domain: sdv chim

text:
Saida Majdoub

Dr Zoltán Garda

Dr Alexandre C Oliveira

Dr Inga Relich

Dr Agnès Pallier

Dr Sara Lacerda

Dr Christelle Hureau

] Carlos

F G C Geraldes

Jean-François Morfin

Éva Tóth

] S Majdoub

Dr J.-F Morfin

Dr C F G C Geraldes

Concentration-dependent interactions of amphiphilic PiBderivative metal complexes with amyloid peptides A and amylin

Keywords: amyloid peptide, A, amylin, metal complex, micellar aggregation

Metal chelates targeted to amyloid peptides are widely explored as diagnostic tools or therapeutic agents. The attachment of a metal complex to amyloid recognition units typically leads to a decrease in peptide affinity. We show here that by separating a macrocyclic GdL chelate and a PiB targeting unit with a long hydrophobic C10 linker, it is possible to attain nanomolar affinities for both A1-40 (Kd = 4.4 nM) and amylin (Kd = 4.5 nM), implicated respectively in Alzheimer's disease and diabetes. The Scatchard analysis of surface plasmon resonance data obtained for a series of amphiphilic, PiB derivative GdL complexes indicate that their A1-40 or amylin binding affinity varies with their concentration, thus micellar aggregation state. The GdL chelates also affect peptide aggregation kinetics, as probed by thioflavin-T fluorescence assays. A 2D NMR study allowed identifying that the hydrophilic region of A1-40 is involved in the interaction between the monomer peptide and the Gd 3+ complex. Finally, ex vivo biodistribution experiments were conducted in healthy mice by using 111 In labelled analogues. Their pancreatic uptake, ~3 %ID/g, is promising to envisage amylin imaging in diabetic animals.

Introduction

Correct folding is essential for proteins in order to maintain longterm stability and biological function. [1] Today, a wide range of pathologies are known to be directly linked to protein misfolding and amyloidogenesis, including prion disease, amyotrophic lateral sclerosis, cerebral amyloid angiopathy, type II diabetes and Parkinson's, Huntington's and Alzheimer's diseases. [2] Amyloidogenesis involves a cascade of processes that start with the unfolding or misfolding of originally folded globular proteins and lead to the formation of soluble and insoluble toxic oligomer/polymer cross-β-sheet fibrillary aggregates, referred to as amyloids. [3] Cytotoxicity is increased by the concomitant generation of reactive oxygen species during amyloidogenesis. Metal ions, typically Cu 2+ , Zn 2+ and Fe 3+ , are also implicated in the aggregation processes and metal ion coordination to the amyloid peptide plays a further role in inducing oxidative stress. [4] It was evidenced early on that amyloidogenesis occurs well before the earliest clinical symptoms of the diseases. Consequently, amyloid peptide deposits have been long considered as relevant biomarkers for diagnostic molecular imaging. [5] Beyond diagnostic purposes, the visualization of amyloid proteins is also important for a better delineation of the molecular mechanisms underlying the diseases or for monitoring potential therapeutic approaches. The greatest progress, attaining clinical applications, has been achieved in the context of Alzheimer's disease (AD). Today several 18 F labelled Positron Emission Tomography (PET) tracers (florbetapir, flutemetamol and florbetaben) are clinically available to detect the presence of senile amyloid A plaques in the brain. [6] These molecular imaging probes have the advantage of possessing blood brain barrier (BBB) permeability, which, together with the low quantities required for PET detection, largely facilitated their translation into clinical application.

Metal complexes provide great versatility for the development of molecular imaging agents in general, including various imaging modalities such as magnetic resonance imaging (paramagnetic Gd 3+ or Mn 2+ chelates), nuclear (PET or SPECT with complexes of  + or -emitting metal isotopes, respectively) or optical techniques (luminescent metal complexes). However, the typically larger size and more hydrophilic character of metal complexes, as compared to the previously mentioned small fluorinated PET tracers, severely limit their brain penetration, thus their utility for imaging in Alzheimer's disease. The problem is further complicated for the low sensitivity MRI detection which requires large quantities of the imaging probe. [7] Consequently, in Alzheimer's disease, only few successful examples of in vivo visualization of brain A are known using metal-based imaging agents, and these are mainly restricted to nuclear imaging. [5a] Less effort has been dedicated to the imaging of other types of amyloid peptides relevant to diseases other than AD. Not all amyloid pathologies concern the central nervous system, and in these cases, reaching the target can be easier for the imaging agent, even in larger quantities such as required for MRI. For instance, Cerebral Amyloid Angiopathy (CAA) is characterized by amyloid deposition in the walls of cerebral vasculature, and low brain uptake of the imaging probe is even important to differentiate CAA from AD. 99m Tc-hydroxamamide complexes of multivalent ligands bearing stilbene and benzothiazole amyloid recognition units were used to highlight amyloid in CAA. [8] Another example concerns SPECT detection of amylin in the pancreas with a 99m Tc complex. [9] Amyloid deposits in pancreatic islets appear in >90% of type 2 diabetes mellitus (T2DM) patients and their quantity has been recognized as a direct indicator of disease severity. Amylin or islet amyloid polypeptide (IAPP) is a 37-amino-acid peptide co-secreted with insulin by β-cells. The inhibition of amylin aggregation contributed to better glucose homeostasis in animal model studies. [10] Moreover, recent studies evidence a crosstalk between AD and diabetes via the implication of amyloid peptides. [11] Metal complexes have been also widely explored for therapeutic purposes, mostly in relation to Alzheimer's disease. The interaction of metal chelates with the A peptide is able to influence the aggregation pathway, modulate oxidative stress and toxicity. Despite the difficulties associated with the lack of sufficient BBB permeability, complexes of many transition metal ions, including V(V), Mn(II), Re(I), Re(V), Fe(II), Fe(III), Ru(II), Co(III), Rh(III), Ir(IIII), Pt(II) have been studied in this context. [12] In the past, we described a series of DOTA or DOTA-monoamide ligands bearing a PiB unit as A-targeting vector. These macrocyclic scaffolds form stable complexes with a variety of metals, including Gd 3+ , 111 In 3+ or 68 Ga 3+ , which were proposed respectively as potential agents to detect A amyloid peptide in MRI, SPECT or PET imaging. [13] These studies revealed that the conjugation of a metal complex to the PiB unit leads to a significant affinity decrease towards A. BBB permeability of the chelates was limited, similarly to analogous Gd 3+ complexes reported by Bort et al. [14] Brain penetration could be slightly improved through non-covalent attachment of the complexes to carbon nanotubes. [15] In general, most of the metal complexes proposed either for imaging or for therapeutic purposes are amphiphilic, composed of a rather hydrophilic metal-coordinating unit and a hydrophobic amyloid-targeting moiety. Depending on the concentration and the surrounding medium, such amphiphilic molecules tend to form micelle-like aggregates, in which the peptide-recognition moiety can be more or less shielded, thereby likely impacting the interaction with the amyloid peptide. Despite the abundant literature on amyloid-binding metal chelates, this aspect has not been so far addressed. The present study had three major goals. On a long-term perspective, we aim to propose potential MRI probes for the detection of islet amyloid polypeptide in the context of type 2 diabetes. In contrast to the constraints in brain delivery, the pancreas can be more easily accessible for large quantities of Gd 3+ complexes. Thanks to its excellent resolution and noninvasiveness, MRI could be then the imaging modality of choice to better understand the set-in of diabetes and to monitor its early stage evolution. However, no amylin-targeted small Gd 3+ complexes have been so far reported, and while several organic molecules have been compared with respect to their affinity to amylin and A, no data exist for Gd 3+ complexes. The second objective was to approach nanomolar affinity of metal complexes by further optimizing their chemical structure by elongating the linker between the DOTA and the biovector. Finally, in a more general consideration, we wanted to assess how micellar aggregation promoted by the amphiphilic nature of amyloidtargeted metal complexes influences their interaction with amyloid peptides. In these objectives, we have investigated three Gd 3+ chelates bearing a PiB unit, attached via either an amide or an ether oxygen to an alkyl (C5, C6 or C10) linker (Figure 1). Ligands L1 [13e] and L2 [15] have been previously described, while L3 was specifically designed here to space the PiB at a further distance from the macrocycle and increase affinity. We have assessed micellar aggregation of the Gd 3+ complexes by relaxometry (when possible) and UV-Vis spectroscopy. By using surface plasmon resonance, we have quantitatively characterized the affinity of the chelates to A and amylin present in a controlled aggregated form, as well as to human serum albumin. The interactions were further assessed by fluorescence and relaxometric studies and by high resolution NMR. Finally, ex vivo biodistribution data have been acquired in mice to assess the biological fate of these complexes. While these studies evidence first of all the large complexity of the systems, they also indicate that careful molecular design allows for very high peptide affinities. In a more general perspective, they point out that concentration-dependent aggregation phenomena should not be neglected when considering the interaction of biomolecules with amphiphilic metal complexes.

