 136.6, 136.2, 133.1 (CH_ar), 132.9 (CH_ar), 129.9 (CH_ar), 128.0, 6, 46.8 (CH₂), 37.9 (CH₂), 37.8 (CH₂). HR-MS calculated for C₁₂H₁₆BrN₄S₂: m/z 358.9994, m/z found 358.9995.

Compound 7 was synthetized following method 1 using 2-bromo-1,3-bis(bromomethyl)-benzene (165 mg, 0.48 mmol, one eq) and imidazolidine-2-thione (111 mg, 1.1 mmol, 2.2 eq) in ethanol (8 ml). The residue was purified by preparative HPLC (gradient from 100% A to 80/20 A/D in 28 min, t_R = 8% D) to afford 7 (141 mg, 0.23 mmol, 48%) as a white solid (Appendix 1—figure 7).

RP-UPLC: t_R = 1.17 min (method A). ¹H NMR (300 MHz, DMSO): δ 7.60 (2H, d, J = 7.5 Hz, H_ar), 7.48 (1H, t, J = 7.3 Hz, H_ar), 4.67 (4H, s, CH₂), 4.00 (8H, s, CH₂). ¹³C NMR (101 MHz, MeOD): δ 170.9, 136.3, 133.0 (CH_ar), 130.0 (CH_ar), 128.0, 46.8 (CH₂), 37.9 (CH₂). HR-MS calculated for C₁₄H₁₈BrN₄S₂: m/z 385.0151, m/z found 385.0142.

In a three-necks flask, dibenzofuran (2.0 g, 11.9 mmol, one eq) was dissolved in dry diethyl ether (100 ml) under argon atmosphere. The temperature was cooled down to – 78°C before the addition of TMEDA (4.3 ml, 29.5 mmol, 2.6 eq). sec BuLi (21.2 ml, 1.4 M, 29.7 mmol, 2.5 eq) was added slowly (dropwise). The mixture was stirred 16 hr and allowed to heat at room temperature during the night. Iodomethane (3.80 ml, 61.0 mmol, 5.0 eq) was added. The reaction mixture was stirred at room temperature for 20 hr. Then NH₄Cl sat (70 ml) was added to quench the reaction. The product was extracted with diethyl ether (3 times 100 ml). The organic layers were combined, dried over Na₂SO₄, filtered and concentrated. The residue was recrystallized from methanol to afford 8 (846 mg, 4.3 mmol, 36%) as a white solid (Chafeev et al., 2008).

¹H NMR (400 MHz, CDCl₃): δ 7.75 (2H, dd, J = 6.4, 2.6 Hz, H_ar), 7.25–7.20 (2H, m, H_ar), 2.62 (6H, s, CH₃). ¹³C NMR (75 MHz, CDCl₃): δ 155.1, 128.0 (CH_ar), 124.2, 122.6 (CH_ar), 122.0, 118.2 (CH_ar), 15.4 (CH₃).

8 (200 mg, 1.0 mmol, one eq) was dissolved in acetic acid (3 ml). Br₂ (0.11 ml, 2.1 mmol, 2.1 eq) was added. The mixture was stirred at room temperature overnight. Br₂ (0.4 ml, 0.82 mmol, 0.8 eq) was added and the solution was stirred at room temperature for 24 hr. Water was added. A precipitate appeared, was filtered and kept. DCM was added in the filtrate. Product was extracted with DCM. Organic layers were combined, washed with water, dried over Na₂SO₄ and concentrated. All compound was recrystallized from ethyl acetate and then purified by flash column chromatography on silica gel (Hexane) to afford 9 (330 mg, 1.1 mmol, 65%) as an off-white solid (Chafeev et al., 2008).

RP-GCMS: t_R = 9.50 min (from 50°C to 280°C in 8 min, then stay at 280°C). ¹H NMR (300 MHz, CDCl₃): δ 7.58 (2H, d, J = 8.4 Hz), 7.50 (2H, d, J = 8.3 Hz), 2.64 (s, 6H). ¹³C NMR (101 MHz, CDCl₃): δ 155.3, 127.2 (CH_ar), 123.3, 123.0, 122.9, 118.8 (CH_ar), 15.7 (CH₃).

