# Iron(III) chelation: Tuning of the pH dependence by mixed ligands

A.-M. Albrecht-Gary, Sylvie Blanc, F. Biaso, F. Thomas, P. Baret, G. Gellon, J.-L. Pierre, G. Serratrice

► To cite this version:

A.-M. Albrecht-Gary, Sylvie Blanc, F. Biaso, F. Thomas, P. Baret, et al. Iron(III) chelation: Tuning of the pH dependence by mixed ligands. European Journal of Inorganic Chemistry, 2003, 14, pp.2596-2605. 10.1002/ejic.200200643. hal-01458134

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Iron(III) Chelation: Tuning of the p[H] Dependence by Mixed Ligands

A.-M. Albrecht-Gary,[a]* S. Blanc,[a] F. Biaso,[b] F. Thomas,[b] G. Serratrice,[b]* P. Baret,[b] G. Gellon[b] and J.-L. Pierre[b]

[a] Laboratoire de Physico-Chimie Bioinorganique, UMR CNRS 7509, European School of Chemistry, 25 rue Becquerel, 67200 Strasbourg, France,

Fax: 33 (0)3 90 24 26 39

E-mail: amalbre@chimie.u-strasbg.fr

[b] Laboratoire de Chimie Biomimétique, LEDSS, UMR CNRS 5616, Université Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France

Fax: 33 (0)4 76 51 48 36

E-mail: guy.serratrice@ujf-grenoble.fr

**Keywords:** Tripodal ligands / Iron / Thermodynamics / UV-Vis spectroscopy / Siderophores

**Summary**

The iron(III) chelating properties of two heteropodands with 8-hydroxyquinoline and catechol binding groups were examined and compared to those of the corresponding homopodal analogues, O-TRENSOX and TRENCAMS. Like the parent homopodands, the two heteropodands are based on the TREN scaffold and the chelating units are connected by amide groups, TRENSOX2CAMS having two 8-hydroxyquinoline and one catechol arms and TRENSOXCAMS2 one 8-hydroxyquinoline and two catechol moieties. The aqueous coordination chemistry of these ligands was examined using potentiometric and spectrophotometric methods in combination with ¹H NMR. The respective p[Fe(III)] values evidenced a cooperative effect of the mixed chelating units. Moreover, the p[Fe(III)] dependence on p[H] showed that the mixed ligands exhibit a higher complexing ability, compared to the parent ligands, over the p[H] range 5-9 which is of biological relevance. This result could be of great interest for medical applications.
Introduction

Siderophores are iron chelating agents that are excreted by microorganisms to render iron soluble in the environment and favour the uptake of this metal. Iron chelation by some natural or synthetic chelators can be applied to human diseases characterised by iron overload. Although the coordination chemistry of a great number of water-soluble synthetic iron chelators have been described, [1] the p[H] dependence of the coordination abilities have not been thoroughly investigated. Most of the comparative data of the literature are allowed by the use of the p[Fe(III)] values (calculated for [Fe(III)]<sub>tot</sub> = 1 µM, [L]<sub>tot</sub> = 10 µM at p[H] =7.4) and, of course, p[Fe(III)] reflects only the complexing ability of a given ligand at physiological p[H]. Owing to the fact that p[H] can vary in a relatively large range between the different biological compartments, the p[H] dependence knowledge of the p[Fe(III)] value may be a decisive criterion for the understanding of the in vivo behaviour of a given iron chelator. On another hand, the design and the synthesis of iron chelators with high p[Fe(III)] values in a large range of p[H], constitute an interesting challenge for the scientists. We previously reported the dependence of p[Fe(III)] on p[H] for two homopodands based on catechol and on 8-hydroxyquinoline bidentate units.[2,3] We showed that the triscatechol ligand (TRENCAMS) is a stronger ferric chelator than the trishydroxyquinolinate analogue (O-TRENSOX) at p[H] > 7 and that the opposite sequence is observed at p[H] < 7. In order to obtain an efficient chelator of iron(III) in a large range of p[H], we have synthesized mixed tripodal siderophores with both catechol and 8-hydroxyquinoline moieties. Recently, the synthesis of mixed tripodal ligands based on (i) two hydroxypiridinonate and one catechol moieties, and (ii) two hydroxypiridinonate and one 2-hydroxyisophtalamide groups has been published.[4] We report in this paper the synthesis, the acid-base properties, and the ferric complexation of two monotopic tripodal mixed ligands TRENSOX2CAMS (L¹) and TRENSOXCAMS2 (L²) (Figure 1). These molecules belong to a family of tripodes with a tris(2-aminoethyl)amine group anchoring arms which bear bidentate coordination subunits (namely either 5-sulfo-8-hydroxyquinoline-7-carbamoyl group or 5-sulfo-2,3-dihydroxybenzoyl unit). The ligands differ by the number of catechoylamide groups, none for ligand L⁰ (O-TRENSOX), one for ligand L¹, two for ligand L² and three for ligand L³ (TRENCAMS) (Figure 1). Combining potentiometric and absorption spectrophotometric titrations with ¹H NMR, we were able to characterize in water the protonated and ferric species formed with ligands L¹ and L² in a large range of p[H].

<caption>Figure 1</caption>
# Results and Discussion

## Synthesis

The synthesis of the heterotripodal hexadentate ligands L¹ and L² is presented in Figure 2. The two catecholate subunits of ligand L² were connected to the tripodal scaffold by direct condensation[5] of two equivalents of 2,3-dimethoxybenzoic chloride with one equivalent of tris(2-aminoethyl)amine (TREN) [path (a')], then the oxinate subunit was grafted onto the free primary amine group by coupling with activated (CDI) 7-carboxy-8-hydroxyquinoline. The synthesis of L¹ required protection of one arm of TREN with a trityl group [paths (a, b, c)]. Phenol groups were protected with benzyl groups (oxine) and methyl groups (catechol) respectively; deprotections were performed with BBr₃. Finally, regiospecific sulfonation in position 5 of both oxinate and catecholate subunits afforded the hydrosoluble ligands L¹ and L².

<caption>**Figure 2**</caption>

## Acid-Base Properties of Ligands

The protonation constants of L¹ and L² were determined by combination of potentiometric and spectrophotometric titrations (see details in experimental section). Both ligands possess totally ten protonable functions. However, protonation of the three sulfa groups that are moderately strong acids (log K^H < 2)[6] was not considered under our conditions. Seven stepwise protonation constants were determined for each ligand and their values and corresponding error estimations are given in Table 1.

<caption>**Table 1**</caption>

The protonation constants of L¹ were determined by potentiometry except for log K₁^H = 11.5 and log K₇^H = 1.8 which were determined spectrophotometrically. As for L², the values in Table 1 represent averages of values determined by both methods, except for log K₅^H = 6.56 (determined by potentiometry) and log K₁^H = 12.5 (determined by spectrophotometry).

The protonation constants were attributed to L¹ and L² by comparison with the log K^H values of the ligand L⁰, bearing three hydroxyquinoline groups,[2] and with those of the tris(catecholate) ligand L³[3] (Table 1). The highest protonation constants (11.5 for L¹; 12.5 and 11.41 for L²) can be assigned to the *meta* hydroxyl groups of the sulfocatechoyl moieties. These values are in agreement with those reported under similar conditions for some sulfocatechol derivatives such as 1,2-dihydroxybenzene-4-sulfonic acid (log K^H = 12.16),[7] 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron; log K^H = 12.5)[8] or N,N-dimethyl-2,3-dihydroxy-5-sulfonatobenzamide (DMBS; log K^H =
11.5).[9] Values of the lowest protonation constants (1.8 and 2.89 for L¹; 2.34 for L²) correspond to the pyridine nitrogen of the sulfohydroxyquinoline moieties. Compared to 8-hydroxyquinoline-5-sulfonic acid (log K^H = 3.93),[10] the protonation constants determined for L¹ and L² are two orders of magnitude lower. This could be explained by the electron withdrawing effect of the carbonyl linker in position 7, which is similar to that of nitro group in 7-nitro-8-hydroxyquinoline-5-sulfonic acid (log K^H = 2.01).[11] The hydroxyl protonation constant of 8-hydroxyquinoline-5-sulfonic acid[10] is equal to 8.42 and those of L⁰ varied between 7.44 and 8.62.[2] We attribute the values 8.78 and 7.85 for L¹ and 7.93 for L² to the protonation of the 8-hydroxyquinoline OH groups.