Results and Discussion

Synthesis. The synthesis of DO3A-PiB, [13c] L1 [13d] and L2 [15] have previously been described and L3 has been prepared as shown in Scheme 1. Compound 1 is obtained following the procedures described in the literature. [15] Then, the C10 linker is introduced by O-alkylation of N-(10-Bromodec-1-yl)phthalimide, giving compound 2, and the phtalimide protecting group is quantitatively removed with hydrazine hydrate. Acylation of amine 3 is performed with chloroacetyl chloride and the product 4 obtained is engaged in a nucleophilic substitution reaction with commercial DO3A-tBu to afford the protected ligand 5. L3 is finally obtained after the removal of the tert-butyl protecting groups in acidic conditions. Gadolinium complexes are prepared by adding GdCl3 at a controlled pH of 5.5. Lipophilicity of the complexes. Lipophilicity, typically characterized by the water-octanol partition coefficient, logPoct/water, is an important parameter to predict the capability of molecules to cross biological membranes, including the BBB. A certain lipophilicity is also important for amyloid peptide affinity; though obviously it is not the only determinant. [16] LogPoct/water values were measured by the "shake flask" method for GdL2 and GdL3, as it was previously done for GdL1. [13d] They increase in the order of GdL1, GdL2 and GdL3 (0.03, 0.63 and 1.46, respectively). Between GdL2 and GdL3, the strong lipophilicity increase reflects the elongation of the alkyl chain from C6 to C10. GdL1 and GdL2 differ first of all in the orientation of the PiB, which by itself, does not affect lipophilicity as it was evidenced for Re complexes. [16] Therefore, the lower logPoct/water determined for GdL1 with respect to GdL2 is mainly the consequence of the hydrophilicity of the amide function linking the PiB to the C5 chain. In overall, these values fall in the range reported for metal complexes appended with a PiB or other recognition units for amyloid peptides. [13e, 17] Micellar aggregation of the metal chelates. In aqueous solutions of amphiphilic metal chelates such as GdL1, GdL2 and GdL3, micellar aggregates can form when the concentration exceeds a threshold, commonly called the critical micellar concentration (cmc). However, premicellar aggregates can exist already at 2-3 orders of magnitude lower concentrations, often called critical premicellar concentration. [18] Such premicelle formation has been observed in solution of amphiphilic Gd 3+ complexes. [19] Premicelle formation as well as the transition between premicelles and micelles affect many physical-chemical properties (conductivity, optical parameters, etc) which can be used to determine the critical premicellar or micellar concentration. For paramagnetic complexes, the water proton relaxation rate is an additional parameter that can be exploited to assess the cmc. [20] The relaxation rate is linked to the motional dynamics of the system, thus showing an important change from a monomeric to a micellar state of the complex. By plotting the paramagnetic relaxation rates vs. the concentration, a break in the curve is observed at the cmc. By this relaxometric method, cmc = 1.0 mM was previously determined for GdL1. [13d] When we performed the same experiment for GdL2 and GdL3, we did not observe any break in the concentration range accessible for relaxation rate measurements (> 0.1 mM; below the measurements are not reliable), indicating that the cmc for these systems is below 0.1 mM and its determination is not possible by relaxometry. Therefore, we took advantage of the optical properties of the PiB unit in the complexes to assess micellar aggregation. Concentration-dependent UV-Vis absorption spectra have been recorded for the three Gd 3+ complexes. (Figures S1-S3 in ESI). The absorption band centred at ~350 nm undergoes a hypsochromic shift of ~12 nm upon increasing the concentration, a change that we can ascribe to the aggregation process. The plot of the relative absorbances at two wavelengths, characteristic of this hypsochomic shift, allowed determining cmc values, which are 15, 30 and 5 M for GdL1, GdL2 and GdL3, respectively. The cmc previously obtained for GdL1 from relaxometry (1 mM) is well above the one determined by UV-Vis. The two techniques give access to concentration ranges which do not overlap, thus they allow assessing different aggregation steps. Therefore, the combined UV-Vis and relaxometric results suggest that, as a function of the concentration, different micellization regimes exist in GdL1 solution. They can be likely associated to premicellar aggregation and micelle formation, each characterized by a "threshold" concentration which we denote as cmc. Analogously to GdL1, a UV-Vis study performed on the La(DO3A-PiB) complex (Fig. S4) yielded a cmc of 53 M, while another cmc of 1.5 mM was previously obtained by relaxometry. [13c] Particle size has been measured by dynamic light scattering as well as particle charge assessed at different concentrations. The charge measurements confirm the neutrality of the particles, as expected. On the other hand, light scattering data indicate extremely polydisperse systems for the three compounds and under all conditions, with some proportion of very large aggregates even at low concentrations (Table S1 andS2).

Interaction of the Gd 3+ complexes with A amylin and human serum albumin

Surface plasmon resonance. A number of experimental techniques, including thioflavin T fluorescence assay, NMR and EPR spectroscopy, mass spectrometry, circular dichroism spectroscopy, etc. have been reported to investigate interactions between metal complexes and amyloid peptides, mostly A. [12] Radioactive competition experiments and surface plasmon resonance (SPR) are also widely used for quantitative assessment. One advantage of SPR is that the peptides can be immobilized on the sensorchip in a controlled aggregation state. For this, we have followed a recent protocol established by Lee et al. [21] based on ultrasound treatment-driven decomposition of the amyloid into shorter fibrils, which are then conjugated to the sensorchip, followed by reconstruction to mature fibrils by successive monomer addition and heating. Two different immobilization levels (~4000 RU and ~9000 RU) have been tested. Typically, low immobilization is interesting to prevent steric effects and non-specific interactions. On the other hand, given the relatively small molecular weight of our complexes with respect to the peptides, higher immobilization levels can allow for better sensitivity and for the assessment of interactions at very low GdL concentrations. The SPR data have been analyzed according to a 1:1 Langmuir model to yield dissociation constants, Kd, for the interactions of all three complexes with aggregated A1-40 and amylin, as well as with human serum albumin (HSA). In the case of GdL1 and GdL2 with both A1-40 and amylin, similar results were obtained independently of the immobilization level on the chip (Fig. S5, S6 in ESI). In contrast, for GdL3, the higher immobilization revealed an additional interaction in the nanomolar concentration range, with a Kd of 4.4 nM and 4.5 nM for A1-40 and amylin, respectively (Fig. 2). All dissociation constants are listed in Table 1. The dissociation constants of GdL1, GdL2 and GdL3, calculated from the entire concentration range used in the SPR experiments, are all in the M range, varying little from one complex to the other; though GdL3 has slightly better affinities. The chelates do not show selectivity for any of the peptides, the Kd values are practically identical for A1-40 and amylin in the case of GdL2 and GdL3. This holds also true for Gd(DO3A-PiB) for which the amylin binding has been now assessed as well to complete the previous study with A1-40 (Table 1). GdL1 has a tenfold higher affinity for amylin than for A1-40; the reason for this is difficult to identify at this point. The dissociation constants of GdL1, GdL2 and GdL3 are lower than those we previously reported for analogous PiBderivative lanthanide complexes with A1-40 (67-160 M). [13e] The reason is likely the lack of control of the A1-40 aggregation state in those previous experiments. 4.5±0.9 nM (only from data between 0 and 62.5 nM) -Gd(DO3A-PiB) 170 µM (c] 154±10 µM 1100 µM [d] [a] immobilization: ~4000 RU. [b] immobilization: ~9000 RU. [c] measured for La(DO3A-PiB), ref. [13e] , [d] measured by relaxometric titration, ref. [13a] .

Importantly, the very high peptide immobilization level allowed revealing an additional interaction in the nanomolar range between GdL3 and both amyloid peptides. This affinity, similar to that of the PiB itself (Ki = 4.7 nM obtained from radiocompetition experiments), [22] is particularly remarkable, and shows the importance of very long and hydrophobic spacers between the targeting unit and the metal complex to retain high affinity. We should note that non-specific interactions between the complexes and the peptides can be likely neglected. Indeed, SPR experiments performed with a GdDO3A-monoamide chelate bearing a C12 alkyl chain did not show any interaction with the amyloid peptides. Bort et al. have reported a series of DOTA-and PCTA-complexes with 2-arylbenzothiazole, 2-arylbenzoxazole or stilbene functions. [14] They determined Ki values for A1-42 interactions by radiocompetition experiments with the radiolabelled molecule

[ 125 I]IMPY ([ 125 I]6-iodo-2-(40-dimethylamino)-phenyl-imidazo[1,2- a]pyridine
). This technique also allows for accessing to a very low concentration range, thus potentially very high affinities.

Nevertheless, the Ki values obtained for the complexes were between 220 nM and >1000 nM, corresponding to considerably lower affinities than those attained by GdL3. This difference can be likely attributed to the much shorter and more hydrophilic spacers used by them, which do not keep sufficiently far from the amyloid recognition the metal chelate moiety.

Concerning the interaction between metal complexes and amylin, less data are available. Yoshimura et al. reported 99m Tc complexes bearing a pyridyl-benzofurane (PBF) attached via a triethylene glycol spacer. [9] The best affinity, determined by radiocompetition, was Ki = 146 nM, two orders of magnitude below the Ki of the 125 I-labelled PBF (2.66 nM), indicating a strong affinity loss induced by the Tc complex. Here the spacer (9 atoms) was almost as long as in our GdL3 (10 atoms), however the hydrophilicity of the PEG seems to limit peptide binding. HSA is the most abundant plasma protein, capable of binding molecules of very different structures. HSA-binding can hence modulate their pharmacokinetics. The affinity of our complexes to HSA is strongly increasing in the order of GdL1, GdL2 and GdL3, but it is always 1-2 orders of magnitude lower than the affinity to the amyloid peptide aggregates (Fig. S7 and Table 1). Such moderate HSA affinity can be interesting in order to extend the blood circulation time of the complexes and to facilitate for them to reach the target. The case of GdL3, where a very high affinity interaction could be also identified at low concentrations, revealed the limits of a simple Langmuir model to describe the SPR data in the entire concentration range. Scatchard linearization is often used to identify multiple binding. For this, the maximum SPR response divided by the concentration (RUmax/c) is plotted as a function of RUmax and gives a straight line with a slope of -1/Kd. We have reanalysed all the SPR data (the interaction of the three complexes with A1-40, amylin and HSA). Interestingly, most of the Scatchard plots show more than one regimes, each characterized by welldefined straight lines (Figure 3).

Our complexes, as previously shown, form micellar aggregates at higher concentrations. An inspection of the Scatchard plots shows that the breakpoints correspond to GdL concentrations which, for a given chelate, are independent of the nature of the peptide (A1-40 or amylin). More interestingly, these concentrations are in the same order of magnitude as those identified as cmc values (Table 2). The GdL1-A1-40 system does not show any breakpoint; and indeed, we note that the concentration range used for SPR (30 µM -1 mM) does not include the cmc values for GdL1 (13 M and 1 mM). We have also analysed the SPR data for previously studied complexes, including Gd(DO3A-PiB) [13e] or the tetraacetate derivative Gd(DOTAGA-PiB). [13a] Again, the Scatchard plots present two straight lines with a breakpoint corresponding to the cmc value of the complexes (Fig. S8 and Table 2). All these findings seem to indicate that the different aggregated forms of the GdL complexes interact differently with the amyloid peptides and with HSA (see Fig. S9 for HSA plots). We should note that in general, a break in the Scatchard plot indicates multiple binding. This could be a potential explanation for our systems as well. Nevertheless, the fact that the breakpoint concentrations are more characteristic of the GdL complex itself than of the amyloid peptide, and their similarity with the cmc strongly suggest that these breaks are related to the transition to a differently aggregated state of GdL. These differently aggregated micellar states seem to have different peptide affinities.

