V Simonet

B Grenier

F Villain

A.-M Flank

G Dhalenne

A Revcolevschi

J.-P Renard

Effect of structural distortions on the magnetism of doped spin-Peierls CuGeO 3

Keywords: . 61.10.Ht X-ray absorption spectroscopy: EXAFS, NEXAFS, XANES, etc. -61.72.-y Defects and impurities in crystals; microstructure -75.10.Pq Spin chain models

The chemical selectivity and great sensitivity of the Extended X-ray Absorption Spectroscopy technique allowed the determination, in the paramagnetic phase, of the structural distortions induced by doping in the spin-Peierls CuGeO3 compound. The distorted environments were analyzed as a function of concentration, magnetic nature of impurity and the substitution site (Ni, Mn and Zn impurities on the Cu site, Si impurity on the Ge site). This has led to estimate the variation of the angles and pair distances, and hence to evaluate the magnetic coupling along the Cu chains in the vicinity of the impurities. The antiferromagnetic interaction between Cu first neighbors in the pure sample is found to be weakened around Ni, almost cancelled in the case of Mn doping, and even to change sign, producing a ferromagnetic coupling for Si doping. More generally, the structural distortions on a local scale are shown to be key parameters for the understanding of the magnetic properties of doped spin-Peierls compounds.

Introduction

The first inorganic spin-Peierls (SP) compound, CuGeO 3 , [1] has already been extensively studied for its original magnetic properties. The possibility of substituting the cationic sites, Cu or Ge, by different magnetic or non-magnetic elements, has brought new information and rose new questions.

The structure of CuGeO 3 can be described as a stacking of CuO 6 octahedra and GeO 4 tetrahedra along the c direction of the structure. Each O belongs to both a Ge tetrahedron and a Cu octahedron. The stacking of distorted octahedra (elongated along the direction of the apical O(1) and with a planar distorted square of basal O(2) in the perpendicular direction) results in chains of magnetic Cu 2+ ions located at the center of edge sharing squares of O(2) running along the c axis (Fig. 1). At room temperature, this compound then consists of uniform chains of spins S=1/2, interacting by superexchange through O(2) atoms leading to an antiferromagnetic (AF) Heisenberg-type coupling (Fig. 1). A magnetoelastic instability produces a dimerization of the chains below a critical temperature T SP of 14.25 K. This leads to a sin- glet (non-magnetic) ground state separated from the first excited triplet state (S = 1) by an energy gap of 2 meV. Below T SP , the magnetic susceptibility drops to zero with a temperature activated behavior, and diffraction experiments reveal new superlattice peaks [2].

Doping, either with Zn, Ni, Mg or Mn on the Cu site [3,4,5] or with Si on the Ge site [6,7], results into a decrease of T SP and, at a lower temperature, the Néel temperature T N , into a transition to an AF long-range ordering (LRO). Some of these impurities are magnetic, like Ni with spin 1 and Mn with spin 5/2 [8], while the others, Zn, Mg and Si, are non magnetic. They also have very different ionic radii whose consequence on the magnetic properties will be discussed in Sec. IV and V. A systematic study of the magnetic properties of single crystals with various doping levels (CuGe 1-x Si x O 3 with 0 < x ≤ 0.08, and Cu 1-y M y GeO 3 with M = Zn, Mg, Ni and 0 < y ≤ 0.1), all synthesized and characterized in the same way, has allowed establishing a quasi-universal phase diagram for the spin-Peierls transition [5]. The variation of T SP versus concentration was found identical for all impurities on the Cu site, including Mn, which has been studied since Ref. [5] (for 0 < y ≤ 0.02), and for Si impurities applying a scaling factor y ≈ 3x. This is illustrated in Fig. 2 of Ref. [5] and in the alternative representation of the present Fig. 2 where no scaling factor y ≈ 3x is applied in view of the discussion of Sec. V.B. Surprisingly, doping outside the spin chains is thus about three times more efficient in destroying the spin-Peierls phase than doping within the chains 1 . In contrast, T N varies with the type of impurity: it is higher for Mn and Si-doping and slightly lower for Ni-doping, in comparison with the value, found identical, for the non-magnetic impurities Mg and Zn. A difference between Ge site and Cu site doping is observed concerning the T N (y, x) transition line which is continuous in the first case and presents a kink between a dimerized AF phase at low doping and a uniform AF phase at larger doping in the second case [12]. This behaviour is characteristic of a second-order and a first-order phase transition, respectively. Another particularity concerns the easy axis of magnetization in the AF phase: it is the a-axis for Ni doping and the c-axis for all other substitutions, which can be explained by the single-ion anisotropy of Ni 2+ in a distorted octahedral environment [13].

These results must be considered in the light of the different couplings along the Cu chains. The magnetic properties are mainly driven by two kinds of AF couplings: A superexchange coupling Cu-O(2)-Cu between first Cu neighbors with an AF exchange constant J 1 = 120 -180 K and a frustrating AF coupling between second neighbors J 2 = 0.24 -0.36 J 1 via the Cu-O(2)-O(2)-Cu path [14,15] (Fig. 1). The inter-chain couplings are one order and two orders of magnitude smaller along the b and a axes, respectively [15]. These inter-chain couplings induce a coherence between the phases of the distortion waves on different chains. In CuGeO 3 , the dimerization pattern is out of phase for adjacent chains in the b and a directions. The strength and nature of the magnetic interactions are strongly dependent on the Cu-O(2) and Ge-O(2) distances and even more drastically on the Cu-O(2)-Cu superexchange angle (denoted α). The Goodenough-Kanamori-Anderson rules [START_REF] Goodenough | Magnetism and the chemical bond[END_REF] state that the exchange is ferromagnetic (FM) for an angle of 90 • . The deviation of the Cu-O(2)-Cu angle from this value (α=99 • in CuGeO 3 ) will drive the exchange towards antiferromagnetism [START_REF] Braden | [END_REF]. However, Geertsma and Khomskii pointed out that calculations taking into account this sole effect still yield a slightly ferromagnetic exchange coupling [18]: The hybridization of Ge orbitals with the 2p y ones of the O(2), which destroys the equivalence of 2p x and 2p y O(2) orbitals, must indeed be taken into account to obtain, for J 1 , an AF exchange with a reasonable value of 135 K.

The aim of the present work is to understand the effect of doping the Cu chains, with magnetic and non-magnetic impurities, and out of the chains in the case of Si-doping, by measuring the induced structural distortions of doped CuGeO 3 . These distortions should strongly influence the magnetic properties, in particular via the variation of the superexchange angle α and thus of the related magnetic coupling J 1 . The EXAFS (Extended X-ray Absorption Fine Structure) spectroscopy technique was chosen for this purpose as a local order probe, with chemical sensitivity, and for its possibility to measure very diluted systems. For the first time with this technique, the influence of Ni, Mn, Zn and Si-doping was investigated in the paramagnetic phase (T > T SP ) but not that of Mg-doping because of the too low energy absorption of the Mg atom.

χ(k) pure CuGeO 3 , Cu K-edge (b) χ(k) k (Å -1 ) pure CuGeO 3 , Ge K-edge 2% Si, Si K-edge
The next section is devoted to the experimental details and to a brief recall of the EXAFS analysis methodology. The EXAFS results are reported in section III for Cu site and Ge site doping. The structural distortions, as deduced from the EXAFS refined parameters, are analyzed in section IV. Their influence is discussed in section V, in relation with the magnetic properties, and a conclusion is given in Sec. VI.