Based on the fact that the species L²H₃ and L²H₄ have the same absorption spectra, suggesting the protonation does not occur on a chromophore, the protonation constant log K₅^H = 6.56 was attributed to the tertiary amine nitrogen of L² anchor. This was confirmed by the ¹H NMR titration of L² which showed an inflection point at pD ≈ 6 and a significant upfield shift of the methylene protons, characteristic of the protonation of its closest acid-base site, i.e. the tertiary amine (Figure 3). Similarly for L¹, the protonation constant log K₄^H = 6.96 was attributed to the tertiary amine.

Figure 3

The values of log K^H of the tertiary amine anchor determined for L¹ and L² are close to that of L⁰ (log K^H = 6.36).[2] They are, however, largely lower than that of the tris(catecholate) ligand L³ (log K^H = 8.01).[3] It has already been reported[12] that the protonation of this site is sensitive to the ability of the tertiary amine to form hydrogen bonds with substituents in β position on the TREN scaffold. Formation of a hydrogen bond between the lone electron pair of the tertiary amine nitrogen and the amide hydrogen atoms decreases strongly its basicity. On the other hand, when the tertiary amine nitrogen is protonated, it can be stabilised via hydrogen bonds formed with the carbonyl oxygens of the amide linkers, which leads to higher basicity of the tertiary amine anchor. Thus, depending on which interactions predominate, the basicity of the tertiary amine can be finely tuned. As can be seen in Table 1, the presence of just one 8-hydroxyquinoline group brings a sufficient stabilisation of the deprotonated form of the amine.

The interactions of the amide linkers with the tertiary amine nitrogen can be further affected by the protonation state of the *ortho* hydroxyl groups either of the 8-hydroxyquinoline or the catechol subunits. Indeed, the protonation of these sites could induce a different geometry around the TREN anchor as it is suggested by the shoulder at pD = 7-8.5 in the curve H_b during the ¹H NMR titration of ligand L² (Figure 3). The isomer shift of the H_c as well as H_d and H_e (Figure 3) observed for L² increases from pD = 6.5 to 9 with an inflection point at pD = 7.5-8 in relation to the protonation of hydroxyl groups (log K^H = 7.35 and 7.93). This pointed out that such a protonation is responsible
for a conformational change around the amide-catechol axis, which induces a shift of the proton resonance.

The remaining protonation constants 7.35 and 5.55 for L¹ and 5.62 for L¹ are attributed to the catechol hydroxyl groups *ortho* with respect to the amide linker. These values are in agreement with those of other tripodal catechoylamide-type ligands.[13]

The calculated electronic spectra of protonated species of L¹ and L² are given in Figure 4. The spectral properties of L¹ and L² are qualitatively in agreement with those of other 8-hydroxyquinoline and/or catechol based ligands. Catechol derivatives show a $\pi \rightarrow \pi^*$ transitions in the UV region (300-400 nm) with the maximum of absorption band shifting to longer wavelengths and the molar absorption coefficient increasing as the hydroxyl groups are deprotonated.[14] Similarly, 8-hydroxyquinoline-5-sulfonic acid displays spectral variations in this region upon protonation.[15] It should be noted that molar absorption coefficients of the heterotripodal ligands (~2 x 10⁴ M⁻¹ cm⁻¹ at around 340 nm and ~5–6 x 10⁴ M⁻¹ cm⁻¹ at around 270 nm for totally deprotonated L¹ and L²) are higher than the value obtained by addition of molar absorption coefficients of simple 8-hydroxyquinolinate or catecholate ligands.[15,16]

**Figure 4**

**Ferric Complexes Characterisation**

Both ligands are hexadentate and form only 1:1 ferric complexes. The potentiometric titration curves recorded for 1:1 metal to ligand ratio (Figure 5) show a large p[H] jump at a = 7, indicating that all ligand's protons are released when ferric ion is bound. Since both L¹ and L² form ferric complexes totally at p[H] < 3 the global stability constant log β<sub>FeL</sub> could not be determined from the potentiometric data.

**Figure 5**

The spectra obtained during the spectrophotometric titrations of ferric complexes of L¹ and L² are shown in Figures S1 and S2. The absorbance data were refined with the Letagrop-Spefo[17,18] and Specfit[19-22] programs and provided values of global stability constants of ferric complexes of both L¹ and L². The spectrophotometric data obtained from the competition for ferric ion between L² and EDTA were treated with Specfit program allowing the determination of the stability constant log β<sub>FeL</sub>. All the results are summarised in Table 2.

**Table 2**
The electronic spectra of differently protonated ferric complexes of L¹ and L² obtained are shown in Figure 6.

Figure 6

The ferric complexes of catechol derivatives generally show strong ligand to metal charge transfer (LMCT) band with maximum at ~700 nm for mono(catecholate) species, at ~560 nm for bis(catecholate) species and finally at ~480 nm for tris(catecholate) complexes. The charge transfer bands observed are due to electronic transitions from the highest valence orbitals of the ligand to the 3d orbitals of iron(III).[23] As the maximum moves to shorter wavelengths with increasing number of coordinating ligands the molar absorption coefficient grows. Electron withdrawing substituents on aromatic rings of catechol moieties increase the molar absorption coefficient but have little effect on the wavelength of maximum. Ferric complexes of 8-hydroxyquinoline-5-sulfonic acid are characterised by two absorption maxima observed in visible region. The maximum at 443 nm is assigned to LMCT band belonging to the phenolic oxygen, while the band at 595 nm is due to charge transfer from pyridine nitrogen to the metal cation.[2] It should be noted that similarly to L⁰ and L³, both L¹ and L² form protonated ferric complexes, indicating that the coordination sites of the complexes can be protonated without loosing the ferric ion. This behaviour was attributed to a change of catecholate or oxinate bonding mode to the salicylate mode (coordination with carbonyl and o-hydroxyl oxygens).

It is of interest to consider the different multiprotonated ferric complexes of L¹ and L² by examining their electronic spectra (Table 2) which can provide information on the coordination modes and their successive deprotonation constants $pK_{FeLH_n}$ (defined in Table 3). These values are calculated from the difference between the values of $log \beta_{FeLH_n}$ and are reported in Table 3 together with the values from tris-catecholate and tris-8-hydroxyquinolinate complexes for comparison. The deprotonation constants of ferric L¹ and L² complexes have been attributed by considering that the deprotonation of a 8-hydroxyquinoline group involved in the ferric coordination occurs at a p[H] largely lower than that of the deprotonation of the catechol group. This is supported by the $pK_{FeLH_n}$ values recently determined for tripodal ferric complexes having a C-pivot scaffold with either 8-hydroxyquinoline (ligand COX)[24] or catechol subunits (ligand CacCAM).[25] Particularly, the values of $pK_{FeLH}$, 2.12 for Fe-COX and 6.59 for Fe-CacCAM has been unambiguously attributed to the coordination of the third chelating arm.