In principle, the slope of the Scatchard plots provides a Kd value. Nevertheless, in this kind of analysis, the low concentration points are overweighed, which can lead to inaccurate Kd. Further, it is obvious that our systems are complex, with potentially different aggregated forms of GdL, and therefore the determination of an individual dissociation constant for each regime would be physically meaningless. We prefer to interpret the "effective" dissociation constants presented in Table 1. At pH 7.4, the GdL1, GdL2 and GdL3 complexes are neutral, while A1-40 is negatively (PI = 5.3) and amylin is positively charged (PI = 8.9). In order to gain insight into the role of the charge in the interaction, we have carried out SPR experiments with the non-complexed ligand L2. At neutral pH, the ligand alone has a negative overall charge, as a result of the three deprotonated carboxylate and two protonated macrocyclic amine functions. For amylin, there is less influence of the charge (Kd values 30 and 17 M for L2 and GdL2, respectively), while with A1-40, the affinity is slightly decreased for the negatively charged L2 (Kd = 68 vs. 20 M for GdL2). In overall, these SPR studies and in particular the Scatchard analysis revealed a large complexity of the systems, which should be always kept in mind when interactions between amphiphilic metal complexes and amyloid peptides are considered. 1 mM [b] 30±10 M [a] 5±1 M [a] 53±3 µM [d] 1.5 mM [b] 500 M [c] a from UV-Vis b from relaxometric data, ref.

[13c] c from relaxometric data, ref. [13a] d from UV-Vis, determined for the La(DO3A-PiB).

Thioflavin-T fluorescence assays

To further probe the interaction of GdL1, GdL2 and GdL3 with the peptides, their impact on the aggregation kinetics of A1-40 was investigated by Thioflavin-T (ThT) fluorescence measurements (Figure 4). This gold standard technique relies on the use of a fluorescent probe that turns-on upon intercalation in β-sheets, the main constituent of amyloid fibrils. The kinetic curve is then described by an s-shape curve according to:

𝐹(𝑡) = 𝐹 0 + 𝐹 𝑚𝑎𝑥 -𝐹 0 1+𝑒 -𝑘(𝑡-𝑡 1/2 ) (1)
where F(t) is the ThT fluorescence intensity at a given time, F0 and Fmax are the starting and final intensity values of the ThT fluorescence, respectively, k is the elongation rate and t1/2 corresponds to the time where the ThT fluorescence intensity equals half of the (maximuminitial) ThT fluorescence intensity. [23] The curve is split into three parts (i) the lag phase during which nuclei are formed, (ii) the growth phase corresponding to the elongation of fibrillary species from the nuclei and (iii) the plateau phase when the β-sheet content of the mixture is stable. The effect of the compounds was investigated at 20 µM A1-40 concentration and at various stoichiometries (0.1-2.0 equivalents) of GdL. The A1-40 aggregation is consistent with previous reports [24] characterized in our experimental conditions by a t1/2 of about 15 hours. With respect to their impact on A1-40 aggregation, GdL1 behaves differently from GdL2 and GdL3. GdL1 accelerates the aggregation process, translated by a shorter t1/2, and enhances the β-sheet content in line with a higher maximum ThT fluorescence. In contrast, both GdL2 and GdL3 induce an increase in t1/2 and a decrease in the maximum fluorescence (Table 3). It is worth noting here that such a decrease in ThT intensity may originate from a competition between ThT and the GdL2 or GdL3 complex for a possibly common binding site. For all compounds, the effects observed on t1/2 and on the maximum fluorescence intensity increase with stoichiometry, although they are observable from 0.1 equiv. of GdL1 and GdL3 and 1.0 equiv. of GdL2 (Figure 4). In addition, it is observed that GdL3 is more potent than GdL2 in delaying the aggregation process and in diminishing ThT fluorescence intensity.

From these experiments, it thus appears that GdL1 has a different and a much less pronounced effect on peptide aggregation as compared to GdL2 and GdL3. This is likely related to the different orientation of the 2-aryl-benzothiazole moiety with respect to the Gd 3+ containing macrocycle. This might indicate that during the aggregation process the interaction with the β-sheets is enhanced by the dimethyl aniline part of the 2-aryl-benzothiazole moiety. In addition, in line with the order of affinity for pre-formed peptide fibrils as evaluated above by SPR, GdL3 has a higher impact on the aggregation kinetics as compared to GdL2. This is particularly obvious at the sub-stoichiometric ratio (Table 3 and Figure 4). [ThT] = 10 µM.

Table 3. Key kinetic parameters describing the aggregation of A1-40 in the presence of GdL1, GdL2 and GdL3. The values represent the average of at least two independent experiments with data recorded in quadruplicate.

t1/2 [a] (hours) Fmax-F0 [a] equiv. GdL 0. [a] Values are normalized with respect to value of the peptide alone.

Relaxometric studies. The efficacy of a paramagnetic complex to act as an MRI contrast agent is related to its capacity to enhance the nuclear relaxation of surrounding water protons. Proton relaxivity, r1, is defined as the increase of the longitudinal water proton relaxation rate induced by the Gd 3+ chelate referred to 1 mM concentration. Relaxivity depends on different dynamic and structural features of the complex, such as the number of water molecules directly coordinated to the metal ion, their exchange rate with bulk water, and the rotational dynamics of the molecule. When the complexes bind to amyloid peptides or to other proteins, their rotational motion becomes slower, and consequently, their proton relaxivity increases. As predicted by the Solomon-Bloembergen-Morgan (SBM) theory of paramagnetic relaxation, this effect is strongly dependent on the magnetic field, being the most important at "intermediate" fields (0.5-1.5 T, corresponding to ~20-60 MHz proton Larmor frequency). Relaxation rate measurements are commonly used to study the binding of paramagnetic complexes to macromolecules, such as proteins. We have recorded Nuclear Magnetic Relaxation Dispersion (NMRD) profiles, which represent relaxivity as a function of the proton Larmor frequency, for GdL2 and GdL3 (Figures S10 andS11), data on GdL1 were previously reported. [13d] Both complexes show a relaxivity hump centred at 20 MHz with a maximum of r1 = 12.5 and 12.3 mM -1 s -1 , respectively (37°C). This is characteristic of slowly tumbling systems and results from micellar aggregation in the samples (cGdL = 0.2 mM). The relaxivities are practically constant between 25°C and 37 °C. Given the complexity of the systems, the fitting of the NMRD curves to the SBM theory seems difficult. Nevertheless, the temperature invariance indicates that, upon temperature increase, the effect of a faster water exchange rate is counterbalanced by the effect of faster rotation. In the presence of equimolar concentration of A1-40, the profile of GdL2 remains very similar (Figure 5). Under these conditions and based on Kd = 16 M, ~75 % of the complex is bound to the peptide. Thus, the similar relaxivities measured in the presence of A1-40 suggest that the rotational dynamics of GdL2 (more precisely the Gd-water proton vector) is not significantly different between the micellar and the peptide-bound state. In contrast, the high field relaxivities of GdL3 double in the presence of A1-40, indicating an important immobilization of the complex in the peptide-bound state (present in 86 % according to Kd = 5 M) with respect to the micellar form (Figure 5). This also seems to be in accordance with the stronger affinity of GdL3 to A1-40, even if it is not straightforward to directly relate the restriction in motional mobility of the chelates, as represented by the relaxivity increase, to their peptide affinity. Unfortunately, it was impossible to investigate the effect of amylin binding, since solutions containing amylin and the complexes in concentrations high enough for relaxometric measurements are prone to precipitation during the relaxometric measurement time. In the presence of physiological concentrations of HSA (0.6 mM), the relaxivity of both GdL2 and GdL3 increases (Figure 5), indicating protein binding, as it has been previously observed for GdL1. [13d] NMR studies NMR was used to study the interaction of GdL2 with the 15 Nlabeled Aβ1-40 peptide in the monomer state and to identify the peptide regions involved in this interaction. For that purpose, the changes in the 1D 1 H and 2D 1 H-15 N-HSQC spectra of 100 µM 15 N-Aβ1-40 were followed upon addition of increasing amounts of GdL2. [13a, 13e] These experiments were performed at low temperature (5 °C) to minimize NMR signal loss due to amide hydrogen exchange with water and the broadening effect resulting from peptide self-association. This peptide has a predominant random coil structure with two residue segments prone to adopt a β sheet conformation (segments 16-24 and 31-36), two regions prone to adopt a poly-proline type II helix (PIIhelix, residues 1-4 and 11-15), and two regions without ordered structure but with high mobility connecting these two structural elements (residues 5-10 and 25-30) (Figure S12). [25] The 2D 1 H-15 N-HSQC spectrum of 15 N-Aβ1-40 in water is shown in Figure 6B, expanded in the region of the 1 H-15 N of the peptide backbone and excluding the 1 H-15 N amide signal of the side chain of the residues Q15 and N27 and the 1 H- 15 Nɛ signal of the R5 side chain. All the cross-peaks from the backbone HN groups appear in this expanded spectrum, except the solvent exchange broadened resonances from D1, A2, H6 and H14. [26] Even though there is some peak overlap, the peak assignments (Figure 6B) correspond to previous reports. [13a, 13e, 25, 27] Addition of 0.5 and 1 equivalents of GdL2 to the 15 N-Aβ1-40 peptide caused only slight signal broadening with no shifts in the 1D and 2D spectra of 15 N-Aβ1-40 (Figures 6A andS2). Figure 6C shows the superposition of the 2D 1 H-15 N HSQC spectra of the alone and in the presence of one equivalent of GdL2, showing that there are no significant changes in the spectrum. However, in the presence of 2 and 4 equivalents of GdL2, a selective broadening of some signals is observed (Figure 6A, D andS13). In the presence of 4 equivalents of GdL2, these broadening effects became drastic, causing the disappearance of some of the signals. (Figure 6D). These signals correspond to residues concentrated in the hydrophilic F4-F20 region of the peptide (highlighted in Figure S12), and the signals from some residues outside this region (E22 and G25) are also broadened to a lesser extent. However, the G29-V40 hydrophobic region is not affected. These selective signal broadening effects result from specific interactions of the paramagnetic GdL2 NMR relaxation probe (present in solution predominantly in micellar form) with the monomeric peptide, which is strongly dependent on the distance between the Gd 3+ ion and the peptide 15 N-1 H nuclei (r -6 ). The interaction with GdL2 could induce conformational changes in the peptide occurring in the μs-ms time scale, producing signal broadening, as noted before for the interaction of Aβ1-40 with diamagnetic surfactants such as sodium dodecyl sulfate, [28] congo red [29] and hydrophobic molecules like lacmoid, [27] where the signal loss was interpreted by the formation of heterogeneous aggregates able to interconvert in the NMR time scale. In our case, the large broadening observed at higher GdL2 excess are more likely dominated by paramagnetic relaxation effects. These data evidence a relatively strong interaction (Kd estimated in the few hundred μM range) of GdL2 with the hydrophilic F4-F20 region of the monomeric Aβ1-40. Similar interaction with the hydrophilic region of the peptide has been observed previously with the negatively charged Gd(DOTAGA-PiB), [13a] while the interaction was weaker for the neutral Gd(DO3A-PiB), where the Gd 3+ -chelate is very close to the PiB moiety. [13e] Therefore, the interaction of the Gd 3+ -PiB conjugates with the Aβ1-40 monomer does not seem to depend on the charge or on the length and the nature of the spacer, as opposed to specific effects observed for their interaction with Aβ1-40 aggregates.