Experiment

The EXAFS measurements at the Cu and Ge absorption K-edges, in transmission detection mode, and at the Ni, Mn and Zn absorption K-edges, in fluorescence detection mode, were carried out at LURE (Orsay) on the D42 and D44 beamlines of the DCI storage ring. The experiments were performed at 78 K, in the paramagnetic phase, above the spin-Peierls transition. The incident energy was varied by 2 eV steps using a Si(111) single crystal monochromator. The EXAFS oscillations were obtained in transmis-sion detection mode by measuring the X-ray beam intensities with ionization chambers, before (I 0 ) and after (I 1 ) the sample, in an energy range close to the absorption K-edge of Cu (λ Cu K =8979 eV) and Ge (λ Ge K =11103 eV). The samples studied in the transmission detection mode were pellets of homogeneous thickness made of a mixture of about 15 mg of single crystal ground into very fine powder and 35 mg of cellulose. In the fluorescence detection mode, the powder obtained from ground doped crystals was spread on an adhesive tape. The EXAFS spectra were measured in the fluorescence detection mode with a multielements detector around the impurities K-edges in the 2% and the 6% Ni-doped samples (absorption K-edge λ N i K = 8333 eV), 1.5% Mn-doped sample (λ Mn K = 6539 eV), and the 4% Zn-doped sample (λ Zn K = 9659 eV). The Si K-edge EXAFS experiment was performed on the SA32 beamline of SUPERACO using a 2 InSb crystals monochromator. A thin slice of 2% Si-doped single crystal (absorption K-edge λ Si K = 1839 eV) was cleaved and stuck with Ag glue on an indium covered sample-holder. The absorption signal was recorded with a monoelement detector in the fluorescence detection mode.

The EXAFS oscillations were analyzed using the FEFF package [19]. The EXAFS oscillations, χ(k), were extracted from the experimentally measured absorption coefficient µ(E) using the AUTOBK program with k, the wavenumber of ejected photoelectron, equal to:

k = 2m(E -E 0 )/h 2 (1)
where E 0 is the absorption energy for the K-edge and m the electron mass.

For an absorbing central atom surrounded by N identical atoms, when considering only single scattering paths, EXAFS oscillations created by the backscattering of the photoelectron from the neighboring atoms are given by:

χ(k) = -S 2 0 N kr 2 |F (k, π) | e -2k 2 σ 2 e -2r/λ(k) sin [2kr + 2δ(k) + Φ(k)] (2)
where r is the mean pair distance, σ 2 is the associated Debye-Waller factor which takes into account the structural and dynamical disorders, and S 2 0 is the amplitude reduction factor accounting for many-body effects within the absorbing central atom. The other parameters in Eq. ( 2) are the phase Φ(k) and the modulus of the complex function F (k, π), which both depend on the nature of the backscattering atom, the mean free path λ(k) and the absorbing atom phase shift δ(k). They are derived from the theoretical ab-initio multiple scattering calculations by the FEFF program using crystallographic data. In order to describe a real environment, the surrounding atoms are gathered into atomic shells (atoms at approximately the same distance from the absorber), themselves subdivided into several subshells, for each kind of chemically and crystallographically equivalent neighbors.

Representative measured EXAFS oscillations χ(k) are shown in Fig. 3. Note that the spectra of the doped samples, recorded at the impurity K-edge, are more noisy than those recorded at the Cu and Ge K-edges because of the small amount of absorbers and of the fluorescence detection mode. In consequence, the EXAFS analysis could not be performed up to such large k values at the impurity K-edges compared to the Cu and Ge ones. For the Si Kedge spectrum, an additional source of noise was the low fluorescence yield and the small photon flux at this low energy. The complex Fourier transforms (denoted FT) of the k 3 weighted χ(k) functions were then calculated using a Hanning window. The amplitude of this FT can be associated with a pseudo-radial atomic distribution around the absorber in the r-space. Lastly, the EXAFS oscillations of the atomic shells of interest have been isolated by a subsequent inverse Fourier-transform (denoted IFT) performed in a restricted appropriate r-range and leading to a Fourier filtered k 3 χ(k) function. The FT and IFT were simulated with the FEFFIT program by summing the contributions given in Eq. ( 2) for all subshells obtained from the structural model. The multiple scattering paths, whose contribution was checked to be weak, were not included in the calculation. In the fit, the pair distances, the Debye-Waller factors and the energy shift ∆E 0 were allowed to vary 2 . For the analysis of the EXAFS oscillations recorded at the 2 ∆E0 accounts for inaccuracies in the FEFF determination of the absorption threshold energy E0. Different values of ∆E0 were refined for the O, Cu and Ge neighbors. These extra shifts impurity absorption edges, the FEFF files for each scattering path were recalculated with the impurity replacing Cu or Ge absorber within the same structure.

3 Results

Pure sample

The structure of CuGeO 3 in the paramagnetic state was initially refined by Völlenkle et al. [20] and later by Braden et al. [START_REF] Braden | [END_REF]21] (space group P bmm, a = 4.796 Å, b = 8.466 Å, c = 2.940 Å at 295 K), from Rietvelt refinement of X-ray and neutron powder diffractograms 3 . The crystallographic data of Braden et al. [START_REF] Braden | [END_REF] were used as input are meant to correct errors introduced by FEFF in the calculation of the scattering phase shifts. 3 The structure was revised by M. Hidaka et al., J. Phys.: Condens. Matter 9, 809 (1997). They found a different space group P 212121 and a larger unit cell 2a × b × 4c than Braden et of the FEFF program in our EXAFS analysis. These structural parameters yield, for the atomic distribution around Cu atoms, a first shell with the 4 square planar O(2) at 1.942 Å and a second shell including the 2 apical O(1) at 2.78 Å, the 2 chain neighbors Cu at 2.95 Å and 4 Ge at 3.30 Å (top of Fig. 4). The atomic distribution around Ge atoms is made of a first shell with the 4 O of the tetrahedron (2 O(2) at 1.737 Å and 2 O(1) at 1.778 Å) and a second shell including 2 Ge at 2.946 Å and 4 Cu at 3.30 Å (cf. bottom of Fig. 4). This shell description is summarized in Table I.

The EXAFS fitting procedure was tested on the pure CuGeO 3 sample. The first two atomic shells of the radial distributions at both Cu and Ge K-edges were simultaneously fitted. This implies that the distance and the Debye-Waller factor corresponding to each Ge-Cu paths were hold identical for the two sets of data during the refinement. The k range used for the FT and for the fitting procedure was [3.5, 15 Å-1 ] while the r range used for the IFT was [1, 3.4 Å] at both edges. The fits are in good agreement with the experimental spectra, as shown in Fig. 5, where the contribution of each kind of neighbors is represented. Note that, at the Cu K-edge, the signal from the two apical oxygens O( 1) is already very weak and the further O(1) neighbors were not considered because yielding a negligibly small contribution. However, the presence of the next 4 Ge atoms at about 3.786 Å was found necessary to fit correctly the second shell. The resulting structural parameters obtained at both edges are listed in Table I and the comparison with their initial values shows the consistency of the fit. After this first EXAFS analysis, the same procedure was applied to the doped samples.

Substitution on the Cu site

The Ni and Mn K-edges EXAFS oscillations were analyzed up to k = 12 Å-1 (see Fig. 3). In the case of Zn, an additional difficulty arose from the mixing of Cu K-edge and Zn K-edge EXAFS oscillations due to the closeness of the absorption energies of both elements: 8979 eV for Cu and 9659 eV for Zn. 4 . Therefore, the Zn K-edge spectrum, after correction from the contamination by the Cu K-edge oscillations, could be analyzed in a more limited k range, up to 9 Å-1 (cf. Fig. 3).