Table 3
The electronic spectrum of FeL¹H₅ which exhibits two shoulders at 440 nm (ε = 6940 M⁻¹ cm⁻¹) and 540 nm (ε = 3680 M⁻¹ cm⁻¹) is similar to that of the bis(salicylate) complex of L⁰ [2] involving a coordination with two 8-hydroxyquinoline arms of the ligand through the oxygen atoms of the carbonyl and hydroxyl groups. The spectrum of the FeL¹H₃ complex exhibits an absorption band at λmax = 560 nm (ε = 3970 M⁻¹ cm⁻¹) in agreement with the bis(oxinate) coordination. The oxygen and pyridine nitrogen atoms of two 8-hydroxyquinoline arms are involved in the coordination. The value pK<sub>FeL1H5</sub> = 2.4 (FeL¹H₅/FeL¹H₃ equilibrium) corresponds to the deprotonation of both 8-hydroxyquinoline nitrogens in a two-protons step. The small red shift of λ<sub>max</sub> □ to 580 nm for the formation of FeL¹H₂ without significant change of ε (3930 M⁻¹ cm⁻¹) might be attributed to the deprotonation of the ammonium nitrogen. The value pK<sub>FeL1H3</sub> = 1.9 shows a strong stabilisation of the deprotonated tertiary amine nitrogen by intramolecular hydrogen bonds. The increase of ε to 4490 M⁻¹ cm⁻¹ with the concomitant blue shift of λ<sub>max</sub> to 560 nm for the formation of FeL¹H concords with the coordination of the catechol arm in a salicylate mode. The last deprotonation equilibrium is accompanied with a spectral change (λ<sub>max</sub> □= 550 nm, ε = 4560 M⁻¹ cm⁻¹) that is indicative of a shift to a catecholate coordination and formation of the FeL¹ species. It should be pointed out that the values of pK<sub>FeLH2</sub> and pK<sub>FeLH</sub> (2.7 and 4.8) are significantly lower than those observed for tris-catecholate complexes: 8.34 (two-protons step) and 5.5 for L³, 4.1 and 5.74 for MECAMS[9] (Table 3) while the pK<sub>a</sub>'s of the free ligand are of the same order of magnitude.

The spectral properties of the FeL²H₅ species (ε = 4700 M⁻¹ cm⁻¹ at λmax = 409 nm and a shoulder at 520 nm with ε = 3260 M⁻¹ cm⁻¹) are intermediate between the bis(salicylate) coordination of ferric L³ (two catechols)[3] and ferric L⁰ (two 8-hydroxyquinolines).[2] This confirms that L² forms a bis(salicylate) complex with one catechol and one 8-hydroxyquinoline coordinated to the ferric ion. For the formation of FeL²H₄ the λmax is red shifted to 573 nm which is consistent with the change of coordination from salicylate to oxinate mode for the 8-hydroxyquinoline arm. There is only slight change in ε (3530 M⁻¹ cm⁻¹) indicating that iron is still coordinated with two arms. The value pK<sub>FeL2H5</sub> = 2.2 of the FeL²H₅/FeL²H₄ equilibrium corresponds well to the pyridinium nitrogen deprotonation. The formation of the FeL²H₂ and FeL²H species is accompanied by spectral shifts of λmax from 573 nm to 542 nm and 525 nm, respectively, and an increase of ε to 4530 M⁻¹ cm⁻¹ and 4820 M⁻¹ cm⁻¹ is observed. The equilibrium between FeL²H₄ and FeL²H₂ (pK<sub>FeL2H4</sub> = 6.0) is attributed to the coordination of the third arm (catechol) in a salicylate mode and the change of coordination from salicylate to catecholate for the coordinated catechol arm. The equilibrium between FeL²H₂ and FeL²H corresponds to the shift of bonding mode from salicylate to catecholate of the third catechol arm (pK<sub>FeL2H2</sub> = 3.0). As for Fe-L¹ complexes, the deprotonations of the
catechol groups upon complexation occur at lower pH than for tris-catechol ligands cited above. The last equilibrium leading to $FeL^2$ species does not induce any spectral changes. It is likely the deprotonation of the tertiary amine nitrogen that is involved. The value of $pK_{FeL2H} = 3.4$ is higher than the corresponding constant for ferric $L^1$ complex ($pK_{FeL1H3} = 1.9$).

**p[Fe(III)] values.** Since the ligands are weak acids, proton competition occurs depending on their protonation constants and the p[H]. The $p[Fe(III)] = -\log [Fe(III)]$ is thus a better measure of the relative efficiency of the ligands under given conditions of p[H], $[Fe(III)]_{tot}$ and $[L]_{tot}$. The p[Fe(III)] values have been calculated for p[H] over the range 2 - 10 and are reported on the plot p[Fe(III)] versus p[H] presented in Figure 7 together with the plots calculated for $L^0$ and $L^3$.

Figure 7

The important result, as can be seen in Figure 7, is that the mixed ligands $L^1$ and $L^2$ are stronger iron chelating agents than $L^0$ and $L^3$ over the p[H] range 5 - 9 and 6 - 9, respectively. This shows that mixed ligands lead to enhanced affinity for the ferric ion at p[H] above 5 ($L^1$) or 6 ($L^2$) relatively to ligand with three identical arms. This is the direct result of different basicities of the binding subunits. Catechol is more basic than 8-hydroxyquinoline, which leads to a stronger competition of protons with ferric ions for catechol ligands at low p[H]. Hence, 8-hydroxyquinoline is a better chelating agent at low p[H], while catechol is stronger chelator at high p[H]. The plot in Figure 7 show that the highest values for p[Fe(III)] are obtained with $L^0$ (tris-8-hydroxyquinoline ligand) at p[H] < 5 and with $L^3$ (tris-catechol ligand) at p[H] > 9. Our results on $L^1$ and $L^2$ show that these properties are combined in mixed ligand with 8-hydroxyquinoline and catechol subunits to give the best efficiency to complex iron(III) over the biologically relevant p[H] range 5 - 9. Furthermore, greater $pK_a$ difference between the proton dissociation of the catechol in the free ligand and that of the same engaged in the complex is observed for $L^1$ and $L^2$. This indicates that the coordination of a 8-hydroxyquinoline with Fe(III) favors proton loss of the free catechol and thus increases the affinity of this group for Fe(III). We have also reported in Table 2 the p[Fe(III)] values calculated under standard conditions (p[H] = 7.4, $[L]_{tot} = 10^{-5}$ M, $[Fe(III)]_{tot} = 10^{-6}$ M) for $L^1$ (31.6) and for $L^2$ (32.3). These values are higher than those determined for $L^0$ (29.5) and for $L^3$ (29.6) and among the highest values determined for iron sequestering agent.
## Conclusion

The iron(III) complexation by two heterotripodal ligands L¹ and L² has been characterised. The results are compared to the parent homotripodal ligands allowing to study the variation of the ligand properties from tris(8-hydroxyquinoline) to tris(catechol) ligand. The thermodynamic and spectroscopic study reveals that in the acidic medium the complex formation starts with two arms coordinating the ferric ion in the salicylate coordination mode. Both 8-hydroxyquinoline subunits are involved in the case of L¹ and one 8-hydroxyquinoline and one catecholate subunits in the case of L². This indicates that 8-hydroxyquinoline is a better ligand than catechol in the presence of high concentration of H⁺. Despite the fact that catechol oxygens are better donors than the oxygen and nitrogen of 8-hydroxyquinoline, the competition between H⁺ and metal cation "balance" the complexation efficiency. The striking feature of the mixed ligands is their higher complexing ability than that of the parent ligands over a range of biologically relevant p[H] and especially at neutral p[H]. This can be explained by finely tuned complexation power of 8-hydroxyquinoline and catechol groups. It is to be noted that the complexation efficiency of the tris(8-hydroxyquinoline) L⁰ is less sensitive to p[H] than the tris(catechol) L³ over the p[H] range 2 - 10. The results described in this work illustrate the great interest of the mixed ligands approach which, via a synergistic action, leads to a more efficient iron complexing agent than the homopodate ligands.

## Experimental Section

### Syntheses

*Materials and Equipment:* Solvents were purified by standard techniques. The amine TREN was distilled from CaH₂. All other compounds were of reagent grade and were used without further purification. Mass spectra were obtained on NERMAG R 10 10C or Thermo Finnigan Polaris Q or Brüker Esquire-LC 1.6n (electrospray) mass spectrometers. ¹H and ¹³C NMR spectra were recorded in 5 mm tubes at 25 °C with Brüker AC 200 or WM 250 or Avance 300 spectrometers (δ ppm, TMS reference). Microanalysis were performed by the Central Service of CNRS, Solaise (France). Melting points were determined with a Büchi apparatus and are not corrected.