Biodistribution studies

To evaluate the biological behavior of these potential imaging probes, their Indium-111 analogues were synthesized and an ex vivo biodistribution study was performed in healthy wild-type (C57BL6) mice. L2 and L3 were labelled by reacting ligand solutions with 111 InCl3 for 1h at 50°C and pH 7, and the complexes were obtained with >98% radiochemical yield. Mice (n=3) were injected intravenously in the tail vein (≈2.5 MBq/mouse) and sacrificed at 2 and 30 min post injection (p.i.), the organs of interest were collected, weighted and their activity measured. The biodistribution data, expressed in percentage of injected dose per gram of tissue (%ID/g ± SD), are presented in Figure 7 and Table S3. Table 4 shows the uptake in the most relevant organs. The radiocomplexes studied show fast clearance and no specific organ retention, as expected for small molecular weight complexes in healthy mice. This is important for a potential detection of amyloid peptides in diseased mice. 111 InL2 displays mainly renal elimination, with kidney retention of 17.9±1.8 %ID/g at 2 min which decreases over time, while 111 InL3 shows both kidney uptake (15.1±1.2%ID/g at 2 min) as well as liver accumulation which increases over time (11.4±1.3 and 21.7±2.5 %ID/g at 2 and 30 min, respectively). In contrast to 11 InL2, an uptake is observed for 11 InL3 in the liver, spleen and the intestines, which also increases with time. This indicates rather hepatobiliary/intestinal elimination for 111 InL3, in accordance with the more lipophilic character of trivalent L3 complexes (LogP = 1.46) compared to L2 analogues (LogP = 0.63). The low bone and muscle uptake reveals good in vivo stability for both complexes and excludes potential transmetalation. "Free" 111 In is indeed retained by bone and muscle, with reported values 4-20-fold higher than those of 111 InL2 and 111 InL3, respectively, even at 6h post injection. [30] Regarding the pancreas, an accumulation of 2.9±0.4 %ID/g and 3.7±0.6 %ID/g was obtained at 2 min p.i. for 111 InL2 and 111 InL3, respectively. These uptake values are promising and allow for envisaging the potential detection of amylin when overexpressed in diabetic animals.

A previous biodistribution study of 111 InL2 [15] yielded a rather different biodistribution profile, with uptakes that were higher in the liver, kidney and lung, while lower in the brain. Between the present and the former study, the labelling conditions and the concentration of the samples injected were different; we used micromolar instead of the previous millimolar concentrations.

Given the concentration dependence of the micellar aggregation, the concentration difference can likely account for the different biodistribution. A similar effect on the biodistribution has been already observed for amphiphilic complexes. [19] Concerning PiB-derivative small chelates, previous studies involved ex vivo biodistribution of 111 In(DOTA-PiB) in wild type mice [13c] , and in vivo PET biodistribution of 68 Ga(DOTA-PiB) and 68 GaL1 in control mice as well in a transgenic AD murine model. [13b] 111 In(DOTA-PiB) had fast global elimination, while 68 GaL1 had slower clearance from highly vascularized organs/tissues, in agreement with the hydropholic/lipophilic character and HSA affinity of the complexes. The brain uptake is similar for 111 In(DOTA-PiB) and 111 InL2/L3 (0.36 vs. 0.3 %ID/g at 2 minutes p.i.). This seems to indicate that the increased lipophilicity of 111 InL2 and 111 InL3 is counterbalanced by their higher molecular weight to determine brain uptake. 68 GaL1 accumulated more in the brain of diseased mice with a compromised BBB than in healthy brain (1.3 vs. 0.5 % injected dose per volume).

For amylin-imaging, two 99m Tc-labeled pyridyl-benzofuran derivatives have been recently proposed and their biodistribution was assessed in normal mice as well as in a mouse model with human amylin transplanted in the pancreas. [9] In normal mice, both complexes had low pancreatic uptake (0.74%ID/g and 1.37%ID/g at 2 min p.i.), which increased by ~50 % in the disease model mice, but remained still at a much lower level than and 111 InL3.

Conclusion

We have investigated a series of macrocyclic Gd 3+ complexes conjugated to the amyloid targeting PiB unit via different linkers. Depending on the concentration, these amphiphilic complexes form different micellar aggregates. Surface plasmon resonance studies evidence that nanomolar binding affinities, unprecedented for a metal chelate, are possible to attain with both A1-40 and amylin when the amyloid recognition unit is separated from the chelate by a long, hydrophobic C10 spacer. The Scatchard analysis of the SPR data indicate different affinity regimes as a function of GdL concentration, which could be related to the presence of different micellar aggregates. The large complexity of these systems prevents from finely characterizing the aggregated GdL micellar structures and attributing individual affinity constants to them. It is very likely that the monomeric form of the chelates has the highest affinity. Further, each of the different premicellar and micellar aggregates can be characterized by a different affinity. In overall, these studies are important as they clearly indicate that micellar aggregation of metal chelates in general should always be kept in mind when interactions between amphiphilic metal complexes and amyloid peptides are considered. In the future, it would be interesting to extend these studies to other amyloidogenic proteins such as Tau, α-synuclein, PrP or insulin fibrils.

The effect of the chelates on the amyloid peptide aggregation kinetics has been also investigated by Thioflavin-T fluorescence assays. The GdL complexes can either accelerate or slow down the aggregation of A1-40 and this effect seems to depend on the orientation of the 2-aryl-benzothiazole moiety with respect to the Gd 3+ containing macrocycle. Further, a 2D 1 H- 15 N-HSQC study helped identify that the hydrophilic peptide region is primarily involved in the interaction between GdL2 and monomeric A1-40. Finally, L2 and L3 have been labelled with 111 In and an ex vivo biodistribution study has been carried out in healthy mice. The radiocomplexes have fast clearance and no specific organ retention, but their uptake in the pancreas looks sufficiently high to envisage amylin detection in diabetic animals. To maximize the contact time, the flow rate was kept at 30μL /min. GdL1, GdL2 and GdL3 solutions were prepared at a concentration of 500μM with the running buffer. 150μL of these solutions was injected with an association and dissociation time equal to 300s each. This procedure was repeated for every complex and every concentration studied. For the regeneration of the surface, a solution of 100mM glycine -HCl in 10mM Tris (tris(hydroxymethyl)aminomethane) buffer at pH 9 was used with a contact time of 18s and a flow rate of 100μL /min. Then the flow rate is reduced to 30μL/min, the regeneration solution is re-injected if necessary and the flow cell is washed with the running buffer for 5min before the next injection.

The injection system was checked with every new chip, and primed to change the buffer when necessary. Biacore 3000 control software (version 4.1) was used to record the time dependence of the RUs. The response at equilibrium was plotted versus concentrations. The Langmuir binding isotherm was used to fit the binding plots and obtain the dissociation constant (Kd), with GraphPad Prism 7 software.

Aggregation kinetics. ThT fluorescence assays for A1-40 aggregation were performed on a ClarioStar microplate reader system (BMG Labtech) at 37°C. Thioflavin-T (ThT) was used to probe β-sheet structure formation [31] (fluorescence measured at 490 nm upon excitation at 440 nm). ThT fluorescence was measured every 5 min for about 100 hours, after 15 s of shaking at 200 rpm. 384-well microplates were used with a total volume of 100 µL for each sample. Different equivalents of GdL compounds NMR studies. NMR spectra for characterizing the interaction of GdL2 with the Aβ1-40 peptide were recorded on a Bruker Avance III spectrometer operating at 14.09 T 600.13 MHz for 1 H), equipped with a cryoprobe with three channels ( 1 H, 13 C e 15 N) and z-gradients. The 15 N-labeled A 1-40 peptide, purchased from AlexoTech (Umeå, Sweden), was stored in solid form at -20ºC and warmed to room temperature before use. 15 N-Aβ1-40 was initially dissolved as a concentrated 1 mM stock solution in DMSO-d6 (Aldrich), and was diluted 10-fold into a buffer containing 10 mM K2HPO4 and 10 % D2O (99.9%, Cambridge Isotope Labs, UK). The initial pH of the solution was about 8.2 and it was carefully adjusted to 7.2 by adding very small amounts of deuterated acetic acid (Aldrich). The final Aβ1-40 concentration was about 100 μM. During sample preparation, the peptide and the solvents were kept on ice. 1D 1 H (16 scans per spectrum) and 2D 1 H-15 N HSQC [32] spectra (matrix = 1024 for the 1 H dimension and 128 for the 15 N dimension, number of scans = 8, sweep width = 10 ppm for 1 H and 23 ppm for 15 N) were registered at 5 °C. A gradient-based Watergate module for solvent suppression was employed for the 1D spectrum and no solvent suppression was done for the 2D HSQC experiments. Spectra were Fourier transformed and analyzed using Topspin 2.1. The 1 H shifts were referenced to DMSO, and the 15 N chemical shift was referenced indirectly to the DMSO derived 1 H signal based on the relative gyromagnetic ratios of these nuclei. [33] Synthesis of 111 In complexes. 111 InCl3 was purchased from Mallinckrodt/Curiumpharma (Le Petten, Netherlands). The pH of 150 µL aliquots of 111 InCl3 (112 -130 MBq) were adjusted to 7 by addition of freshly prepared NaOH solution. Radiolabelling of L2 and L3 was performed as follows: a) 1 µL of a 1.54 mM solution of L2 was mixed with 150 µL of 111 InCl3 (133 MBq) giving a 13 µM final concentration; b) 50 µL of an aqueous 1.29 mM solution of L3 was mixed with 150 µL of 111 InCl3 (112 MBq) giving a 323 µM final concentration. After readjusting the pH to 7 (with NaOH), the solution was stirred for 1h at 50°C. A radiochemical yield > 98% was obtained and no further purification was performed. Labelling efficiency was followed by ascending silica gel ITLC (Polygram, Macherey-Nagel) developed with the mobile phase MeOH:H2O:NH4OH (2:4:0.2). The TLCs were exposed by impregnation on a multisensitive phosphor screen (Packard, Perkin Elmer, Meriden, USA, and revealed on a Cyclone Storage phosphor system Packard, Perkin Elmer, Shelton, USA). In this system the 111 In-complexes migrate with Rf = 0.9, while 111 InCl3 remains at the origin. Prior to injection, the radiocomplex solutions were diluted with a PBS/saline buffer yielding a final concentration of 1.9 µM for L2 (26.0 MBq) and 39.5 µM for L3 (19.7 MBq). Biodistribution studies. 8-weeks-old C57BL/6JRj wild type males were purchased from Janvier Labs (Le Genes Saint Isle, France). All animal experiments were carried out in accordance with the guidelines for animal experiments and under permission number 19861, from the French "Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation". A group of six mice per ligand (25.5±3.3 g and 27.1±2.3 g for 111 InL2 and 111 InL3 respectively) were injected intravenously (tail vein) with 2.08±0.15 MBq of 111 InL2 and 3.10±0.07 MBq of 111 InL3 (in 200µL) and an ex vivo biodistribution study was performed at 2 and 30 min post-injection. After 2 and 30 min, mice were sacrificed by cervical dislocation and the organs of interest (pancreas, brain, kidneys, lungs, heart, liver, spleen, intestine, muscle and bone (femur)) were harvested, weighted and their activity measured using a Carpintec gamma counter as well as a NaI based γ-camera (the acquired spectra are treated with WinTMCA program). Calibration of both equipment was done by measuring a set of 9 solutions with activities between 0 and 2 MBq in both equipment, under the same conditions of the organs' measurements (distance and time and energy range of measurement acquisition), yielding a good correlation: y = 3.943x10 6 ꭓ, R 2 = 0.9999. All data were corrected for the activity decay, bringing the activity to the injection time of each animal, as well as converted to MBq (Carpintec) using the calibration curve obtained. Biodistribution data are presented as % of injected dose per weight of organ (% ID/g) ± standard deviation (n=3).