First, the structural results derived at the Cu K-edge in the doped samples were found identical to those of the pure sample, even for doping levels as high as 6% (Ni). The distortions induced by the impurities were then deduced from the comparison of the structural parameters al. [START_REF] Braden | [END_REF], who nonetheless reconfirmed the initial structure one year later [21]. 4 To correct the Zn K-edge spectrum, the Cu K-edge oscillations χ(E) of the pure sample, recorded up to 9998 eV, were converted into χ(k) using Eq.( 1) with the absorption energy E0 of Zn. The Cu EXAFS contribution, whose amplitude at the Zn edge was obtained from the ratio of both absorption jumps recorded in a unique scan in the doped sample, was then subtracted from the Zn spectrum.

k (Å -1 ) 2% Ni Filtered k 3 (k) k (Å -1 ) FT(k 3 (k)) FT(k 3 (k)) r (Å) r (Å) 6% Ni Cu K-edge Ni K-edge 6% Ni Ni K-edge Filtered k 3 (k) k (Å -1 ) 2% Ni FT(k 3 (k)) r (Å)
Fig. 6. EXAFS results for the 2% and 6% Ni-doped CuGeO3. Amplitude and imaginary part of the FT of k 3 χ(k) (right) and Fourier filtered k 3 χ(k) (left). Comparison of the measured spectra at the Ni K-edge for both doping levels and at the Cu K-edge for 6% Ni sample (top). Comparison of the measured spectra (circles) and of the fits (solid lines) at the Ni K-edge for 2% doping (middle) and 6% doping (bottom).

obtained at the impurity and at the Cu K-edge in the same sample and using the same k and r-ranges of analysis for the EXAFS oscillations. Note that we did not try to take into account impurity neighbors in our structural model (although there can be some for high doping levels) because it is most probably impossible to distinguish them from Cu neighbors in the EXAFS analysis. The FT of k 3 χ(k) and Fourier filtered k 3 χ(k) are shown in Fig. 6 for the 2% and 6% Ni-doped compounds and in Fig. 7 for the 1.5% Mn and 4% Zn-doped compounds. The spectrum recorded at the impurity K-edge in each compound is compared to its fit and to the reference spectrum at the Cu K-edge. The structural parameters extracted from the fits are reported in Table II. The fits in k and r spaces are of rather good quality. For all doping values, the amplitude of the FT, for the first O shell, is smaller at the impurity K-edge than at the Cu one, which is related Table 1. Structural parameters of the atomic radial distribution up to 3.95 Åcentered on Cu and Ge atoms, from the crystallographic data of Braden et al. [START_REF] Braden | [END_REF] and from the EXAFS simulations of the pure CuGeO3 sample at both Cu and Ge K-edges. The atom labels are those of Fig. 4. Crystallographic data of Braden et al. [START_REF] Braden | [END_REF] imply two slightly different Ge-O distances for the two pairs of oxygens within the tetrahedron. The parameters reported with no error bar were held fixed during the fit. The error bars only account for the statistical uncertainties and are therefore probably underestimated. The pair distances r and Debye-Waller factors σ 2 are in Å and Å2 respectively. The quality of the EXAFS fit is given by the R-factor: Rf =0.002 at the Cu K-edge and Rf =0.008 at the Ge K-edge. The refined S 2 0 values are 0.927 at the Cu K-edge and 0.953 at the Ge K-edge.

Cu center

Ref. [ )

Impurity K-edge Cu K-edge Filtered k 3 χ(k) 4% Zn k (Å -1 ) FT(k 3 χ(k)) r (Å) FT(k 3 χ(k)) r (Å)
Fig. 7. EXAFS results for 1.5% Mn (top) and 4% Zn (bottom) doped CuGeO3 : amplitude and imaginary parts of the FT of k 3 χ(k) (right) and Fourier filtered k 3 χ(k) (left) of the measured data (full circles) and their fits (solid lines) at the impurity K-edge. The Cu K-edge spectra measured on the same sample are also shown (dashed lines), for comparison.

to an increase of the Debye-Waller factor, in agreement with a doping-induced structural disorder. An increased Debye-Waller factor is also observed for the second shell in the case of the 6% Ni and 1.5% Mn-doped compound. Note that the 4% Zn-doped compound shells are less welldefined because of the smaller k range of integration. For all compounds, the first shell is shifted towards larger r values. The distance from the absorber to the O(2) first neighbors increases more and more when replacing Cu by Ni (∆r/r = 1.4% and 2.2% for 2 and 6% doping resp.), Zn (∆r/r = 4.5%) and Mn (∆r/r = 7.3%). Interestingly, the structural distortion varies also for a same impurity as a function of its doping level, as shown by the increase of the Debye-Waller factor and first Ni-O(2) distance from 2% to 6% Ni. For Ni and Zn impurities, there is an ambiguity concerning the position of the next nearest neighbors, the apical oxygens O(1). They are found either closer or further away from the impurity than from Cu (see parameters of the equally good EXAFS fits in Table II). At the Mn K-edge, there is no such ambiguity since the EXAFS spectra is 10 times better fitted when the O(1) atoms are closer from the impurity than further away, as compared to the Cu environment. Lastly, there is a clear variation of the distances to the next Cu and Ge neighbors with respect to the Cu environment (1.6% and 1% increase, respectively) in the case of the Mn impurity only.

Substitution on the Ge site

The EXAFS oscillations, recorded at the Si K-edge in a 2% Si-doped sample, are limited to 8.7 Å-1 and allow getting information only about the first O tetrahedral shell. Note that, due to the small range of fitting, the Debye-Waller factor could not be accurately refined and was fixed to 0.002 Å2 , i.e. to the value found in the pure sample at the Ge K-edge. This may be one reason for the small discrepancies (R-factor value of 0.011) observed between the fit and the experimental data (Fig. 8). The results, obtained using S 2 0 =0.81 in the fitting procedure, indicate a strong contraction of the Si-O distance of about 8.4% with respect to the Ge-O distance. In addition, a more symmetric environment is found around the Si than around the Ge atoms, that can be described by a small regular O tetrahedron with distance from center to corner of 1.61(2) Å (instead of 1.74 and 1.78 Å for the two pairs of oxygen forming the distorted tetrahedron surrounding the Ge atoms).

Table 2. Structural parameters obtained from the simulations of the EXAFS spectra at the impurities K-edges of the Cusite doped CuGeO3 samples: R-factors (quality of the fit), relative distance variations ∆r/r and Debye-Waller factors σ 2 . The relative distance variations induced by impurities are calculated with respect to the results obtained for the Cu K-edge spectrum of the same sample analyzed using the same k and r ranges (see Table 1). For Ni and Zn impurities, two roughly equivalent solutions of the EXAFS fits are reported with a different distance from the impurity to the apical oxygens O(1), while only one solution is relevant for Mn. The results given in bold characters correspond to the solutions validated by the analysis presented in Sec. 4.1. Note that the parameters presented with no error bar were held fixed during the fit and equal to the Cu K-edge refined value in the same sample. The S 2 0 factors at all the impurities K-edges were fixed to the value of 0.927 obtained at the Cu K-edge. 4 Analysis

2%

Comparison with ionic radii

The ionic radius for each chemical species, which depends mainly on its oxydation and spin states, on its coordination number (the radius increases with the number of neighbors) and on the polyhedral description of its environment, can be roughly estimated as a function of these sole criteria and independently of the detailed structure [22]. It is therefore interesting to compare the results of the present experiment concerning the structural distor-tion of the first shell around the impurities with the pair distances evaluated from these ionic radii (tabulated in Table I of Ref. [22]). This qualitative analysis allows checking the validity of the EXAFS analysis and lifting some remaining ambiguities.

In practice, the M -O pair distance (M denoting the absorbing ion on the Cu or Ge site) is evaluated by summing the ionic radii of M and of its O 2-first neighbors for comparison with the distances obtained from EXAFS. The results are summarized in Table III. The simplest case concerns the Ge site in which Ge 4+ and Si 4+ ions are surrounded by a slightly distorted and a regular tetrahedron, respectively. The calculations of the Ge-O and Si-O distances determined from EXAFS are in excellent agreement with the distances derived from the ionic radii [22].