### 2: N¹-(2-amino-ethyl)- N¹-[2-(trityl-amino)-ethyl]-ethane-1,2-diamine

Under argon at room temperature, triphenylchloromethane (2.65 g, 9.5 mmol) in dry CH₂Cl₂ (150 mL) was added dropwise in a solution of ris(2-aminoethyl)amine (Tren) 1 (5.56 g, 38 mmol) in CH₂Cl₂ (100 mL). The mixture was stirred overnight and then successively washed with NaOH 10% (ca. 100 mL). The organic phase was dried on sodium sulfate and concentrated under vacuum. 2 was obtained pure as a yellowish solid (3.7 g, yield : 9.5 mmol, 100%).

mp 63 - 65 °C.
¹H NMR (200 MHz, CDCl₃): δ = 7.5 (m, 6H; ArH), 7.3 - 7.1 (m, 9H, ArH), 2.9 (brd s, 5H; NH), 2.7 (t, ³J (H-H) = 6 Hz, 4H, CH₂), 2.6 (t, ³J(H-H) = 6 Hz, 2H, CH₂), 2.4 (t, ³J (H-H) = 6 Hz, 4H, CH₂), 2.2 (t, ³J(H-H) = 6 Hz, 2H, CH₂).

¹³C NMR (250 MHz, CDCl₃): δ = 146.1 (Cq), 128.5 (CH), 127.7 (CH), 126.1 (CH), 70.6 (Cq), 57.4 (CH₂), 55.0 (CH₂), 40.8 (CH₂), 39.9 (CH₂).

MS (DCI, NH₃ - i Butane): *m/z*: 389 [M+H]⁺, 243 [C₁₉H₁₅]⁺.

**3a: [2-(trityl-amino)-ethyl]-bis{2-[(8-benzyloxy-quinoline-7-carbonyl)-amino]-ethyl}-amine**

Under a stream of argon in order to remove CO₂ as and when it is produced, 7-carboxy-8-benzyloxyquinoline (3.65 g, 13 mmol) in dry THF (150 mL) was treated with a solution of carbonyldiimidazole (CDI) (2.2 g, 13.6 mmol) in THF (40 mL), under reflux for 2 h. Tripode 2 (2.535 g, 6.5 mmol) in THF (50 mL) was added dropwise and the mixture was stirred overnight under reflux. The solvent was evaporated off and CH₂Cl₂ was added onto the residue. The solution was washed successively with saturated NH₄Cl and brine and then dried. Concentration afforded a dark oil which was chromatographed (silica, cyclohexane/CH₂Cl₂ gradient, then CH₂Cl₂/methanol 1 to 2%). **3b** was obtained as a yellow foam (3.7 g, 4 mmol, 62%).

¹H NMR (250 MHz, CDCl₃): δ = 8.95 (dd, ³J (H-H) = 1.6, 4.0 Hz, 2H; ArH), 8.14 (m, 4H; ArH), 8.07 (brd t, ³J (H-H) = 4.8 Hz, 2H; NH amide), 7.6-7.0 (m, 29 H; ArH), 5.5 (s, 4H; CH₂ benzyl), 3.2 (dt, ³J (H-H) = 6.3, 4.8 Hz, 4 H; CH₂), 2.47 (t, ³J(H-H) = 5.5 Hz, 2 H; CH₂), 2.21 (t, ³J (H-H) = 6.3, 4.8 Hz, 4 H; CH₂), 2.13 (t, ³J(H-H) = 5.3 Hz, 2 H; CH₂), 2.0 (brd s, 1H; NH).

¹³C NMR (250 MHz, CDCl₃): δ = 165.1 (CO), 153.8 (Cq), 149.5 (CH), 146.1 (Cq), 142.6 (Cq), 136.7 (Cq), 136.0 (CH), 131.3 (Cq), 128.8-128.5 (unresolved CH's), 127.6 (unresolved CH's), 126.0 (CH), 125.3 (Cq), 123.1 (CH), 122.3 (CH), 78.3 (CH₂), 70.7 (Cq), 53.9 (CH₂), 52.5 (CH₂), 40.5(CH₂).

MS (DCI, NH₃ - i Butane): *m/z*: 911 [MH]⁺, 243 [C₁₉H₁₅]⁺; 91 (C₇H₇)⁺.

Elemental analysis calcd (%) for C₅₉H₅₄N₆O₄ (911.1): C 77.78; H 5.97, N 9.22; found C 77.40, H 5.92, N 9.31.

**4a: bis{2-[(8-benzyloxy-quinoline-7-carbonyl)-amino]-ethyl}-amine**

Under argon at room temperature, **3a** (3.61 g, 4 mmol) in CH₂Cl₂ (100 mL) was stirred with CF₃CO₂H (3.7 g, 32 mmol) for 12 h. The mixture was treated with NaOH 10%, then washed with brine, dried and concentrated. Column chromatography (silica, CH₂Cl₂ then CH₂Cl₂/ methanol 5%/isopropylamine 1%) afforded **4b** as a beige foam (2.15 g, 81%).

¹H NMR (300 MHz, CDCl₃, 22°C, TMS): δ = 8.95 (dd, ³J (H-H) = 1.7, 4.1 Hz, 2H; Hquin), 8.22 (brd t, ³J (H-H) = 5.6 Hz, 2H; NH amide), 8.11 (dd, ³J<sub>AB</sub> (H-H) = 1.7, 4.1 Hz, 2H; Hquin), 7.97 (d,
$^3J$ (H-H) = 8.6 Hz, 2H; Hquin), 7.5-7.3 (m, 14 H; ArH and Hquin), 5.5 (s, 4H; CH₂ benzyl), 3.25 (dt, $^3J$ (H-H) = 5.6, 6.4 Hz, 4 H; CH₂), 3.2 (brd t, 2H; NH₂), 2.59 (m, 2H; CH₂), 2.50 (m, 2H; CH₂), 2.36 (t, $^3J$ (H-H) = 6.4 Hz, 4 H; CH₂).

$^{13}$C NMR (300 MHz, CDCl₃): $\delta$ = 165.5 (CO), 153.7 (Cq), 149.6 (CH), 142.4 (Cq), 136.7 (Cq), 136.1 (CH), 131.3 (Cq), 128.9 (CH), 128.8 (CH), 128.7 (CH), 127.3 (CH), 125.3 (Cq), 123.2 (CH), 122.3 (CH), 78.5 (CH₂), 55.1 (CH₂), 53.5 (CH₂), 38.9(CH₂), 37.9(CH₂).

MS (DCI, NH₃ - i Butane): *m/z* : 669 [M+H]⁺.

**5a: [2-(2,3-Dimethoxybenzoylamino)-ethyl]-bis{2-[(8-benzyloxy-quinoline-7-carbonyl)-amino]-ethyl}-amine**

A solution of 2,3-dimethoxybenzoic chloride (0.685 g; 3.4 mmol) previously prepared from the corresponding acid, **4a** (2.07 g, 3.4 mmol), triethylamine (0.35 g, 3.5 mmol) in dry CH₂Cl₂ (150 mL) was stirred at room temperature under argon. The mixture was treated successively with NaOH 25% and brine and then dried and the solvent was evaporated off. Column chromatography (silica, CH₂Cl₂ then CH₂Cl₂/methanol 1 to 5%) gave **5a** as a white foam (1.665 g, 2 mmol, yield = 59%).

$^1$H NMR (250 MHz, CD Cl₃): $\delta$ = 8.94 (dd, $^3J$ (H-H) = 1.6, 4.0 Hz, 2H; Hquin), 8.2 - 8.1 (m, 7H; Hquin + NH amide), 7.6 - 7.3 (m, 15H, ArH), 7.01 (t, $^3J$ (H-H) = 7.9 Hz, 1H; H catech.), 6.89 (dd, $^3J$ (H-H) = 1.6, 6.4 Hz, 1H; H catech.), 5.55 (s, 4H; CH₂ benzyl), 3.80 (s, 3H; OCH₃), 3.75 (s, 3H; OCH₃), 3.41 - 3.25 (m, 6H; CH₂), 2.6 (m, 2H; CH₂), 2.4 (m, 4H; CH₂).