Figure 1 :

 1 Figure 1: Structure of the ligands discussed.

Scheme 1 .

 1 Scheme 1. Synthesis of L3. Reagents and conditions were as follows: a) N-(10-Bromodec-1-yl)phthalimide, K2CO3, acetone, reflux, 72h, 77%; b) NH2NH2, reflux, 2h, 95%; c) chloroacetyl chloride, NEt3, THF, 0°C, 3h, 85%; d) DO3A-tBu, K2CO3, CH3CN, reflux, 16h, 72%; e) HCl, 1,4-dioxane, RT, 3h, 67%;

Figure 2 .

 2 Figure 2. SPR binding plots fitted as Langmuir isotherm functions for the interaction of GdL3 with A1-40 (A and C) and amylin (B and D). Peptide immobilization was 4000 RU (A, B) and 9000 RU (C, D). For C and D, points recorded only at low concentrations are represented.

Figure 3 :

 3 Figure 3: Scatchard linearization of SPR experiments for the interaction between GdL complexes and A1-40 or amylin. The complex concentrations corresponding to each breakpoint are also indicated.

Figure 4 .

 4 Figure 4. Average of representative kinetic experiments of A1-40 aggregation as monitored by ThT fluorescence in the presence of GdL1 (A), GdL2 (B) and GdL3 (C) as a function of the GdL:peptide stoichiometry (black = apo peptide; blue = 0.1 equiv. of GdL ; light blue = 0.5 equiv. of GdL ; purple = 1.0 equiv. of GdL and pink = 2.0 equiv. of GdL). [A1-40] = 20 µM, [Hepes] = 20 mM, pH = 7.4. [ThT] = 10 µM.

Figure 5 .

 5 Figure 5. Proton NMRD profiles of GdL2 (top) and GdL3 (bottom) at 37 °C in water (), in the presence of 0.2 mM A1-40 (■) or 0.6 mM HSA (▲). cGd = 0.2 mM

Figure 6 .

 6 Figure 6. Interaction of GdL2 with 100 µM 15 N-Aβ1-40 in water studied by NMR. A) 1D 1 H spectra of the Aβ1-40 peptide (black line), with addition of 0.5 equivalents (blue line), 1 equivalent (green line), 2 equivalents (orange line) and 4 equivalents (red line) of GdL2; 2D 1 H-15 N HSQC spectra of 15 N-Aβ1-40 B) alone with the signal assignment based on previous publications, and overlap spectrum with that in the presence of C) 1 equivalent (green) and D) 4 equivalents of GdL2. All spectra were recorded at 5 °C in 10 mM K2HPO4 at pH 7.20 in 90 % H2O, 10 % D2O on a 600 MHz Bruker spectrometer.

Figure 7 .

 7 Figure 7. Ex vivo biodistribution profiles of 111 InL2 (a) and 111 InL3 in healthy mice (n=3) at 2 (black) and 30 min (gray) post intravenous injection. Values are presented in % ID/g ± SD.

  The synthesis of ligand L3 is described in the supporting information. Complex preparation. The purity of the ligands was assessed by adding an excess of ZnCl2 solution to a ligand solution, followed by complexometric titration of the non-complexed Zn 2+ with EDTA. GdL complexes were prepared by mixing equimolar quantities of a GdCl3 and ligand solutions under continuous pH adjustment to 5.5. The absence of free Gd 3+ was checked by the xylenol orange test. Gd 3+ concentration was checked by ICP-OES or BMS (Bulk Magnetic Susceptibility) measurements. LogP determination. The shake flask technique was used. Octanol and water were respectively saturated with the other solvent. Concentrations were determined by UV-vis spectrophotometry (using the absorption of the benzothiazol ring at 330nm). Critical micellar concentration. For cmc determination, UV-visible absorption spectra were recorded on a PerkinElmer Lambda 19 spectrometer at 25 °C, 1 cm quartz cells. Dynamic light scattering measurements were performed on a Zetasizer Nano Series ZS (Malvern) instrument with temperature control (37°C). Each sample was measured in quintuple; each measurement is the average of 20 data sets acquired for 10 s. Hydrodynamic diameters have been calculated using the internal software analysis from the DLS intensity-weighted particle size distribution. Surface plasmon resonance measurements. A Biacore 3000 instrument (Biacore Life Science/GE Healthcare Uppsala, Sweden) was used for real time studies to assess the binding of the Gd 3+ complexes to immobilized peptides. The immobilization was done on CM5 sensor chips. N-hydroxysuccinimide (NHS) and N-ethyl-N'-[(dimethylamino)propyl]carbodiimide (EDC) were used for the activation of carboxylate functions. Ethanolamine, HCl, vials for samples and caps were purchased from GE Healthcare. HBS-EP (pH 7.4) which is composed of 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA and P20 surfactant (0.0005%) was used as running buffer. Aβ1-40 and amylin were dissolved in acetate buffer pH 5 (0.2mg of peptide in 500μL) and left at 37°C overnight to form aggregates. The immobilization followed standard amine coupling conditions. Activation of the carboxymethyl dextran matrix on the CM5 chips was realized with the injection, during 420s, of 70μL of the mixture EDC/NHS (200μL of 0.05M NHS and 50μL of 0.2M EDC). After sonication, 50μL of the peptide solution was injected into the activated flow cell with a flow of 10μL /min with 300s of contact time. Unreacted NHS ester is used to grab the peptide to the activated carboxylate groups for the amine coupling reaction. The second step of the immobilization is the elongation phase, consisting of injecting ten times a volume of 400μl of the monomeric peptide with a flow rate of 20μl /min leading to a contact time of 1200s. The unreacted NHS esters were capped with 70μL ethanolamine to result in a surface with a final change in resonance units (RU) equal to 9126.4RU. Knowing that 1.0 pgmm -2 of bound ligand results in 1000RU, it means that the total immobilized mass of Aβ1-40 is 9.1264 pg.mm -2 .

1 H

 1 (1 equiv. corresponds to 20 µM solution for A1-40) were added to the peptide in the presence of 10 µM of ThT in 20 mM HEPES buffer (pH 7.4) and placed in a 384-well microplate. Note that the experiments were performed 2-5 times (depending on the stoichiometry conditions) on 3 different batches of peptide, by 3 persons, and that in each experiment each condition was recorded at least in quadruplicate. The effects observed always followed the same trend. Purification of A1-40 for aggregation experiments. The A1-40 synthetic peptide was bought from GeneCust (Dudelange, Luxembourg), with purity grade > 95%. Stock solutions of the A1-40 (sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) were prepared by dissolving the powder (~ 3 mg) in 500 µL of Tris-HCl (0.1 M) with Guanidinium chloride (6 M). The solutions were incubated at 20°C overnight and purified by Fast Protein Liquid Chromatography (FPLC) (column Superdex 75, elution solvent NaOH 15 mM with NaCl 150 mM, flow rate 0.5 mL/min). The peptide concentration in the recovered fractions (500 µL) was then determined by UV-visible absorption of Tyr10 considered as free tyrosine (at pH 12, (ε293-ε360) = 2400 M -1 cm -1 ). NMRD. NMRD profiles of GdL2 (0.2 mM, pH 7) and GdL3 (0.25 mM pH 7) were recorded on a Stelar SMARTracer Fast Field Cycling relaxometer (0.01-10 MHz) and a Bruker WP80 NMR electromagnet adapted to variable field measurements (20-80 MHz) and controlled by a SMARTracer PC-NMR console. The temperature was monitored by a VTC91 temperature control unit and maintained by a gas flow.

Table 1 .

 1 Dissociation constants, Kd, determined by fitting the SPR data to 1:1 Langmuir isotherms.

	Kd	A1-40	amylin	HSA
	GdL1	71±9 µM	8.3±0.9 µM	1700±300 µM
	GdL2	16±2 µM	17.2±0.7 µM	300±8 µM
	L2 -	68±5 µM	30±3 µM	-
	GdL3 a	5±0.2 µM	3±0.9 µM	36±7 µM
	GdL3 b	4.4±0.9 nM (only
		from data between
		0 and 125 nM)

Table 2 .

 2 Concentrations corresponding to the breakpoints of the Scatchard plots for the different GdL-peptide interactions and cmc values of the GdL complexes

	conc. at	GdL1	GdL2	GdL3	Gd(DO3	Gd(DOT
	breakpoints				A-PiB)	AGA-PiB)
	A1-40	-	27 M	6 M	55 M	500 M
				98M
	amylin	21 M	27 M	4 M,	36 M	n.d.
				115 M
	HSA	-	100 M	148 M	-
	cmc	13±3
		M [a]

Table 4 .