The case of the Cu site is more complex. The Cu 2+ is surrounded by an elongated octahedron made of a distorted basal square of 4 O(2) and 2 apical O(1) at a much larger distance. The calculated Cu-O distance from the ionic radii, in the hypothesis of square planar configuration, yields a very good agreement with the measured Cu-O(2) distance indicating that the orbital configuration of the Cu is mainly sensitive to the 4 closer O(2). According to the EXAFS results, there are two indistinguishable solutions for the geometry of the oxygen octahedra around Ni 2+ and Zn 2+ impurities on the Cu site. The apical O(1) would get closer to the impurity or further away as compared to the Cu environment, corresponding to an environment closer to a regular octahedron or closer to a square planar environment, respectively. Note however that a square planar configuration exists only for Cu and Ni. Concerning the Ni, the only way to reproduce the observed increase of the Ni-O(2) distance, as compared to the Cu one, is an orbital configuration compatible with an O octahedron around the Ni and excludes a square Table 3. Distances to the first O neighbors, in Å, calculated from the ionic radii ( a , from Ref. [22]) in octahedral and in square planar environments (when possible) for the Cu site, and in tetrahedral environment for the Ge site. The distances in bold are compatible with those derived from EXAFS and reported below ( b ) with the corresponding elongation ratio of the octahedron. The 2% and 6% Ni-doping results are reported. The last line of both tables gives the superexchange α angle in degrees, estimated from these EXAFS results (see Sec. 4 planar description. This conclusion also holds for Zn, for which only an octahedral environment is expected to exist. For Mn 2+ , the EXAFS analysis was unambiguous. A Mn-O distance close to the one derived from the EXAFS analysis is indeed found when the ionic radius of Mn 2+ is evaluated assuming an octahedral environment and a high spin state (i.e. S=5/2), as inferred from the fine structure splitting of the ESR spectra at low temperature [8]. Note that the M -O distances for Ni, Zn and Mn, are slightly smaller than the one derived from the ionic radii because the octahedron is distorted. Some characteristic features of elongated octahedral environments are also observed in the X-ray Absorption Near Edge Structure spectra measured at the Cu K-edge in the pure compound and at the Cu-site impurities K-edge in the doped compounds, in agreement with the present analysis. These considerations allow to validate the EXAFS solutions for the M -O distances and to reproduce qualitatively their increase when substituting Cu by Ni, Zn and Mn, in this order. For the non-magnetic Mg impurity, extensively studied but for which no EXAFS measurements could be done, the following prediction can be made based on the same ionic radii analysis: the distortions around Mg are expected to be intermediate between those induced by Ni and Zn. Note that the distortion reduction of the oxygen octahedron, when replacing Cu by the other impurities, could be expected since the axial elongation in the Cu 2+ case is related to a strong Jahn-Teller effect, usual for this ion. Its substitution with non-magnetic or non Jahn-Teller 3d ions, like Ni 2+ or high spin Mn 2+ , should reduce this effect. The remaining octahedral distortion is then only induced by the structural environment.

To summarize, i-the Cu environment is closer to a square planar one (r Cu-O(1) /r Cu-O(2) ≃ 1.4) while substituting this site yields an environment closer to a regular octahedron (r M-O(1) /r M-O(2) ≃1.14-1.18 for M = Ni, Zn, Mn) with the first distance increasing with respect to Cu in the following order: M = Ni, Mg, Zn, Mn, ii-Si-doping on the Ge site induces a contraction of its surrounding oxygen tetrahedron, as strong as the pushing away of the O(2) first neighbors around the Mn impurity.

Superexchange angle calculation

The distance variations in the impurities environment determined from the present experiment can now be used to calculate a structural parameter relevant for the magnetic properties: the superexchange angle α, connecting 2 adjacent Cu sites through the O(2) atom. In the case of doping on the Cu site, this angle α = M -O(2)-Cu will be modified within one chain on both sides of the impurity M , while in the case of doping on the Ge site, this angle α = Cu-O(2)-Cu involves the O(2) connected to the Si impurity and will thus be altered on two adjacent chains along the b direction (see Fig. 4).

Concerning the Cu-site impurities M = Zn, Ni and Mn, two hypothesis for the direction of displacement of their nearest neighbors can be made in order to estimate α from the measured r M-O(2) and r M-Cu distances. Note that the distance r M-Cu is altered only in the case of Mn doping. In the first hypothesis, the O(2) are pushed away from the impurity in the same direction than the initial Cu-O(2) one (cf. Fig. 9(a), arrow (1)). This also produces a slight modification of the distance between the O(2) and the first Cu neighbor of the impurity. In the second hypothesis, this Cu-O(2) distance is held identical to that in the pure sample, provided a small modification of the M -O(2) direction is made (cf. Fig. 9(a), arrow ( 2)). This assumption is suggested by the rigidity of the Cu-O(2) bond whose length is found unchanged in the otherwise largely distorted CuSiO 3 compound [23,24]. The calculated α values with both hypothesis are 98-97.7 • for 2% Ni, 97.6-97.2 • for 6% Ni, 96.1-95.5 • for 4% Zn, and 95.7-96 • for 1.5% Mn respectively, yielding the average values listed in Table III. The angle decreases for all impurities on the Cu site, with respect to their value in the pure compound (99 • ), but this effect is smaller in Ni-doped compounds than in the Zn and Mn-doped compounds.

In the case of substitution by Si on the Ge site, a complication arises from the fact that the whole geometry is not planar i.e. the β angle between the Cu-O(2) ribbons and the Ge-O(2) segment is equal to 159.5 • (cf. Fig. 9(b)). A simple assumption for the Si-induced distortion is a contraction of the O tetrahedron around the impurity without change of bond direction with respect to the Ge environ-

(a) (b) Ge/Si O(2) α β Cu α Cu Cu/M Cu O(2) (1)
(2) ment. In this case, the O(2) atom is pulled towards the Si atom by 0.13 Å (arrow in Fig. 9(b)). This leads to an increase of the β angle (161.7 • ) and a decrease of the α angle down to 94.1 • . Note that a different variation of β towards larger (resp. smaller) values would increase (resp. decrease) the value of α (estimated variation within ± 1 • ). A possible contraction of the Cu-Cu distance was not taken into account in this simple estimation of α, while it is suggested from the study of Si-doped polycrystals by X-ray diffraction (variation of the c parameter) [25]. This would decrease even more the Cu-O(2)-Cu angle. In conclusion, the reduction of the α angle should be even stronger for Si than for Zn and Mn-doping.

Discussion

State of the art

The next step is to relate the EXAFS results, and especially the above determination of the superexchange angle, to the magnetic properties of doped CuGeO 3 . Let us recall that there is a universal decrease of T SP versus the doping concentration with y ≈ 3x in (Cu 1-y M y )(Ge 1-x Si x )O 3 , whereas the lower temperature onset of an AF LRO occurs at different T N values depending on the impurity [5].

Two observations have stimulated a lot of theoretical work [26,27,28,29,30,31]: the coexistence of both AF and SP LRO for small Si, Zn, Ni and Mn doping, as shown by neutron scattering studies [32,33,4,34], and the apparent absence of critical concentration of impurities for the onset of this AF LRO [35]. From the different models that have been proposed, a generally admitted description of the magnetic excitation of these dimerized chains emerges in terms of topological defects connecting regions of opposite dimerization parity. These defects, that can be introduced through doping, are then constituted by a soliton, pinned to the Cu substituent, followed on the same chain by an antisoliton which restores the interchain phasing.