$^{13}$C NMR (250 MHz, CDCl₃): $\delta$ =165.2 (CO), 165.0 (CO), 153.8(Cq), 152.3 (Cq), 149.5 (CH), 147.4 (Cq), 142.4 (Cq), 136.7 (Cq), 135.9 (CH), 131.2 (Cq), 128.6 (unresolved CH's), 127.4 (CH), 126.8 (Cq), 125.0 (Cq), 123.9 (CH), 123.0 (CH), 122.4 (CH), 122.2 (CH), 114.9 CH), 78.3 (CH₂), 61.1 (CH₃), 55.7 (CH₃), 52.7 (CH₂), 52.4 (CH₂), 37.5 (CH₂), 37.2 (CH₂).

MS (FAB, NBA matrix): *m/z* : 833 [M+H]⁺, 541, 305 [C₁₉H₁₇N₂O₂]⁺.

Elemental analysis calcd (%) for C₄₉H₄₈N₆O₇ (832.9) : C 70.66, H 5.86, N 10-09; found C 70.21, H 5.83, N 9.96.

**6a: [2-(2,3-Dihydroxybenzoylamino)-ethyl]-bis{2-[(8-hydroxy-quinoline-7-carbonyl)-amino]-ethyl}-amine**

Under argon at 0 °C, BBr₃ (5.3 g, 21 mmol) in CH₂Cl₂ (30 mL) was added dropwise to a solution of **5a** (1.52 g, 1.8 mmol) in CH₂Cl₂ (250 mL). After stirring overnight at room temperature, the mixture was cooled to 0°C and treated with methanol (50 mL). After 4 h the solution was concentrated and then repeatedly evaporated with methanol to remove the borate. The residue was treated with NaOH 4N and extracted with CH₂Cl₂ to remove the impurities. The aqueous solution
was acidified with HCl 4N to give a beige precipitate which was washed with water and then dried under vacuum. **6** was obtained pure (0.937g, 1.5 mmol, 83%).

¹H NMR (300 MHz, DMSO): $\delta$ = 9.0 - 8.7 (m, 5H; NH + ArH), 8.3 - 8.1 (m, 2H; ArH), 8.0 - 7.8 (m, 2H; ArH), 7.6 - 7.5 (m, 2H, ArH), 7.3 - 7.1 (m, 4H; ArH), 6.9 - 6.8 (m, 1H; ArH), 3.5 - 3.3 (m, 6H; CH₂), 2.8 - 2.7 (m, 6H; CH₂).

¹³C NMR (250 MHz, CDCl₃): $\delta$ = 169.6 (CO), 167.7 (CO), 156.7 (Cq), 151.2 (Cq), 149.4(Cq), 148.7 (CH), 146.2 (Cq), 139.1 (Cq), 135.9 (CH), 130.4 (Cq), 125.2 (CH), 118.5 (CH), 117.7 (CH), 117.6 (CH), 116.6 (CH), 112.8 (Cq), 52.9 (CH₂), 52.8 (CH₂), 37.3 (CH₂), 37.2 (CH₂).

MS (DCI, NH₃ - i-Butane): m/z: 625 [M+H]⁺.

### L¹: [2-(2,3-Dihydroxy-5-sulfo-benzoylamino)-ethyl]-bis{2-[(8-hydroxy-5-sulfo-quinoline-7-carbonyl)-amino]-ethyl}-amine

In portions, **6a** (0.7 g, 1.1 mmol) was added to oleum SO₃ - H₂SO₄ (15 mL) while stirring vigorously. After stirring overnight at room temperature, the mixture was carefully poured onto ice to give a beige precipitate. Filtration and washing with cold water gave the product which was recrystallized in minimum amount of water. The pure product (acidic form) was thoroughly dried under vacuum at 30°C. A yellow powder was obtained (0.347 g, 0.4 mmol, 36.5%).

¹H NMR (300 MHz, D₂O-NaOD): $\delta$ = 8.65 (dd, ³J(H-H) = 1.6, 8.6 Hz, 2H; Hquin), 8.56 (dd, ³J(H-H) = 1.5, 4.1 Hz, 2H; Hquin), 8.40 (s, 2H; Hquin), 7.63 (d, ³J(H-H) = 2.5 Hz, 1H; Hcatech.), 7.47 (dd, ³J(H-H) = 4.1, 8.6 Hz, 2 H; Hquin), 7.03 (d, ³J(H-H) = 2.6 Hz, 1H; Hcatech.), 3.54 (m, 6H; CH₂), 2.85 (m, 6H; CH₂).

¹³C NMR (300 MHz, D₂O-NaOD): $\delta$ = 171.3 (Cq), 170.7 (Cq), 170.2 (Cq), 161.3 (Cq), 148.9 (Cq), 147.4 (CH), 145.1 (Cq), 134.6 (CH), 129.1 (CH), 127.8 (Cq), 125.9 (Cq), 123.9 (CH), 119.6 (Cq), 119.0 (CH), 116.1 (Cq), 111.4 (Cq), 111.0 (CH), 53.2 (CH₂), 36.9 (CH₂).

MS (Electrospray, Methanol-water, mode+): m/z: 865 [M+H]⁺.

Elemental analysis calcd (%) for C₃₃H₃₂N₆O₁₆S₃, 3.5 H₂O: C 42.72; H 4.24, N 9.06, S 10.37; found C 42.51, H 3.85, N 8.49, S 9.62.

### 4b: bis[2-(2,3-Dimethoxybenzoylamino)-ethyl]-amine

Under argon, a mixture of 2,3-dimethoxybenzoic acid (5.28 g, 29 mmol), CDI (5.16 g, 31.9 mmol) in dry CH₂Cl₂ (350 mL) was refluxed for 1.5 h. TREN (2.11 g, 14.5 mmol) in CH₂Cl₂ (40 mL) was added dropwise and reflux was maintained overnight. The solution was filtered and washed with NaOH 4N then brine. After drying on anhydrous Na₂SO₄, the solvent was eliminated to afford an oil. Chromatography (silica, CH₂Cl₂/methanol 1 to 8% and iPr NH₂ 1%) gave **4b** as a pale yellow oil (6.53 g, 13.7 mmol, 95%) pure enough for the following steps.
¹H NMR (200 MHz, CD Cl₃): δ = 8.22 (brd t, ³J(H-H) = 5 Hz, 2H; NH), 7.61 (dd, ³J(H-H) = 1.6, 8 Hz, 2H; ArH), 7.11 (t, ³J(H-H) = 8 Hz, 2H; ArH), 7.01 (dd, ³J(H-H) = 1.6, 8 Hz, 4 H; ArH), 3.88 (s, 6H; OCH₃), 3.87 (s, 6H; OCH₃), 3.60 (dt, ³J(H-H) = 5.8, 6.2 Hz, 4 H; CH₂); 2.75 (m, 8H; CH₂), 2.64 (m, 2H; CH₂), 2.2 (brd s, 2H; NH₂).

¹³C NMR (200 MHz, CDCl₃): δ = 165.3 (CO), 152.3 (Cq), 147.2 (CH), 126.7 (Cq), 124.1 (CH), 122.3 (CH), 115.1 (CH), 61.1 (OCH₃), 57.0 (CH₂), 55.87 (OCH₃), 53.5 (CH₂), 39.7 (CH₂), 37.6 (CH₂).

MS (DCI, NH₃ - i-Butane): *m/z*: 475 [M+H], 432, 280, 208, 182.

**5b: {2-[(8-Benzyloxy-quinoline-7-carbonyl)-amino]-ethyl}-bis[2-(2,3-dimethoxybenzoylamino)-ethyl]-amine**

Under argon a solution of 7-carboxy-8-hydroxyquinoline (2.30 g, 12 mmol) and CDI (2.27 g, 14 mmol) in dry THF (200 mL) was refluxed for 3 h. **4b** (6 g, 12 mmol) in THF 80 mL was added dropwise and the reflux was maintained during 12 h. The THF was evaporated off and the residue was taken in CH₂Cl₂ and treated with NH₄Cl salt and brine. Drying, evaporation of the solvent successively afforded an orange foam which was chromatographed (silica, *iPrNH₂* 1 °C, CH₂Cl₂, MeOH 1 to 5%); **4b** (5.8 g, 9 mmol, 75%) pure enough for the following step.