 4 Ex vivo uptake of111 In complexes of L2 and L3 in most relevant organs (%ID/g ± SD).

			111 InL2		111 InL3
		2 min	30 min	2 min	30 min
	Brain	0.3±0.1	0.14±0.02	0.31±0.04	0.21±0.01
	Pancreas	2.9±0.4	1.0±0.3	3.7±0.6	1.8±0.2
	Kidneys	17.9±1.8	7.4±1.5	15.1±1.2	10.5±1.1
	Liver	3.6±0.3	2.8±0.2	11.4±1.3	21.7±2.5
	Intestines	2.15±0.05	1.1±0.1	3.0±0.1	5.4±0.7
	Lungs	8.0±0.8	3.2±0.2	10.7±1.6	5.0±2.2
	Bone	2.3±0.1	0.9±0.1	2.0±0.4	1.8±0.2

Acknowledgements

The authors thank the French National Research Agency (grant DIVA ANR-16-CE18-0022-01) for funding. We thank Stéphanie Lerondel and Julien Sobilo, Centre of Small Animal Imaging (CIPA), Orléans, for the biodistribution studies. CH and IR thank Xudong Lin and Lucie de Cremoux for their participation to the aggregation experiments and Dr. Béatrice Mestre-Voëgtlé for her involvement in the DIVA project. CH also acknowledges ERC StG 638712 for funding research on A1-40 aggregation. ACO and CFGCG thank the Fundação para a Ciência e a Tecnologia (FCT), Portugal, for funding of the Coimbra Chemistry Centre through the programmes UIDB/00313/2020 and UIDP/00313/2020, also co-funded by FEDER/COMPETE 2020-EU. ACO thanks the FCT for the PhD grant SFRH/BD/120934/2016 and PTNMR and Dr. Eurico Cabrita for access to the Bruker Avance III 600 NMR spectrometer.

Entry for the Table of Contents

Amyloid-targeted, amphiphilic metal chelates can form different micelles in aqueous solution and this influences their affinity to amyloid peptide aggregates. The separation of the targeting PiB unit from the metal binding macrocycle by a long hydrophobic chain allows for obtaining nanomolar affinities to both A1-40 and amylin aggregates. The GdL complexes also affect the aggregation rate of A1-40.

Nanomolar affinity

Probe concentration

Probe aggregation

Micromolar affinity


Teddy Roy

Julie Rousseau

Antoine Daudin

Gerhard Pirngruber

Benedicte Lebeau

Jean-Luc Blin
email: jean-luc.blin@univ-lorraine.fr

Dr Sylvette Brunet
email: sylvette.brunet@univ-poitiers.fr

Deep hydrodesulfurization of 4,6-dimethydibenzothiophene over CoMoS/TiO 2 catalysts: Impact of the TiO 2 treatment

Keywords: Mesoporous titania, Brønsted acidity, Hydrodesulfurization, Dismutation, Isomerization, 4, 6-Dimethyldibenzothiophene

Mesostructured titania as support for the CoMoS active phase in deep hydrodesulfurization (HDS) of 4,6-dimethydibenzothiophene (4,6-DMDBT) leads to an increase of the intrinsic HDS activity and a higher selectivity for direct desulfurization (DDS) for HDS reaction in contrast with the conventional CoMoS/alumina catalyst. The temperature treatment of the mesostructured TiO 2 support, modifies the catalyst's activity for the transformation of 4,6-DMDBT. The higher total and HDS activities were obtained after treatment at 380°C corresponding to the higher specific surface area and to a mesostructured TiO 2 material with a semi-crystalline anatase framework. Beyond 550°C, the specific surface area decreases strongly corresponding to a complete crystallization of the mesopores walls into anatase structure. Moreover, the temperature under which the support is treated prior its impregnation has no impact on the selectivity of the transformation routes of the sulfur compound.

Introduction

One of the major issue of our society is the reduced quality of air due to atmospheric pollution, mainly in urbans area. Emissions from transports are one of the most important factors contributing to the atmospheric pollution due to nitrogen oxides (NO x ) and sulfur oxides (SO x ). In order to deal with this issue, stringent regulations have been set up and are challenging researchers to develop highly active and stable catalysts to produce cleaner fuels. Indeed, since 2009, the regulation imposed sulfur level in gas oils lower than 10 ppm [1]. To cope with this legislation, refiners have developed the deep hydrodesulfurization (HDS) for removing sulfur-containing compounds to obtain ultra-low sulfur fuels. It is well established that the sulfur compounds present in diesel fuels at sulfur levels of less than 250 ppm are mainly dibenzothiophenes and alkyldibenzothiophenes, with 4,6-dimethydibenzothiophene (4,6-DMDBT) usually used as model molecule for HDS refractory compounds [2][3][4][5][6][7][8]. The transformation pathways of the latter are also well established (Scheme 1). Three routes have been reported in the literature: the hydrogenation (HYD) route, hydrogenating a benzene ring before the cleavage of C-S bond, the direct desulfurization (DDS) route, directly cleaving the C-S bond and the acidic route (DDS Acid) including isomerization and/or dismutation reactions of the methyl groups. The latter route is only observed on supported catalysts containing Brønsted acidity, particularly zeolites alone or in mixture [9][10]. HDS is commonly performed at medium temperature (340 °C) and under hydrogen pressure (3)(4) MPa) over a heterogeneous catalyst, usually CoMo over alumina. The active phase of the catalyst consists of supported MoS 2 slabs on γ-alumina promoted by cobalt. Over a conventional catalyst CoMo/Al 2 O 3 , the main transformation pathway of 4,6-DMDBT is following the HYD route. Much researches have been reported in order to optimize this process, one option is to design different supports. Indeed, the experts in the field are aware of the fact that the surface chemistry of alumina is not really optimal for the preparation of highly active promoted MoS 2 catalysts. The strong interaction of transition metals such as Mo, Ni or Co with the alumina support impedes full sulfidation and hence limits activity [11].

Other supports like silica, zeolites, carbon, titania, and mixed metal oxides are reported to provide significant improvements compared to γ-Al 2 O 3 [12][13][14][15][16][17]. Among all these supports, TiO 2 is particularly intriguing [18][19][20][21]. For example, Klimova et al. [20] have shown that NiMo catalysts supported on titania nanotubes allowed reaching high conversions of 4,6-DMDBT with a much lower proportion of hydrogenated product than using NiMo/Al 2 O 3 catalyst. Castillo et al. [21], have also reported that the addition of TiO 2 to the surface of alumina involves the dispersion of the well-sulfided active phase and hence in catalytic activity. However, it is rarely used as a support mainly because of its low specific surface area (50 m² g -1 against 230 m² g -1 for γ-Al 2 O 3 ), thermal stability and mechanical strength. A simple method, combining the sol-gel process and the surfactant templating method, to prepare mesostructured titania with high specific area (> 250 m² g -1 ) has been reported [20-23 22-25].

In a recent paper, we have shown that a higher DDS selectivity for the HDS of 4,6-DMDBT was observed with CoMoS supported over mesostructured TiO 2 [19]. This shift towards DDS selectivity in the transformation of 4,6-DMDBT is unprecedented in literature. Moreover, it involves Brønsted acidity of the support, which likely influences the nature of the formed intermediates. In addition, isomerization and dismutation reactions, which are catalyzed by Brønsted acid sites also participate to the shift towards DDS selectivity as observed on a zeolite support [9]. This paper deals with the identification of the parameters involved in the change of selectivity of the various pathways (HYD and DDS) in the HDS of the sulfur model compound (4,6-DMDBT) over CoMoS supported over mesostructured titania catalysts. In this way, the effect of the thermal treatment of the support prior the deposition of the active phase has been studied. The results are compared with the ones obtained using the conventional CoMoS/Al 2 O 3 catalyst and the CoMoS/P25 one, used as a reference titania support. In order to explain the catalytic results, characterizations of the active phase and Brønsted acidity were investigated.

Materials and methods

Cobalt

Supports and catalyst preparation.

Mesostructured titania have been synthesized according to the procedure reported in previous work using the triblock copolymer P123 (EO) 20 (PO) 70 (EO) 20 (labeled as P123) as surfactant [18][19]. To investigate the effect of thermal treatment temperature on the support properties and thus on the catalyst performance, the final mesostructured TiO 2 support (after Soxhlet extraction using ethanol) is treated at different temperatures from 380°C to 650°C for 1h under synthetic air atmosphere. Supported CoMo catalysts over as-synthesized mesostructured titania and commercial P25 (powders) were prepared by incipient wetness impregnation using cobalt(II) nitrate hexahydrate (Co(NO 3 ) 2 .6H 2 O, 99.00 % Sigma-Aldrich) and ammonium heptamolybdate [(NH 4 ) 6 Mo 7 .4H 2 O, 99.98 % Sigma-Aldrich] precursors in aqueous solution. The targeted number of Mo atoms per nm² and the Co/Mo ratio were fixed to 3 and 0.54, respectively. The precursors were then decomposed at 380°C under air during 5 hours.

Catalytic measurements:

Dimethyl disulfide (99% purity), 4,6-DMDBT (95% purity) and n-heptane (99% purity) from Aldrich Chemicals were used without further purification.

As reported in previous papers [18,19], the catalysts are sulfided in situ in a fixed flow reactor using a sulfiding feed made of 5 % wt of dimethyl disulfide (DMDS) in n-heptane as solvent at a temperature of 350°C under a 4.0 MPa of total pressure during 14 hours. The temperature was then lowered to the reaction temperature (340°C).

Then, HDS of 4,6-DMDBT was carried out at 340°C and 4.0 MPa of total pressure after the in situ sulfidation of the catalyst according to the procedure described above. The feed was composed by 4,6-DMDBT (500 ppm S) diluted in n-heptane into which dimethyl disulfide (9500 ppm S) was added to generate H 2 S. Different activities (A) were considered. They are defined as the number of moles of sulfur compounds transformed by each route for the transformation of 4,6-DMDBT per gram of catalyst, per m² and per molybdenum atom. A Total corresponds to the total activity, the sum of acid activity and hydrodesulfurization activity. The different activities are defined as follow:

A acid represents the activity for isomerization and dismutation of 4,6-DMDBT into alkyldibenzothiophenes. A HDS is related to the total activity for 4,6-DMDBT corresponding to desulfurized products (DDS and HYD routes). A HYD means the activity for the HDS hydrogenation route. A DDS stands for the total activity for the HDS direct desulfurization pathway from 4,6-DMDBT and from isomerization and dismutation reactions. A direct DDS refer to the amount of products resulting from the HDS direct desulfurization. A DDS Acid concerns the amount of HDS desulfurized products resulting from the isomerization and the dismutation of 4,6-DMDBT.