Although mobile in the isolated chains, the antisoliton remains confined close to the impurity site when interchain couplings are accounted for in order to minimize the size of the defective segment which presents an outof-phase dimerization with respect to the 3D pattern [36,28,30,31]. Such a description of the solitonic defects, in terms of soliton-antisoliton pairs carrying an uncompensated spin 1/2, leads to large AF fluctuations coexisting with underlying reduced dimerization. In pure samples, the spin-Peierls phase is established when the critical size and phase coherence of the dimerized domains is reached, allowing the system to collapse into a long-range ordered state. This average size of coherent dimerized domains is limited by thermally activated and/or doping-induced solitonic defects. This qualitatively explains the decrease of T SP with increasing impurity concentration. On the other hand, in the presence of inter-chain coupling, the onset of an AF LRO occurs when the AF correlated regions associated with these doping-induced soliton-antisoliton pairs overlap.

The magnetic behavior under doping is thus qualitatively explained for non-magnetic impurities within the Cu chains. They simply cut one dimer, thus releasing one spin 1/2 solitonic defect confined close to the impurity. However, this is less obvious in the case of magnetic impurities on the Cu site, which may still be magnetically coupled to the neighbor Cu spins, and even less for Si impurities that are introduced outside the Cu chains. The nature of the released soliton-antisoliton pair through doping is a key feature in the understanding of the establishment of the AF LRO. Its nature will crucially depend on the strength of the local magnetic interaction. It is therefore necessary to relate the structural parameters determined in the vicinity of the impurity in the previous sections with the value of the Cu-M exchange interaction. The variation of the in-chain nearest neighbor constant, J 1 , with the Cu-O(2)-Cu superexchange angle α was inferred from the pressure dependence of the susceptibility in CuGeO 3 and found to be equal to: δJ 1 /(J 1 δα) ≈5.8% [37,38]. Various calculations based on the analysis of microscopic magnetoelastic coupling [39] and microscopic structural models [START_REF] Braden | [END_REF] yielded different estimations: δJ 1 /(J 1 δα) ≈ 10% and 22%, respectively. A value of δJ 1 /(J 1 δα) ranging between 10 and 20% was also found in microscopic calculations of the exchange constants carried out using different approaches: perturbation theory, exact diagonalization of Cu 2 O 2 clusters, and band calculations [38]. The general result of these studies is that α reaches its critical value, corresponding to the cancellation of J 1 , i.e. to the transition from AF to FM coupling, between 90 and 95 • . Other structural parameters can also affect J 1 like the angle β between the Cu-O(2) ribbons and the Ge-O(2) segment, or the pair distances involved in the exchange path, and more generally, all the parameters that may influence the strength of the orbital hybridization.

Antiferromagnetism vs distortions

In-chain magnetic impurities

Concerning the magnetic impurities on the Cu site, let us recall that they drive the system towards an AF LRO at a temperature T N different from that induced by nonmagnetic Cu-site impurities, slightly lower for Ni and larger for Mn. From our estimation of the local α angle (Table III), the magnetic local coupling J loc 1 between Ni 2+ (spin 1) and its Cu 2+ neighbors in Ni-doped CuGeO 3 is expected to be still AF but smaller than the coupling between Cu neighbors (J 1 = 120 -180K). Both an analysis of the thermal dependence of the magnetic susceptibility [5] and a resonance model for ESR results [13] have indeed evidenced the formation of Ni-Cu pairs with an AF coupling J loc 1 weaker than J 1 (most probable value of J loc 1 estimated to be around 75 K [13]). The spin 1 of the Ni impurity and the released spin 1/2 of the Cu therefore form an effective spin 1/2 solitonic defect at low temperature.

In the case of Mn-doping, the α value for the Mn-O(2)-Cu path (Table III) suggests that J loc 1 , although still AF, becomes very weak, which could be reinforced by the frustrating J 2 next nearest neighbor interaction. Here again, some ESR measurements [8] support this result with the observation of an hyperfine spectrum of the 55 Mn (nuclear spin I=5/2) with fully developed 6 lines for very low Mn concentration (0.1 %). An upper value of J loc 1 ≃4 mK can be estimated from the splitting of the hyperfine lines. The detection of quasi free 5/2 spins between T SP and T N thus demonstrates that the effective coupling between Mn 2+ and Cu 2+ is close to zero. Hence, the onset of the AF LRO is mainly due to a collective behaviour of the spins 1/2 solitonic defects, decoupled from the free 5/2 spins of the Mn impurities. It is indeed excluded for the direct Mn exchange to be responsible for the transition to AF LRO, due to the very large distances between the Mn atoms.

Therefore, the AF LRO transition observed for all the in-chain impurities presently studied seems to be mainly due to the cooperative behavior of soliton-antisoliton pairs (one released per impurity) carrying a spin 1/2, whatever the magnetic state of the impurity. Now, another parameter can be considered in order to explain the differences between the various T N (y) transition lines. The present study has revealed differences in the amplitude of the distortion around each kind of impurities related to their ionic radii. For instance, the first neighbor distances to the impurity increases with respect to the Cu environment following the sequence Ni/Mg/Zn/Mn (section 4.1). The strongest local distortion, in the case of Mn, clearly affects further neighbors (more than two Cu-Cu distances). It is interesting to note that the T N values somehow vary in the same way. The distortions induced by the impurities along the Cu chains can be very large (Table II) compared to the atomic displacements produced by the dimerization itself : 0.2% relative variation of the first Cu-Cu distance. In consequence, they will destroy locally the in-chain dimerization and will render the energetic criterion for inter-chain phase coherence ineffective. This could in turn affect the spatial distribution of the soliton-antisoliton pairs along the chain axis, i.e. their overall size or/and the mobility of the antisoliton. It would become easier for the antisoliton to accommodate its distance from the impurity and the global size of the soliton-antisoliton pair could be enhanced. As a result, this additional degree of freedom may favor the overlap of the AF correlated regions and thus increase the T N . This mechanism provides an explanation for the reported higher T N value for Mn with respect to Zn due to the larger structural distortion of its environment. On the contrary, for Ni, the distortions are the smallest implying the lowest T N , as observed. Furthermore, the AF coupling between the spin 1 Ni and the spin 1/2 Cu may pin the global spin 1/2 soliton-antisoliton pair closer to the impurity. As a result, the overlap between the AF correlations is reduced and the T N is lowered. Lastly, close enough to T N , one can not rule out an indirect polarisation of the AF correlated regions by the Mn 2+ spin (via the inter-chain interactions for instance), which could accelerate the transition towards the AF LRO.

Out-of-chain Si impurity

An unsolved issue remains concerning the strong influence of the out-of-chain Si-doping on the magnetic properties of CuGeO 3 . The present study has allowed us to determine the large local decrease of the superexchange angle α due to Ge substitution by the much smaller Si ion. α was found to be smaller than the one calculated in the case of Mndoping for which an almost vanishing J loc 1 was inferred (Table III). This strongly suggests that J loc 1 , coupling the spins of each Cu pair on both chains facing the Si impurity, is indeed ferromagnetic. This is in agreement with theoretical predictions of a weakly ferromagnetic value of J loc 1 , which was calculated using realistic structural parameters: α=95 • and Si-O bond length smaller by 0.13 Å than the Ge-O one [18,38]. The authors have invoked additional parameters enhancing this trend towards ferromagnetism: the further weakening of the hybridization of the 2p y O(2) orbitals with Si, with respect to Ge, and the influence of frustrating J 2 next nearest interaction. This result is also comforted by the structure and magnetic properties of an isostructural CuSiO 3 single crystal [23,24]. The α angle is equal to 94-95 • in this compound [23,40], i.e. close to the one determined locally around Si impurities in doped CuGeO 3 (cf. section IV.B). The analysis, from magnetometry measurements and a neutron powder diffraction experiment, of the peculiar AF LRO arising around 8 K in CuSiO 3 , suggests a weak ferromagnetic nearest neighbor exchange interaction J 1 and a stronger antiferromagnetic next nearest neighbor interaction J 2 , similar to the CuGeO 3 one [23,40].