¹H NMR (20 MHz, CD Cl₃): δ = 8.82 (dd, ³J(H-H) = 1.6, 4.2 Hz, 1H; Hquin), 8.19 (brd t, 3H; NH), 8.11 - 8.02 (m, 1H; Hquin), 7.56 - 7.44 (m, 3H, ArH + Hquin), 7.27 - 6.86 (m, 6H, ArH + Hquin), 3.83 (s, 6H; OCH₃), 3.76 (s, 6H; OCH₃), 3.65 - 3.56 (m, 6H; CH₂), 2.94 - 2.82 (m, 6H; CH₂).

¹³C NMR (250 MHz, CDCl₃): δ = 167.2 (CO), 165.5 (CO), 154.4 (Cq), 152.2 (Cq), 148.1 (CH), 147.1 (Cq), 138.9 (Cq), 135.6 (Cq), 130.1 (Cq), 126.7 (Cq), 126.1 (CH), 124.0 (CH), 122.8 (CH), 122.2 (CH), 116.7 (CH), 114.9 (CH), 112.8 (Cq), 61.1 (COCH₃), 55.7 (COCH₃), 53.7 (COH₂), 53.2 (CH₂), 37.8 (CH₂), 37.7 (CH₂).

**6b: {2-[(8-Hydroxy-quinoline-7-carbonyl)-amino]-ethyl}-bis[2-(2,3-hydroxybenzoylamino)-ethyl]-amine**

Under argon at 0 °C, BBr₃ (9.04 g, 36 mmol) in CH₂Cl₂ (30mL) was added dropwise to a solution of **5b** (1.94 g, 3 mmol) in CH₂Cl₂ (300 mL). After stirring overnight at room temperature, the mixture was treated with methanol (50 mL) at 0 °C. The solution was concentrated and then repeatedly evaporated with methanol. The residue was treated with NaOH 4N and extracted with CH₂Cl₂. The aqueous solution was then acidified to pH = 7.6 with HCl 4N to give a beige precipitate which was washed with water and dried under vacuum. **6b** was obtained pure enough for the following step (1.65 g, 2.8 mmol, 93%).
¹H NMR (200 MHz, DMSO): $\delta$ = 8.9 (m, 1H; Hquin), 8.7 (m, 3H; NH), 8.32 (d, $^3J$(H-H) = 8.2 Hz, 1H; Hquin), 7.94 (d, $^3J$(H-H) = 8.0 Hz, 1H; Hquin), 7.6.3 (dd, $^3J$(H-H) = 4.80, 4 Hz, 1 H; Hquin), 7.37 (d, $^3J$(H-H) = 28.2 Hz, 1H; Hquin.), 7.20 (d, $^3J$(H-H) = 7.4 Hz, 2H; Hcat.), 6.84 (d, $^3J$(H-H) = 7.4 Hz, 2H; Hcat.), 6.54 (t, $^3J$(H-H) = 7.4 Hz, 2H; Hcat.), 3.42 (m, 6H; CH₂), 2.77 (m, 6H; CH₂).

¹³C NMR (250 MHz, DMSO): $\delta$ = 169.7 (CO), 167.9 (CO), 156.4 (Cq), 149.4 (Cq), 149.0 (CH), 146.1 (Cq), 139.0 (Cq), 136.1 (CH), 130.5 (Cq), 125.3 (CH), 123.4 (CH), 118.8 (CH), 117.9 (CH), 117.3 (CH), 116.8 (CH), 115.1 (Cq), 112.7 (Cq), 52.6 (CH₂), 36.8 (CH₂).

MS (DCI, NH₃ - i-Butane): *m/z*: 590 [M+H].

**L²: {2-[(8-Hydroxy-5-sulfo-quinoline-7-carbonyl)-amino]-ethyl}-bis[2-(2,3-hydroxy-5-sulfobenzoylamino)-ethyl]-amine**

In portions, **6b** (1.49 g, 2.5 mmol) was added to oleum (SO₃ - H₂SO₄) (20 mL) while stirring vigorously. After stirring at room temperature for 12 h, the mixture was carefully poured onto ice to give a brown precipitate. Filtration and washing with cold water afforded the product which was recrystallized in a minimum amount of water. The pure product (acidic form) was dried under vacuum at 30 °C. A yellowish powder was obtained (0.672 g, 0.8 mmol, 32%).

¹H NMR (250 MHz, D₂O-NaOD): $\delta$ = 8.48 (m, 1H; Hquin), 8.48 (m, 1H; Hquin), 8.23 (s, 1H; Hquin), 7.30 (m, 1H; Hquin), 7.14 (m, 2H; Hcatech), 6.49 (m, 2H; Hcatech), 3.30 (m, 6H; CH₂), 2.62 (m, 6H; CH₂).

¹³C NMR (250 MHz, D₂O-NaOD): $\delta$ = 174.3 (Cq), 174.0 (Cq), 173.1 (Cq), 170.2 (Cq), 163.2 (Cq), 150.1 (CH), 147.8 (Cq), 137.0 (CH), 131.6 (CH), 130.3 (Cq), 128.6 (CH), 126.5 (Cq), 122.1 (Cq), 117.6 (Cq), 115.5 (CH), 115.4 (CH), 113.8 (Cq), 55.9 (CH₂), 39.3 (CH₂), 39.2 (CH₂).

MS (Electrospray, methanol-water, mode+): *m/z*: 830 (M + H)⁺, 750 (M-SO₃).

Elemental analysis calc.(%) for C₃₀H₃₁N₅O₁₇S₃, 5H₂O: C 39.17; H 4.49; N 7.61; S 10.46, ; found C 39.29, H 4.59, N 7.64, S 9.65.

**Potentiometric and Spectrophotometric Measurements.**

The solutions were prepared with boiled de-ionised water, de-oxygenated and flushed continuously with argon (purified by a Sigma Oxiclear cartridge) in order to prevent the presence of CO₂ and O₂. The ionic strength was maintained at 0.1 M with sodium perchlorate (Prolabo, Puriss or Merck, p.a.) and all measurements were carried out at (25.0±0.2) °C. Perchloric acid (70%, Fluka; ~ 0.1 M) and sodium hydroxide (Normex, Carlo Erba; ~ 0.1 M) concentrations were determined respectively by titration of sodium tetraborate solution (purris. p. a., ACS, Fluka, ~0.1 M) in presence of methyl red as indicator (Pointet Girard, France) and potassium hydrogenphthalate (purris. p. a., Fluka, ~0.1 M), with phenolphthalein as indicator (Prolabo, France). The ligands L¹
and L² were dissolved in aqueous 0.1 M NaClO₄ medium and their concentrations were calculated by weight. Stock iron(III) solutions (Fe(ClO₄)₃ · 9 H₂O, pract., Fluka or Aldrich, ~0.01 M), prepared under acidic conditions (0.1 M HClO₄), were back titrated with Th(NO₃)₄ · 5 H₂O (Merck, ~0.1 M) in presence of excess EDTA (Titrisol, Merck, ~0.1 M) and xylenol orange (Merck) as endpoint indicator.[27] The concentration was also controlled spectrophotometrically by using the molar extinction coefficient[28] of 4160 M⁻¹ cm⁻¹ at 240 nm in 0.1 M HClO₄. The acid in excess was evaluated by a potentiometric titration with standardised 0.1 M NaOH solution using quantitative formation of a complex with maltol (99%, Aldrich).[29] L¹ and L² ferric complexes were prepared by mixing adequate volumes of stock ferric perchlorate and stock ligand solutions. The free hydrogen concentrations were measured with a glass-Ag/AgCl combined electrode (Metrohm or Tacussel High Alkalinity, filled with 0.1 M NaCl and saturated with AgCl). The electrode was calibrated in order to read the p[H] according to the classical method (neutralisation of 0.1 M HClO₄ by 0.1 M NaOH).[30]

The potentiometric titrations were performed into a jacketed cell, thermostated by the water flow of a Haake thermostat, using either a digital millivoltmeter (Tacussel Isis 20,000) with a piston-fitted microburette (Gilmont) or an automatic titrator system (Metrohm, DMS Titrino 716) connected to an IBM Aptiva microcomputer. The titrations of the ligands ([L] ≈ 10⁻³ M, 2.5 < p[H] < 10.5 for L¹ and 3.9 < p[H] < 9.8 for L²) and of their iron(III) complexes ([Fe(III)]tot = [L]tot ≈ 10⁻³ M, 2.2 < p[H] < 11.0 for L¹ and 2.3 < p[H] < 8.9 for L²) were carried out by addition of known volumes of standardised sodium hydroxide. The data obtained during the potentiometric titrations were fitted with Superquad[31] or Hyperquad[32] software.