The contact time, defined as the ratio between the volume of the catalyst and the gas flow of the reactant, was adapted in order to obtain a conversion of reactant of around 25% for all the catalysts. Owing to the high boiling point of the reactant and products, on-line analysis of the reaction products was not convenient. Consequently, the reactor effluents were condensed and liquid samples were periodically collected to be analyzed by gas chromatography. Gaseous products were not found except for methane which was produced by dimethyl disulfide decomposition. Analyses were carried out with an Agilent 7820A equipped with a 30 m BP1 capillary column (inside diameter: 0.32 mm; film thickness : 5 m) with a temperature program from 50 to 70 °C (4°C/min) then from 70 to 250°C (15 °C/min).

Characterization

Bare and impregnated supports have been characterized by SAXS nitrogen adsorption desorption analysis, pyridine adsorption desorption followed by IR, TEM and XPS.

Small angle X-Ray scattering (SAXS) measurements were carried out on a "SAXSess mc²" instrument (Anton Paar), using line-collimation system. This instrument is attached to a ID 3003 laboratory X-Ray generator (General Electric) equipped with a sealed X-Ray tube (PANalytical, λ Cu, Kα = 0.1542 nm) operating at 40 kV and 50 mA. Each sample was introduced between two sheets of Kapton® then placed inside an evacuated sample chamber and exposed to X-Ray beam. Scattering of X-Ray beam was recorded on a CCD detector (Princeton Instruments, 2084 x 2084 pixels array with 24 x 24 µm² pixel size, sample-detector distance = 309 mm). Using SAXSQuant software (Anton Paar), the two-dimensional image was integrated into one-dimensional scattering intensities I(q) as a function of the magnitude of the scattering vector q = (4π/λ) sin(θ), where 2θ is the total scattering angle. Thanks to a translucent beamstop allowing the measurement of an attenuated primary beam at q = 0, all measured intensities can be calibrated by normalizing the attenuated primary intensity. All data were then corrected for the background scattering from the cell and for slit-smearing effects by a desmearing procedure from SAXSQuant software, using Lake method.

Powder X-ray diffraction patterns of mesostructured TiO 2 supports were recorded at wide angles using a Panalytical X'Pert PRO diffractometer equipped with a Cu X-ray tube (λ Cu (Kα) = 0.15418 nm) operating at 45 kV and 40 mA and an X'Celerator detector. The powder patterns were collected at 25°C in the range 3<2θ<70, step= 0.017°2θ, time/step=220s.

The porosity and the specific surface area have been evaluated by nitrogen adsorption desorption at -196 °C using Micromeritics TRISTAR 3000 equipment. Isotherms were determined over a wide relative pressure range from 0.01 to 0.995. The pore diameter and the pore size distribution were obtained by applying the BJH (Barret, Joyner, Halenda) method [26] to the adsorption branch of the isotherm. Prior to analysis, 100 mg of solid is degassed at 350 °C during one night.

Raman Scattering Spectra were collected on a Jobin-Yvon T64000 spectrometer equipped with an optical microscope in confocal mode. The excitation beam (514.5 nm) was focused using a long-frontal x50 objective (numerical aperture 0.5) on an area of about 3 m 2 . The laser power on the sample was approximately 10 mW. The spectral resolution was 3 cm -1 , with a wavenumber precision better than 1 cm -1 .

The measurement of the acidity (Lewis and Brønsted) by adsorption of pyridine followed by FTIR spectroscopy was carried out with a ThermoNicolet NEXUS 5700 spectrometer at a resolution of 2 cm -1 and collected 128 scans per spectrum. Catalyst samples were pressed into thin pellets (10-60 mg) with diameter of 16 mm under a pressure of 1-2 t.cm -2 and activated in situ during one night under nitrogen at 380°C. After cooling down the samples until room temperature, a background spectrum was collected after removal of the excess of pyridine with a vacuum pump. Lewis and Brønsted acid sites were quantified from the area of the band at 1445-1450 cm -1 for the Lewis acidity and at 1540 cm -1 for the Brønsted acidity [27,28]. All spectra were normalized to sample mass (20 mg).

A JEOL JEM2100F transmission electron microscope (TEM) was used to characterize the morphology of MoS 2 phase. XPS spectra were collected on a Kratos Axis Ultra (Kratos Analytical, U.K.) spectrometer with a hemispherical energy analyzer and using a monochromatic Al Kα source (1486.6 eV).

All spectra were recorded at a 90° take off angle, with the analyzed area being currently about 0.7 x 0.3 mm. Survey spectra were acquired with 1.0 eV step and 160 eV analyzer pass energy and the high-resolution regions with 0.1 eV step and 20 eV pass energy (instrumental resolution better than 0.5 eV). The, Co 2p, Mo 3p, Mo 3d, S 2s and S 2p binding energies were referenced to the C1s line situated at 284.6 eV, i.e. the value generally accepted for adventitious carbon surface contamination. The XPS data, reported in Table 3, of the sulfidation rate of molybdenum (TSMo), the global sulfidation degree (TSG), promotion rate 1). It should be noted that after impregnation regardless the surfactant used, the mesostructured TiO 2 kept a high specific area of 200 m²/g after being calcined at 380°C. The second important fact is that the CoMo supported on mesostructured TiO 2 has higher specific surface area than the one supported on P25 (commercial TiO 2 ) and the reference catalyst CoMo supported on Al 2 O 3 .

However, looking at the effect of the calcination temperature, we noted a decrease of both the specific surface and the pore volume with the increase of the calcination temperature for the bare and the impregnated supports. Moreover, the mesostructure collapsed after 550°C. Indeed, after heating at 650°C no reflection was detected any longer on the SAXS pattern (Figure 1). X-ray peaks characteristic of anatase are observed on the mesostructured TiO 2 thermally treated at 380, 450 and 550°C (Figure 2). Their intensity increase when the temperature of the thermal treatment increases, confirming a higher crystallization degree. A halo due to amorphous phase in the 2θ range 25-32° is clearly observed after thermal treatment at 380°C and is barely visible after thermal treatment at 550°C. Meantime, in Raman the shift from 147 to 143 cm -1 of the Eg mode in the Raman spectra of mesostructured TiO 2 (Figure 3) indicated that the crystallization of the walls was enhanced when the temperature was raised. Indeed, it has been reported that the position of this vibration is dependent of the TiO 2 crystallite size [29][30][31]. The Eg mode appears at 143 cm -1 in bulk anatase and shifts to higher wavenumbers in nanocrystalline materials. So the anatase particles grew when the temperature increased from 380 to 650°C. After treatment at 650°C the appearance of the vibration at 448 cm -1 (Eg), revealed the presence of rutile. The other vibrations at 143 (Eg), 196 (Eg), 398 (B1g), 520 (A1g+B1g) and 642 cm -1 (Eg) were due to anatase. Wide-angle XRD and Raman data indicate that starting with the amorphous phase and the anatase one, the TiO 2 was transforming into anatase and rutile with the increase of the temperature. The specific surface area dropped to 12 m² g -1 after calcination at 650°C, strengthened the growth of the anatase particles and led to a collapse of the mesostructure. This phenomena can also explain the results of IR-pyr (Table 2), used to characterize the acidity properties of the titania materials. Looking at the mesoporous titania prepared according to the sol-gel method, quantification of the acidity (Table 2) reveals that before and after impregnation of Mo and Co Brønsted acid sites were present. The latter can be related to the semi crystalline features of the walls. Some hydroxyls groups, responsible for the Brønsted acidity, were present on the surface of the amorphous phase. Increasing the temperature treatment, the amount of Brønsted sites decreases because of the crystallization of the walls and after treatment at 650°C, no Brønsted is detected, reflecting the complete crystallization of the titania framework into anatase and rutile phase. This evolution is in accordance with literature. Indeed, Afanasiev et al. have shown only strong Lewis acid sites on anatase and rutile surfaces [32]. Alumina and P25 (75% of anatase and 25% of rutile), have only Lewis sites at their surface. The results reported below clearly show that the Brønsted acidity observed for the mesostructured titania is attributed to the presence of the amorphous phase.

It is also worth noting that for any materials the Lewis acidity is superior to the Brønsted acidity. It should finally be noted that the commercial Al 2 O 3 and P25 were impregnated under the same conditions as the mesostructured titania ones.

Characterization of the sulfide phase

The parameters closely associated to the formation of the active phase such as the S/Mo, Co/Mo atomic ratios, the promotion by cobalt (PR), S/Mo atomic ratios, the sulfidation rate of molybdenum (TSMo) and the global sulfidation rate (TSG) are given in Table 3 from XPS analysis. From this table, we can conclude that whatever the support, MoS 2 has been formed.

In addition, taking into account the error on the calculation, about 20%, we can note that for the mesostructured TiO 2 , whatever the temperature, the sulfidation rate of molybdenum (TSMo), global sulfidation degree (TSG), promotion rate (PR), atomic S/Mo and Co/Mo ratios are very close. This shows that the temperature of calcination of the mesostructured did not impact the formation of MoS2 phase and its promotion by the cobalt. For example, the global sufidation degree and the S/Mo ratio vary from 62 to 61 and from 1.6 to 1.2, respectively, when the calcination temperature increased from 450 to 650°C. The typical slabs of MoS 2 crystallites are observed by transmission electronic microscopy (Figure 4). From this figure, it appears that the temperature treatment affected neither the morphology nor the stacking of the slabs, which are constituted by 1 or 2 layers. By contrast the length of the slabs increased with the temperature (Figure 5A) and the slabs' length distribution became broader (Figure 5B). This variation occurred in parallel to the decrease of the Lewis and Brønsted acidities of the impregnated supports.

The characterizations indicate an effect of the thermal treatment of the mesostructured TiO 2 support on the specific surface area as well as on the acidic properties and on the slabs' length.