9 (196 mg, 0.56 mmol, 1.0 eq) and NBS (214 mg, 1.2 mmol, 2.2 eq) were dissolved in CCl₄ (41 ml) under argon. Benzoyl peroxide (one spatula) was added. The mixture was stirred at 60°C overnight. NBS (212 mg, 1.2 mmol, 2.2 eq) and benzoyl peroxide (1.5 spatula) were added. The mixture was stirred at 60°C overnight before cooled down to 0°C. The white precipitate was filtered, and the solvent was removed. The residue was recrystallized from Hexane/Ethyl acetate (50:50) to afford 10 (134 mg, 0.56 mmol, 47%) as an off-white solid (Chafeev et al., 2008).

¹H NMR (300 MHz, CDCl₃): δ 7.73 (2H, d, J = 8.3 Hz), 7.60 (2H, d, J = 8.3 Hz), 5.00 (4H, s, CH₂). ¹³C NMR (101 MHz, CDCl₃): δ 154.9, 128.4 (CH_ar), 123.6, 123.6, 122.8, 121.8 (CH_ar), 26.0 (CH₂).

To a suspension of paraformaldehyde (150 mg, 5.0 mmol, two eq) in acetic acid (2.5 ml), mesitylene (0.35 ml, 2.5 mmol, one eq) was added. HBr (1 ml, 31% in acetic acid) was added quickly. The mixture was stirred at 80°C for 8 hr. Water (10 ml) was added and the white precipitate was filtered and dried in vacuo. The product was purified by flash chromatography on silica gel (Hexane) to afford 11 (530 mg, 1.7 mmol, 69%) as a white solid (Van der Made and Van der Made, 1993).

¹H NMR (400 MHz, CDCl₃): δ 6.90 (s, 1H), 4.57 (s, 4H), 2.45 (s, 3H), 2.38 (s, 6H). ¹³C NMR (101 MHz, CDCl₃): δ 138.2, 137.3, 132.8, 130.9 (CH_ar), 29.9 (CH₂), 19.6 (CH₃), 14.8 (CH₃).

To a suspension of paraformaldehyde (80 mg, 2.7 mmol, two eq) in acetic acid (2.5 ml), 1,3,5-triethylbenzene (0.25 ml, 1.3 mmol, one eq) was added. HBr (0.7 ml, 31% in acetic acid) was added quickly. The mixture was stirred at 80°C for 8 hr. Water (10 ml) was added and product was extracted three times with EtOAc. The combined organic layers were dried over Na₂SO₄ and concentrated. The residue was purified by flash chromatography on silica gel (Hexane) to afford 12 (42 mg, 0.1 mmol, 7%) as a white solid (Van der Made and Van der Made, 1993).

RP-UPLC: t_R = 1.91 min (method A). ¹H NMR (400 MHz, CDCl₃): δ 6.97 (1H, s, H_ar), 4.60 (4H, s, CH₂), 2.94 (2H, q, J = 7.6 Hz, CH₂CH₃), 2.77 (4H, q, J = 7.6 Hz, CH₂CH₃), 1.32 (9H, m, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ 144.9, 143.6, 131.5, 127.7 (CH_ar), 28.7 (CH₂), 26.0 (CH₂), 22.5 (CH₂), 15.9 (CH₃), 15.1 (CH₃).

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

This research was supported by the Swiss National Science Foundation (SNF) through the National Centre of Competence in Research TransCure and the SNF grant 310030_182272 to Matthias A Hediger. Data collection was performed at the X06SA and X06DA beamlines at the Swiss Light Source of the Paul Scherrer Institute and we thank the beamline staff for support during data collection. All members of the Dutzler lab are acknowledged for help in all stages of the project.

1. Received: September 16, 2019
2. Accepted: November 22, 2019
3. Accepted Manuscript published: December 5, 2019 (version 1)
4. Version of Record published: December 17, 2019 (version 2)

© 2019, Manatschal et al.

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