Spectrophotometric measurements were carried out on a double-beam UV-vis spectrophotometers, either Uvikon 941 (Kontron) or Perkin-Elmer (Lambda 2), equipped with a thermostating cell holder (Perkin Elmer PTP-1) and connected to an IBM PC 340 microcomputer for data acquisition (UV Winlab software, Perkin Elmer). Absorption spectra of the free ligands ([L]tot ≈ 10⁻⁴ M, 240 nm < λ < 400 nm, 2.5 < p[H] < 10.5 for L¹ and 2.6 < p[H] < 12.5 for L²) and of their iron(III) species ([L]tot = [Fe(III)]tot ≈ 10⁻⁴ M, 400 nm < λ < 800 nm, for L¹ 2.5 < p[H] < 10.5 and 1.0 < p[H] < 9.0 for L²) were measured at different p[H] using quartz spectrophotometric cells of 0.2 or 1 cm optical path length (Hellma) against 0.1 M aqueous NaClO₄ solution as a reference. Absorption spectra recorded as a function of p[H] for ferric complexes of L¹ and L² are given in supplementary materials (Figure S1).

A batch competition titration between the ferric L² complex and EDTA (99.5%, A.C.S. reagent, Aldrich) was carried out under conditions of [L²]tot = 5.4 × 10⁻⁵ M, [Fe(III)]tot = 4.5 × 10⁻⁵ M, [EDTA]tot = 9.21 × 10⁻⁴ M and p[H] values adjusted with perchloric acid in a span from 3.9 to 5.5
(with a step of 0.1 p[H] unit). The solution were equilibrated for 2 days and then absorption spectra were recorded (400 - 800 nm).

The simultaneous potentiometric and spectrophotometric data were processed with both Specfit[19-22] and Letagrop-Spefo[17,18] programs.

## NMR Titration

The proton NMR titration was carried out for L² over the pD range 3.5 - 9.2 in order to attribute protonation constants to different protonable sites of the ligand. The ligand was dissolved in D₂O. The pD was adjusted with DCl or NaOD solutions. pH measurements were performed with a Tacussel PHN 850 apparatus equipped with a microelectrode Radiometer XC61. pD values were calculated according to $pD = pH_{meas} + 0.4$.[33]

## Acknowledgements

This work has been supported by the Centre National de la Recherche Scientifique (UMR 7509 and 5616, PCV program), the Faculty of Chemistry, University Louis Pasteur, Strasbourg (France) and the University Joseph Fourier, Grenoble (France). The authors thank Professor C. Béguin (LEDSS) for his interest in these studies.
Graphical abstract

<figure><img src="image_2.png" /></figure>

pFe<sup>III</sup> = 32.3 (pH = 7.4; [L]/[Fe] = 10)
## Figure Captions

**Figure 1.** Chemical formulae of the tripodal ligands: (a) L⁰ (O-TRENSOX), (b) L¹ (TRENSOX2CAMS), (c) L² (TRENSOXCAMS2), (d) L³ (TRENCAMS).

**Figure 2.** Synthesis of the heterotripodal ligands L¹ and L².

**Figure 3.** ¹H-NMR chemical shifts variations as a function of pD for the methylene (a) and aromatic (b) protons of L². Solvent: D₂O, [L²] = 5 mM.

**Figure 4.** Calculated electronic spectra of the protonated species of heterotripodal ligands. Solvent: water, *I* = 0.10 M (NaClO₄), *T* = (25.0 ± 0.2) °C. (a) ligand L¹: (1) L¹H₇, (2) L¹H₆, (3) L¹H, (4) L¹; (b) ligand L²: (1) L², (2) L²H, (3) L²H₂, (4) L²H₃ ≡ L²H₄, (5) L²H₅, (6) L²H₆, (7) L²H₇. Charges are omitted for the sake of clarity.

**Figure 5.** Potentiometric titration curves for the heterotripodal ligands and their ferric complexes. Solvent: water, *I* = 0.10 M (NaClO₄), *T* = (25.0 ± 0.2) °C. (a) titration of L¹ (1 mM): (1) free ligand, (2) 1:1 ferric complex; (b) titration of L² (1 mM): (1) free ligand, (2) 1:1 ferric complex.

**Figure 6.** Calculated electronic spectra of the ferric complexes formed with the heterotripodal ligands. Solvent: water, *I* = 0.10 M (NaClO₄), *T* = (25.0 ± 0.2) °C. (a) ferric L¹ species: (1) FeL¹, (2) FeL¹H, (3) FeL¹H₂, (4) FeL¹H₃, (5) FeL¹H₅; (b) ferric L² species: (1) FeL² = FeL²H, (2) FeL²H₂, (3) FeL²H₄, (4) FeL²H₅. Charges are omitted for the sake of clarity.

**Figure 7.** Plot of p[Fe] versus p[H] for the tripodal ligands. Calculated for [L]ₜot = 10⁻⁵ M and [Fe(III)]ₜot = 10⁻⁶ M.

## Supplementary material

S1. UV-vis absorption spectra of Fe³⁻-L1 as a function of pH. [L¹]ₜot = 0.1.mM; [Fe(III)]ₜot = 0.09 mM; *I* = 0.1 M (NaClO₄); 1 : p[H] = 1 ; 2 : p[H] = 6.

S2. UV-vis absorption spectra of Fe³⁻-L2 as a function of pH. [L²]ₜot = 0.1.mM; [Fe(III)]ₜot = 0.09 mM; *I* = 0.1 M (NaClO₄); 1 : p[H] = 1 ; 2 : p[H] = 6.
**Table** Errur! Argument de commutateur inconnu.. Values and attributions of successive protonation constants of the heterotripodal ligands bearing 8-hydroxyquinoline and/or catechol subunits. Solvent: water, *I* = 0.10 M (NaClO₄), *T* = (25.0 ± 0.2) °C. Numbers in parentheses correspond to the estimated errors of the last digit calculated as 3σ.

<table><thead><tr><th>Function</th><th>L<sup>0</sup> [2]</th><th>L<sup>1</sup></th><th>L<sup>2</sup></th><th>L<sup>3</sup> [3]</th></tr></thead><tbody><tr><td rowspan="3">NH pyridinium</td><td>1.8</td><td>1.8(2)</td><td>2.34(6)</td><td></td></tr><tr><td>2.55</td><td>2.89(15)</td><td></td><td></td></tr><tr><td>3.01</td><td></td><td></td><td></td></tr><tr><td rowspan="3">OH quinoline</td><td>7.44</td><td>7.85(9)</td><td>7.93(2)</td><td></td></tr><tr><td>8.18</td><td>8.78(3)</td><td></td><td></td></tr><tr><td>8.62</td><td></td><td></td><td></td></tr><tr><td rowspan="2">OH catechol (ortho)</td><td></td><td>5.62(6)</td><td>5.55(4)</td><td>5.57</td></tr><tr><td></td><td></td><td>7.35(2)</td><td>6.32</td></tr><tr><td rowspan="2">OH catechol (meta)</td><td></td><td></td><td></td><td>7.05</td></tr><tr><td></td><td>11.5(2)</td><td>11.41(8)</td><td></td></tr><tr><td></td><td></td><td>12.5(3)</td><td>12.05<sup>[a]</sup></td></tr><tr><td>NH ammonium</td><td>6.36</td><td>6.96(6)</td><td>6.56(3)</td><td>8.01</td></tr></tbody></table>