Important consequences are expected from a ferromagnetic J loc 1 on the nature of the solitonic defects produced by the Si impurity and thus on the associated magnetic properties. The dimerization pattern imposes that the Si impurity faces one strong bond corresponding to a dimer and one weak bond between two dimers. One can reasonably assume that this local variation of J 1 will alter only the strong bond, freeing two spins 1/2, and will have no effect on the other chain (even locally reinforcing its dimerization). In this case, the two released spins 1/2 of the dimer, ferromagnetically coupled, may combine into an effective free spin 1.

The Curie constant, extracted from the temperature dependence of the susceptibility between T SP and T N , in a series of CuGe 1-x Si x O 3 samples [7], was previously analyzed according to Eq. 3 in a simple model where local spins S=1/2 are associated with the solitonic defects.

χ para (T ) = K para (g µ B ) 2 3k B (T -θ) S(S + 1) (3)
with g = 2.05, the Landé factor, µ B the Bohr magneton, k B the Boltzman constant, θ the Curie-Weiss temperature, and K para = N x with N the number of released effective spins S per impurity. Once the contribution of spins 1/2 due to intrinsic impurities has been subtracted, the number N of spins 1/2 per Si is found close to 3, a puzzling value 5 . Assuming now that the solitonic defects are ferromagnetic pairs of spin 1/2, the susceptibility of one pair is given by :

χ pair (T ) = (g µ B ) 2 k B T 2 3 + exp(-J loc 1 /k B T ) (4)
At low temperature, the expression of χ para (T ) then reduces to Eq. 3 for N such magnetic entities with an effective spin S=1. This analysis yields N ∼ 1.2, which is close enough to the expected value of one ferromagnetic pair of spins 1/2 per Si impurity in this new description. This can be further argued from the analysis of the magnetization measured in a 0.3% Si-doped CuGeO 3 sample (see Fig. 2 of Ref. [41]). The M (H) curve reaches a first saturation plateau M sat = N xgµ B S = 0.0077µ B in the SP phase before entering the incommensurate phase. The analysis of the whole M (H) curve with Brillouin functions, after correction for the intrinsic impurities contribution, yields N ∼ 1.1 spins 1 or N ∼ 2.2 spins 1/2. The first result is in better agreement with the expected number of effective spins 1 and with the previous estimation of N , deduced from susceptibility analysis. The revised analysis of the magnetic measurements in Si-doped compounds is therefore in favor of the release of one solitonic defect per Si with an effective spin 1, on one of both adjacent chains. A consequence of the effective spin 1 of the solitonic defect produced by Si will be to renormalize T N ∝ S(S + 1) by a factor of 2.67 as compared to the case of spin 1/2 solitonic defects released by the same concentration of Cusite impurities. However, this comparison only holds if the spatial extension of the solitonic defect is otherwise identical, thus producing the same overlap between AF correlated regions. The overall size of the soliton-antisoliton pair along the c-axis was indeed estimated to be the same for Si and Zn from X-ray measurements [42]. This supported the origin of the factor ∼3 between the T N of Si and of non-magnetic Cu-site impurities from the different types of the effective spin associated to the solitonic defects, at least for low doping levels (see Fig. 2). The reason why this scaling does not hold at higher x may be due to a different mechanism involved in the onset of AF LRO. The distances between impurities are becoming shorter than the 1D magnetic correlations, and moreover, a first-order transition is observed in the case of Cu-site impurities between a dimerized and a uniform AF LRO, whereas the coherence length of the spin-Peierls ordering decreases progressively in the Si doping case [43].

Spin-Peierls transition vs distortions

Contrary to the sensitivity of the AF LRO to the nature of the impurity, discussed in the previous section on the basis of the EXAFS results, the decrease of the spin-Peierls temperature appears to be more universal. On the Cu site, the decrease of T SP has exactly the same concentration dependence for all studied Cu substituents, whatever the spin impurity and the amplitude of the in-chain local distortions around impurities (see Fig. 2). For Si-doping on the Ge site, T SP decreases ∼ 3 times faster with the concentration, although the size of the solitonic defect induced by Si in the chain direction is similar to the one induced by Zn impurities [42].

Moreover, the average correlation length of the dimerized regions along c does not seem to be so relevant for the appearance of the SP LRO, as shown by the measurements of fluctuations in pure and doped CuGeO 3 for different impurities above the SP transition [44,42]. First, the regime of 1D pretransitional fluctuations along the chain direction in pure CuGeO 3 was shown to start well above T SP , while these fluctuations become 2D and then 3D much closer to T SP . Secondly, the average size of the dimerized regions along c in the pretransitional regime for Si, is comparable to that of Zn despite the factor ∼ 3 in their T SP . Last, the extension of the 1D pretransional fluctuations along c is much smaller for Ni than for Zn or Mg [42], whereas their decrease of T SP has exactly the same doping level dependence.

Therefore, all these experimental results point out that the relevant parameter for the decrease of T SP through doping is not the way the dimerization is affected along the chain direction, but perpendicular to it through the stabilization of the out-of-phase 3D dimerization pattern. This is supported by a major difference observed concerning the spatial extension of the perturbation of the dimerized regions when doping inside and outside of the Cu chains: Only one chain is affected for in-chain doping by Zn instead of three chains in the case of out-of-chain Sidoping [42]. This has provided Pouget et al. [42] with a simple model explaining the factor 3, between Si and Zn doping, in the respective decrease of T SP and in the critical concentration above which the SP LRO is destroyed. According to the present study, we can generalize this last result concerning Zn to the other Cu substituents, Ni and Mg, which produce smaller local distortions. As concerns the Mn impurity, which leads to stronger distortions than Zn, the fact that T SP has the same concentration dependence tends to show that Mn-doping affects only one Cu chain. Note that the furthest analyzed atomic distance probed by our EXAFS experiment is shorter than that to the next Cu on a neighboring chain (4.25 Å). In summary, the universality of the SP transition seems to be related to the transverse weakening of the dimerization which depends therefore essentially on the position of the impurity, outside or inside the chains.

Lastly, let us come back to the difference in the distortions induced by different concentrations of the same impurity. The Ni-O(2) distance to the first neighbors increases more for 6% Ni than for 2% Ni doping with respect to the Cu environment. The higher doped compound, also, has a more disordered environment. It is interesting to note that these concentrations lie before and after the end of the T SP line, and on each side of the transition from dimerized to uniform AF LRO. This result underlines some influence of the disorder in the disappearance of the spin-Peierls phase.

Conclusion

In the present study, the EXAFS technique was used as a powerful probe of the local atomic arrangement around various impurities in the paramagnetic state of doped CuGeO 3 . This technique has allowed us to quantify the distortions in the impurity environment, such as the modification of the shape of the O octahedron around the Cu site, and to evaluate the local modification of the angles and distances relevant for the magnetic properties. An increase of the first neighbor distance to the Cu site was observed when substituting Cu by Ni, Zn and Mn, in this order. An elongation of the distances to the further neighbors was evidenced in the case of Mn-doping which thus produces the largest alteration among the Cu site impurities. The angle α of the Cu-O-Cu superexchange path leading to the AF J 1 interaction between Cu 2+ first neighbors was found to decrease progressively when doping with Ni, then with Zn or Mn, and at last with Si. These results have allowed establishing the local weakening of the J 1 interaction for Ni/Cu substitution, its almost cancellation in the case of Mn/Cu substitution and, lastly, its change of sign for Si doping on the Ge site, leading to a small ferromagnetic coupling between each set of Cu pairs of two adjacent chains.

In a global scheme where each impurity is considered to release magnetic solitonic defects, an analysis of the structural distortions of the doped CuGeO 3 in relation with their magnetic properties could then be proposed. A good description of the magnetic behavior of Si-doped compounds is obtained calling upon ferromagnetic pairs of spins 1/2, which yields in particular a simple explanation for the renormalization of T N for Si with respect to Zn-doping. Another interesting consequence of this study concerns the Cu-site impurities for which the amplitude of the structural distortion, rather than the magnetic moment of the impurity, is found to be correlated to the variation of T N . This last result underlines the importance of the structural distortions in the magnetic behavior of spin-Peierls doped compounds and opens the way to models taking into account realistic structural parameters.