HDS of 4,6-DMDBT

All the catalysts were evaluated for the hydrodesulfurization (HDS) of 4,6dimethyldibenzothiophene (4,6-DMDBT). It should be noted that whatever the catalysts, no deactivation was observed. The catalytic activities were calculated per gram of catalyst, per m² and per Mo atom, respectively reported in Table 4, 5, and 6 in order to have the global activities and to show the contribution of the specific surface area and to determine the modification of the activity per molybdenum atom. The catalytic performances of the different materials are compared in terms of total activity (A T ), hydrodesulfurization activity (A HDS ), acid activity (A Acid ), activities of the different routes (A DDS , A HYD , A direct DDS , A Acid DDS ), selectivity of transformation routes and of HYD/DDS ratio. As reported in Table 4 (specific activity), catalysts with mesostructured TiO 2 thermally treated at 380 and 450°C as supports had a higher total activity (A T ) and HDS activity (A HDS ) than with P25 or Al 2 O 3 as supports. Differences were observed for the activities in HDS (A HDS ) and involving the Brønsted acidity (A acid. ). As reported previously [18,19], mesostructured TiO 2 induced the presence of Brønsted acidity which decreased with the temperature of the treatment of TiO 2 mainly at 550°C and 650°C. In the same way a high decrease of the total activity (A T ), HDS activity (A HDS ) and of Acid activity (A Acid. ) from 550°C to become very low at 650°C was observed. This corresponds also to a more important crystallization of the walls of the support into anatase, to a high decrease of the specific surface area and also to a lower amount of molybdenum (its deposition depended on the specific area: 3atom per nm 2 ). In order to distinguish the impact of these different parameters, we looked at the same activity per m² (Table 5) and at the same amount of molybdenum atom (Table 6). A decrease in total activity can still be seen, but this decrease is only due to a drop of the A Acid , A HDS is constant between 380°C and 550°C (Table 5). The correlation with the physico-chemicals properties (Table 1) explains these results, the diminution of total activity per gram is due to the loss of the specific surface area and the Table 2 shows the importance of the Brønsted acidity because the loss of the latter leads to a decrease of total activity per m² for a thermal treatment from 380°C to 650°C. These observations are confirmed by the Table 6 which allows us to understand that there is no significant change in the Mo atom environment and no change of the active sites for a thermal treatment of the mesostructured titania from 380°C to 650°C. These results were also confirmed taking into account to only the amount of mixed CoMoS phase (not reported here).

Finally, the significant conclusion of this study is that using TiO 2 support prepared by the surfactant templating method, whatever the heating temperature the DDS pathway remains the main route for 4,6-DMDBT desulfurization. On mesostructured TiO 2 , DDS (Direct + Acid) (mainly DDS Direct) became the major pathway in contrast to alumina where HYD is the main route. This change mainly comes from the modification of the direct DDS which contributes to around 50 % of the overall DDS for CoMoS/TiO 2 whatever the temperature of the treatment excepted at 380°C (43%). The other part is due to the presence Brønsted acidity which represents less than 15% of the DDS excepted at 380°C (22%). The consequence is a decrease of a HYD/DDS ratio from 3 in the presence of Al 2 O 3 as support to 0.54 for TiO 2 (treated at 380°C) as support. Only a small HYD/DDS ratio difference was noticed for the different treatment temperatures (from 0.54 to 0.73). The HYD/DDS ratio with P25 as support is intermediate between TiO 2 and Al 2 O 3 as supports. The change of selectivity (HYD/DDS) observed with CoMo/TiO 2 is unique and due to the intrinsic properties of the mesoporous TiO 2 , as the temperature of the thermal treatment does not change the selectivity. A calcination temperature of 380°C seems to be the best temperature of the treatment of TiO 2 leading to the higher contribution of the Brønsted acidity in the HDS activity.

Conclusion

Mesostructured titania used as a support of hydrotreating catalysts, in particular 4 Activity (mmol h -1 m -2 ) x 10

(

  PR) and the promoter [(Co/Mo) slabs ] are calculated as follows : of the impregnated CoMo/TiO 2 catalysts After impregnation of CoMo, except for P25, due to the incorporation of Mo and Co components at the surface and inside the mesopores the values of the specific surface area and of the mesopores volumes decreased by about 33-54% and 25-53%, respectively (Table

, 6 -Figure 1 :

 61 Scheme 1 : Transformation of 4,6-DMDBT over CoMoS/TiO 2 . (HYD: Hydrogenation route,

Figure 2 :Figure 3 :

 23 Figure 2: XRD pattern of TiO 2 at different temperature of thermal treatment

Figure 4 :Figure 5 :

 45 Figure 4: TEM images of the CoMoS/TiO 2 catalysts depending on the temperature of

Table 5 :

 5 Total activity, sum of acid activity and hydrodesulfurization activity A HDS = Total activity of hydrodesulfurization, DDS and HYD pathways A Acid = Total activity of isomerization and dismutation routes leading to no desulfurized products Transformation of 46DMDBT: Effect of the calcination temperature of the mesostructured TiO 2 support, detailed of the total activity (A mmol h -1 m 2 ) compared with reference catalyst (P25 and Al 2 O 3 ) (T = 340°C, P = 40 bars, H 2 /feed = 475 NL/L).

  Total activity, sum of acid activity and hydrodesulfurization activity A HDS = Total activity of hydrodesulfurization, DDS and HYD pathways A Acid = Total activity of isomerization and dismutation routes leading to no desulfurized products A DDS = Activity of direct desulfurization (A DDS Direct) + activity of DDS of the acid route (A DDS Acid) A HYD = Activity of desulfurization by the HYD route

  Total activity, sum of acid activity and hydrodesulfurization activity A HDS = Total activity of hydrodesulfurization, DDS and HYD pathways A Acid = Total activity of isomerization and dismutation routes leading to no desulfurized products A DDS = Activity of direct desulfurization (A DDS Direct) + activity of DDS of the acid route (A DDS Acid) A HYD : Activity of desulfurization by the HYD route

  (II) nitrate hexahydrate (CoN 2 O 6 .6H 2 O, 99.00 % Sigma-Aldrich) and ammonium heptamolybdate [(NH 4 ) 6 Mo 7 .6H 2 O, 99.98 % Sigma-Aldrich] were used as Co and Mo precursors, respectively.

Table 1 :

 1 Specific surface area (S BET ), pore diameter (Ø) and mesopore pore volume (V P ) of the supports before and after impregnation.

								MoO 3	CoO
		Before impregnation	After impregnation
	T calci							(%)	(%)
	Support
	(°C)	S BET	Ø*	Vp*	S BET	Ø*	Vp*	0	0
		(m 2 .g -1 )	(nm)	(cm 3 .g -1 )	(m 2 .g -1 )	(nm)	(cm 3 .g -1 )
	P25	37	-	-	37	-	-	2.6	0.7
	380	300	6.5	0.40	200	6.6	0.33	21.5	4.0
	450	180	6.3	0.30	130	5.6	0.27	12.9	2.6
	TiO 2
	550	130	5.5	0.23	106	4.8	0.15	9.3	2.1
	650	12	-	0.03	6	-	0.02	0.9	0.1

*Values determined from the BJH method applied to the adsorption branch of the isotherm.

Table 2 :

 2 Lewis (L) and Brønsted (B) acidity measurement by FTIR pyridine of TiO 2 after wet impregnation.

	CoMo/	T treatment	n pyr (µmol.g -1 )	n pyr (µmol.m -2 )	B/L
	Support	(°C)	L	B	L	B
	Al 2 O 3	-	140	0	1.1	0	0
	P25	380	140	3	3.8	0.08	0.02
		380	344	85	1.6	0.41	0.25
	TiO 2	450	325	39	2.5	0.30	0.12
		550	276	15	2.6	0.14	0.05
		650	124	0	10.3	0	0

Table 3 :

 3 Sulfidation rate of molybdenum (TSMo), global sulfidation degree (TSG), promotion rate (PR), atomic S/Mo and Co/Mo ratios and promoter ratio [(Co/Mo) slabs ]. Data are obtained from XPS analysis.

									T°C	TSMo	TSG	PR a
			Support			S/Mo Co/Mo (Co/Mo) b	slabs
									(%)	(%)	(%)
									380	53	69	36	1.8	0.3	0.19
									450	47	62	51	1.6	0.3	0.31
				TiO 2
									550	57	84	46	1.8	0.3	0.28
									650	56	61	-	1.2	0.5	-
				P25*			380	40	71	13	2.3	0.4	0.13
			γ-Al 2 O 3 **		-	70	53	26	1.5	0.3	0.10
	* used as TiO 2 reference
	** used as reference for HDS reactions
	a :	PR		   Total Co CoMoS		x	100
	b : The promoter ratio is the Co/Mo ratio in the slabs [(Co/Mo) slabs ]
			  	Mo Co	  	Slabs		 CoMoS 2 MoS	
	-not determined

Table 4 :

 4 Transformation of 4,6-DMDBT : Effect of the calcination temperature of the mesostructured TiO 2 support, detailed of the total activity (A mmol h -1 g -1 ) compared with reference catalyst (P25 and Al 2 O 3 ). The selectivity (%) of each pathway of

	hydrodesulfurization is given (T = 340°C, P = 40 bars, H 2 /feed = 475 NL/L)
			Activity (mmol h -1 g -1 )		Selectivity (%)
	CoMo/ Support	T treatment (°C)	A T	A HDS A Acid HYD DDS Direct DDS Acid HYD/DDS
		380	1.61	0.98	0.39	35	43	22	0.54
	TiO 2	450 550	1.19 0.65	0.89 0.52	0.30 0.13	38 42	48 52	14 6	0.62 0.73
		650	0.09	0.06	0.03	37	50	13	0.59
	P25	380	0.24	0.20	0.04	50	50	0	1
	Al 2 O 3

Table 6 :

 6 Transformation of 46DMDBT : Effect of the calcination temperature of the mesostructured TiO 2 support, detailed of the total activity (A mmol h -1 Mo atom -1 .) compared with reference catalyst (P25 and Al 2 O 3 ) (T = 340°C, P = 40 bars, H 2 /feed = 475 NL/L).

					Activity (mmol h -1 Mo atom -1 .) x 10 -21
	CoMo/ Support	T treatment (°C)	A T	A HDS	A Acid	A HYD A DDS	A DDS direct	A DDS Acid
		380	1.5	1.1	0.4	0.4	0.7	0.5	0.2
	TiO 2	450 550	2.2 1.7	1.6 1.3	0.6 0.3	0.6 0.6	1.0 0.8	0.9 0.7	0.1 0.1
		650	2.5	1.7	0.8	0.6	1.1	0.8	0.2
	P25	380	2.1	1.8	0.3	0.9	0.9	0.9	0.0
	Al 2 O 3

Acknowledgments

We would like to thank the platform "Spectroscopies et Microscopies des Interfaces", Martine Mallet and Aurélien Renard (LCPME) for XPS analyses. The platform "X-ray diffusion" of IS2M is also acknowledged.