<footnote>[a] Average value for the three *meta* catechol OH groups.</footnote>
<table><caption>**Table 2.** Overall stability constants $β_{FeLH_n}$, p[Fe(III)] values and UV-visible spectral characteristics of ferric complexes formed with the tripodal ligands. Solvent: water, *I* = 0.10 M (NaClO₄), *T* = (25.0 ± 0.2) °C. Numbers in parentheses correspond to the estimated errors of the last digit calculated as 3σ. The incertitude of 10% is estimated for the molar absorption coefficients.</caption><thead><tr><th>Ligand</th><th>Species</th><th>log β<sub>FeLH<sub>n</sub></sub><sup>[a]</sup></th><th>p[Fe(III)]<sup>[b]</sup></th><th>λ<sub>max</sub> (nm)</th><th>ε<sub>max</sub> (M<sup>-1</sup> cm<sup>-1</sup>)</th></tr></thead><tbody><tr><td rowspan="4">L<sup>0</sup><sup>[2]</sup></td><td>FeL<sup>0</sup>H<sub>5</sub></td><td>42.2(1)</td><td rowspan="4">29.5</td><td>435</td><td>8200</td></tr><tr><td></td><td></td><td>525*</td><td>3500</td></tr><tr><td>FeL<sup>0</sup>H</td><td>36.5(2)</td><td>443</td><td>5200</td></tr><tr><td></td><td></td><td>595</td><td>5200</td></tr><tr><td rowspan="2">L<sup>1</sup></td><td>FeL<sup>0</sup>H<sub>5</sub></td><td>30.9(1)</td><td rowspan="2">31.6</td><td>443</td><td>5400</td></tr><tr><td></td><td></td><td>595</td><td>5400</td></tr><tr><td rowspan="5">L<sup>2</sup></td><td>FeL<sup>1</sup>H<sub>5</sub></td><td>48.6(2)</td><td rowspan="5">32.3</td><td>440*</td><td>6940</td></tr><tr><td></td><td></td><td>540*</td><td>3680</td></tr><tr><td>FeL<sup>1</sup>H<sub>3</sub></td><td>46.2(4)</td><td>560</td><td>3970</td></tr><tr><td>FeL<sup>1</sup>H<sub>2</sub></td><td>44.3(3)</td><td>580</td><td>3930</td></tr><tr><td>FeL<sup>1</sup>H</td><td>41.6(2)</td><td>560</td><td>4480</td></tr><tr><td rowspan="5">L<sup>3</sup><sup>[3]</sup></td><td>FeL<sup>1</sup>H<sub>5</sub></td><td>36.8(4)</td><td rowspan="5">29.6</td><td>550</td><td>4530</td></tr><tr><td>FeL<sup>2</sup>H<sub>5</sub></td><td>55.91(3)</td><td>409</td><td>4700</td></tr><tr><td></td><td></td><td>520*</td><td>3260</td></tr><tr><td>FeL<sup>2</sup>H<sub>4</sub></td><td>53.7(1)</td><td>573</td><td>3530</td></tr><tr><td>FeL<sup>2</sup>H<sub>2</sub></td><td>47.7(3)</td><td>542</td><td>4530</td></tr><tr><td rowspan="2">L<sup>3</sup><sup>[3]</sup></td><td>FeL<sup>2</sup>H<sup>[c]</sup></td><td>44.7(6)</td><td rowspan="2">29.6</td><td>525</td><td>4820</td></tr><tr><td>FeL<sup>2</sup>H<sup>[c]</sup></td><td>41.3(6)</td><td>525</td><td>4820</td></tr><tr><td rowspan="4">L<sup>3</sup><sup>[3]</sup></td><td>FeL<sup>3</sup>H<sub>5</sub></td><td>64.05(7)</td><td rowspan="4">29.6</td><td>495</td><td>4300</td></tr><tr><td>FeL<sup>3</sup>H<sub>3</sub></td><td>57.4(1)</td><td>520</td><td>4800</td></tr><tr><td>FeL<sup>3</sup>H</td><td>49.1(1)</td><td>500</td><td>5000</td></tr><tr><td>FeL<sup>3</sup></td><td>43.6(1)</td><td>488</td><td>5300</td></tr></tbody></table>

<footnote>[a] $β_{FeLH_n}$ = [FeLH_n] / ([Fe(III)] × [L] × [H⁺]ⁿ). [b] Calculated for [Fe(III)]<sub>tot</sub> = 10⁻⁶ M, [L]<sub>tot</sub> = 10⁻⁵ M, p[H] = 7.4. [c] Species have identical electronic spectra. * shoulder.</footnote>
<table><caption>**Table 3.** Successive deprotonation constants of ferric complexes.[a] Solvent: water, *I* = 0.10 M (NaClO₄), *T* = (25.0 ± 0.2) °C.</caption><thead><tr><th>Ligand</th><th>pK<sub>FeLH<sub>5</sub></sub></th><th>pK<sub>FeLH<sub>4</sub></sub></th><th>pK<sub>FeLH<sub>3</sub></sub></th><th>pK<sub>FeLH<sub>2</sub></sub></th><th>pK<sub>FeLH</sub></th></tr></thead><tbody><tr><td>L<sup>0</sup> <sup>[2]</sup></td><td>5.7<sup>[d]</sup></td><td></td><td></td><td></td><td>5.6</td></tr><tr><td>L<sup>1</sup></td><td>2.4<sup>[b]</sup></td><td></td><td>1.9</td><td>2.7</td><td>4.8</td></tr><tr><td>L<sup>2</sup></td><td>2.2</td><td>6.0<sup>[b]</sup></td><td></td><td>3.0</td><td>3.4</td></tr><tr><td>L<sup>3</sup> <sup>[3]</sup></td><td>3.1</td><td>3.5</td><td>8.34<sup>[b]</sup></td><td></td><td>5.5</td></tr><tr><td>Enterobactin <sup>[26]</sup></td><td></td><td></td><td>2.5</td><td>3.52</td><td>4.95</td></tr><tr><td>MECAMS <sup>[9]</sup></td><td></td><td></td><td>3.46</td><td>4.10</td><td>5.74</td></tr><tr><td>COX <sup>[24]</sup></td><td></td><td>4.8<sup>[c]</sup></td><td></td><td></td><td>2.12</td></tr><tr><td>CacCAM <sup>[25]</sup></td><td></td><td></td><td></td><td>5.15</td><td>6.59</td></tr></tbody></table>

<footnote>[a] K<sub>FeLH<sub>n</sub></sub> = ([FeLH<sub>n-1</sub>] × [H]) / [FeLH<sub>n</sub>]. [b] K<sub>FeLH<sub>n</sub></sub> = ([FeLH<sub>n-2</sub>] × [H]<sup>2</sup>) / [FeLH<sub>n</sub>]. [c] K<sub>FeLH<sub>n</sub></sub> = ([FeLH] × [H]<sup>3</sup>) / [FeLH<sub>4</sub>].</footnote>

<footnote>[d] K<sub>FeLH<sub>n</sub></sub> = ([FeLH] × [H]<sup>4</sup>) / [FeLH<sub>5</sub>].</footnote>
<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>

(a) Ph<sub>3</sub>CCl; (b) 7-carboxy-8-hydroxyquinoline, CDI; (c) Trifluoroacetic acid

(a') 2,3-dimethoxybenzoic acid, CDI; (d<sup>a</sup>) 2,3-dimethoxybenzoyl chloride, Et<sub>3</sub>N

(d<sup>b</sup>) 7-carboxy-8-hydroxyquinoline, CDI; (e) BBr<sub>3</sub>; (f) SO<sub>3</sub> - H<sub>2</sub>SO<sub>4</sub>
<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Figure 5</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure 6</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Figure 7</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Figure S1</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Figure S2</figcaption></figure>
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