We would like to thank B. Canals, P. Monod, R. Mélin, D. Khomskii and N. Laflorencie for fruitful discussions.

Fig. 1 .

 1 Fig. 1. Schematic structure of CuGeO3

Fig. 2 .

 2 Fig. 2. (Temperature -concentration) magnetic phase diagram for CuGe1-xSixO3 and Cu1-yMyGeO3, with M =Zn, Mg, Ni, and Mn.

Fig. 3 .

 3 Fig.3. Comparison of the measured χ(k) functions (a) for pure CuGeO3 at the Cu K-edge, and for 6% Ni, 1.5% Mn and 4% Zn doped samples, at the impurity K-edges and (b) for pure CuGeO3 at the Ge K-edge and for 2% Si-doped sample at the Si K-edge.

Fig. 4 .

 4 Fig.4. Structure of CuGeO3. The Cu and Ge environments are materialized: starting from the dashed Cu and Ge absorbers, the atoms labelled "A","B", ... "F" (for Cu) and "a", "b", ... "g" (for Ge) are their neighbors going from the closest to the furthest ones that were accessible in the EXAFS experiment.

Fig. 5 .

 5 Fig. 5. EXAFS results for the pure CuGeO3 sample. Amplitude of the FT of k 3 χ(k) (left) and Fourier filtered k 3 χ(k) (right) at the Cu (top) and Ge (bottom) K-edges. The measured spectra are represented by full circles, the fit by a thick solid line. The contributions of each kind of neighbors are also shown on the FT plots (left).

Fig. 8 .

 8 Fig.8. EXAFS results for 2% Si-doped CuGeO3. Amplitude and imaginary part of the FT of k 3 χ(k) (right) and Fourier filtered k 3 χ(k) (left) at the Si K-edge of the doped sample (full circles) compared to the fit (solid lines) and to the results obtained at the Ge K-edge on the pure sample (dashed lines).

Fig. 9 .

 9 Fig. 9. Schematic view of the local deformation, due to M /Cu (a) and Si/Ge (b) substitution, assumed for the calculation of the local Cu(or M )-O(2)-Cu angle α (see text for the different scenarii).

  .2).

		Cu	Ni	Mg	Zn	Mn	Ge	Si
	planar square a	1.92	1.84
	octahedron a	2.08	2.04	2.07	2.09	2.18
	tetrahedron a						1.74	1.61
	O(2) b	1.942	1.96/1.98		2.03	2.08	1.737	1.61
	O(1) b	2.78	2.26/2.33		2.36	2.38	1.778	1.61
	r(M/Cu-O(1)) r(M/Cu-O(2))	1.43	1.15/1.18		1.16	1.14
	α angle	98.9(1)	97.9(3)		95.8(3) 95.85(15) 98.9(1) 94(1)

Note that, while for Ni, Zn and Mg, the results of Grenier et al.[5] are in rather good agreement with the work of other groups on crystals of different origin, a large spread of results exists concerning Si-doping. The slope of TSP (x) from the works of Katano et al. [9] and Hiroi et al.[10] is smaller than that found by Grenier et al. while it is similar in the work of Wang et al.[11], leading to the scaling factors y = x, y = 1.5 x and

y ∼ 2 -3 x respectively. However, we are more confident in the results of Grenier et al. concerning the Si-doping data since the weaker effect measured by other groups suggests that not all Si ions enter the CuGeO3 structure and/or part of them segregates (as was already observed in highly doped samples). This leads to overestimate x and thus results in an apparent smaller scaling factor between y and x.

Note that in Ref.[7], the values of Kpara were not corrected for the 0.11% intrinsic spin 1/2 impurities found in pure CuGeO3 and expected to be also present in doped samples.


Kevin Buffet

Iwona Nierengarten

Nicolas Galanos

Emilie Gillon

Michel Holler

Anne Imberty

Susan E Matthews

Sébastien Vidal

Stéphane P Vincent

Jean-François F Nierengarten

Pillar[5]arene-based glycoclusters: synthesis and multivalent binding to pathogenic bacterial lectins

Keywords: Pillar[5]arene, Glycocluster, Click chemistry, Lectin, Glycobiology

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Introduction

Several opportunistic pathogens involved in lung infections in immune-compromised patients have been shown to produce soluble lectins that bind to human oligosaccharide epitopes in lung tissues. [1] Among these, Pseudomonas aeruginosa and the members of the Burkholderia cepacia complex (BCC) are of special interest because of their emerging resistance to antibiotics, and the burden they represent for both cystic fibrosis patients and also patients under ventilation. [2] LecA from P. aeruginosa is a galactose-specific lectin with a tetrameric, calcium-dependent structure. [3] LecA is involved in destruction of the alveolar barrier in animal models of infection [4] and has recently been demonstrated to be necessary for the intracellular uptake of the bacterium. [5] Two soluble fucose-specific lectins have been also characterized from these bacteria. LecB from P. aeruginosa is also a calcium-dependent tetramer with strong affinity for fucose and fucose-containing oligosaccharides such as the Lewis a antigen. [6,7] LecB is a virulence factor that is involved in adhesion on airway epithelial cells. [4] BambL from Burkholderia ambifaria, a member of the BCC, is a trimer that assembles as a -propeller with six-fucose binding sites. [8] BambL binds to blood group oligosaccharides and is a close relative of AFL from, Aspergillus fumigatus, another airborne pathogen that triggers inflammation on cultured epithelial cells. [9] LecB and BambL have very different topologies (Figure 1) and therefore can be expected to bind differently to multivalent ligands. become major macrocyclic host molecules. Exploitation of the πelectron rich core has enabled applications in the preparation of sensors, rotaxanes, supramolecular polymers and vesicles. [32][33][34][35][36][37][38][39][40] Unlike their sister molecules the calixarenes and resorcinarenes, pillararenes offer a uniquely rigid and symmetrical structure with inherent planar chirality and higher levels of functionality. Thus the per-functionalised materials offer 10 or 12 derivatisation points whilst, by using non-symmetrically functionalised alkoxybenzene derivatives, platforms featuring 5 or 6 functionalised positions can also be readily accessed. Additionally a number of different methods including cocyclisation and post-cyclisation modification enable highly selective partial functionalisation. The use of pillararenes in biological applications has to-date been minimal. Hou and co-workers have developed both pillar [5]arene dimers [41] and hydrazide functionalised pillar [5]arenes [42] as transmembrane channel mimics for protons and water molecules respectively. The potential of carboxyl functionalised pillararenes as controlled release drug delivery systems has been demonstrated through the formation of parquet-pillar [6]arene complexes which afforded a reduction in toxicity of the paraquat on complexation but also pH dependent release of the guest. [43] Additionally, the use of cationic functionalised pillararenes for DNA complexation and gene delivery has recently been reported. [44] As part of this research, we have reported the synthesis of a mannose glycocluster based on a per-functionalised pillar [5]arene scaffold which was able to inhibit the adhesion of Escherichia coli to red blood cells through the inhibition of FimH-mediated bacterial adhesion. [45] Subsequently, Huang described the synthesis of pillar [5]arene derivatives featuring 5 galactose moieties and investigated their self-assembly properties into vesicles and on prolonged standing to nanotubes. These assemblies were shown to be of low toxicity to human cells and to facilitate agglutination of Escherichia coli. [46] In this study, we describe the development of a family of pillar [5]arene based glycoclusters incorporating galactose or fucose and their evaluation for binding to the lectins LecA and LecB from Pseudomonas aeruginosa and BambL from Burkholderia ambifaria.

Results and Discussion

Synthesis

The most convenient method for the conjugation of two molecular entities is probably the Cu(I)-assisted azide-alkyne cycloaddition (CuAAC). [47][48][49][50][51][52] The high yields obtained in such 1,3-dipolar cycloadditions are particularly relevant when a high number of reactions must be performed at once on a single molecular scaffold. The synthesis of the pillar [5]arene-based glycoclusters was based on already developed azido-or alkynefunctionalized pillar [5]arene scaffolds. [44,[53][54][55][56] Conjugation of deca-azide 1 [56] with a series of propargylated derivatives (2a-f) provided the acetylated glycoclusters 3a-f (Scheme 1). It is worth noting that a 1:1 mixture of diastereoisomers was obtained upon conjugation of 1 with carbohydrates owing to the chirality of the pillar [5]arene ring system. [45] For all the compounds, their separation was unsuccessful even after several careful column chromatographies. Although these isomeric mixtures could affect the interpretation of the binding studies towards lectins, the spatial arrangements of the carbohydrate epitopes for decavalent scaffolds will not differ greatly and the general presentation of the binding partners to the lectins will be statistically homogeneous. Finally, deprotection of the acetate ester moieties of 3a-f afforded the desired water-soluble glycoclusters 4a-f. While a single galactoside was used in this series, a number of different fucosides were incorporated to investigate the role of both the linker and valency in achieving selectivity and the interpretation of parameters governing the binding properties towards multimeric lectins. Thus fucosides were incorporated without linker arm (4b), with linear and flexible oligoethyleneglycol-based linker arms (4c-d) or with bifurcated oligoethyleneglycol-based linker arms (4e-f) providing up to twenty carbohydrate epitopes. Scheme 1. Synthesis of pillar [5]arene-based glycoclusters using a deca-azido core scaffold. Reagents and conditions: (a) 2a, CuI, iPr2NEt, DMF, 110°C, 15 min µwaves, 56% (3a) or 2b-f, CuSO4•5H2O, sodium ascorbate, CH2Cl2/H2O, 84% (3b), 80% (3c), 72% (3d), 77% (3e), 71% (3f); (b) MeOH, MeONa, r.t., 99% (4a), 79% (4b), 89% (4c), 85% (4d), 84% (4e), 72% (4f).

Additionally, deca-alkyne pillar [5]arene 5 [44] was conjugated with azido-functionalized galactoside 6 to afford the acetylated

O O O O O O O O O O N N N N N N N N N N N N N N N R R R R R N N N N N N N N N N N N N N N R R R R R R H 3a-f (a) O O O O O O O O O N 3 N3 N3 N 3 N3 N3 N3 N3 N 3 N3 O 1 2a-f O O R'O R'O OR' OR' O O OR' OR' R'O H3C O O OR' OR' R'O H 3 C O O O O OR' OR' R'O H3C O O O O O OR' R'O R'O H3C N N N O O O OR' OR' R'O H3C N N N O O O O OR' OR' R'O H 3 C O O O N N N O N N N O O O O OR' OR' OR' H3C O O O 3a R' = Ac 4a R' = H (b) 3b R' = Ac 4b R' = H (b) 3c R' = Ac 4c R' = H (b) 3d R' = Ac 4d R' = H (b) 3e R' = Ac 4e R' = H (b) 3f R' = Ac 4f R' = H (b)
macromolecule 7 and further deacetylation led to the desired hydroxylated glycocluster 8. The post-functionalization of decaalkyne 5 is sensitive to steric effects; all attempts to prepare a per-galactosylated derivative from 5 and 1-azido-1-deoxy--Dgalactopyranoside tetraacetate failed and only partially substituted products were obtained. The introduction of a linker unit between the reactive azide group and the acetylated galactoside moiety overcame the negative steric effects and complete functionalization could be achieved.

A series of two galactosylated and five fucosylated pillar [5]arene-based glycoclusters was thus readily synthesized from the corresponding azido-or alkynyl-functionalized macrocyclic cores. All the new compounds were fully characterized by 1 H and 13 C NMR, and IR spectroscopy. Their structures were further confirmed by mass spectrometry. The monodispersity of the acetylated glycoclusters was also evidenced by their chromatograms recorded on a HPLC equipped with a PLgel size exclusion column (see ESI).

Binding studies

In-depth determination of the binding properties of multivalent ligands towards multivalent receptors is always a complex issue due to the varied binding modes adopted between partners and the bioanalytical techniques used. In order to obtain a broad view of the multivalent interactions involved in such systems, the use of a single assay is not recommended. Thus, the designed glycoclusters were evaluated using up to four complementary techniques including a hemagglutination inhibition assay (HIA), an enzyme-linked lectin assay (ELLA), isothermal titration microcalorimetry (ITC) and surface plasmon resonance (SPR). In this latter case, the affinity was evaluated by the kinetic approach, based on determination of kinetic constants (k on and k off ), and was verified by plotting response at equilibrium against analyte concentration.

LecA Binding studies. The two galactosylated glycoclusters were evaluated as LecA ligands (Table 1). These compounds were compared to the monovalent reference -GalOMe (methyl -D-galactopyranoside).

The initial HIA study highlighted good binding of these multivalent ligands to the lectin although the increase in affinity was not large compared to other known multivalent ligands. [29] The same observation holds for the ELLA assays. A more detailed description of the thermodynamic parameter governing the binding mode was obtained by ITC (Figure 2). Table 1. HIA, ELLA ITC and SPR measurements for binding of pillar [5]arene-based multivalent glycoclusters 4a and 8 to LecA.

Ligand HIA ELLA ITC SPR MIC [a] (µM) IC50 (M)

 [b]  N [c] -H (kJ/mol) -TS (kJ/mol) KD (nM)  [b] KD th (nM) KD cin (nM) kon (M -1 .s -1 ) koff (M -1 .s - 1 )
-GalOMe 6250 658 1 0.8 [d] 39 [d] 15 [d] 70000 [d] 1 n.m. [d] Data from a previous report. [12] [e] n.m. = Not measured.

[f] RU shift was too weak due to the low molecular weight of -GalOMe. [a] Calculated as the ratio of the value obtained for the monovalent reference (methyl -L-fucopyranoside = -FucOMe) to the ligand's value.

 [a]  N [b] -H (kJ/mol) -TS (kJ/mol) KD (nM)  [a] -FucOMe 440 1 0.77±0.03 [c] 41.3±1 [c] 4.9 [c] 430±10 [c]
[b] Stoichiometry of binding defined as the number of glycoclusters per monomer of LecB.

[c] Data from a previous report. [57] Table 3. ELLA, ITC and SPR measurements for binding of pillar [5]arene-based multivalent glycoclusters 4b-f to BambL. [a] Calculated as the ratio of the value obtained for the monovalent reference (methyl -L-fucopyranoside = -FucOMe) to the ligand's value.

Ligand ELLA ITC SPR IC50 (M)  [a]  N [b] -H (kJ/mol) -TS (kJ/mol) KD (nM)  [a] KD th (nM) KD cin (nM) kon (M -1 .s -1 ) koff (M -1 .s -1 ) -FucOMe 2544 1 2.02 [c] 47.8±1.8 [c] 13.5 [c] 960±30 [c]
[b] Stoichiometry of binding defined as the number of glycoclusters per monomer of BambL.

[c] Data from a previous report. [58] [d] n.m. = Not measured.

[e] Compounds 4e and 4f could not be evaluated with the kinetic approach because of their flat dissociation curves.

Both glycoclusters displayed K D values in the sub-micromolar range. The longer linker arms gave slightly better binding when comparing the decavalent glycoclusters 4a and 8. The stoichiometry (N) for the complex generated between the lectin and the glycoclusters (N = 0.16 and 0.20 for 4a and 8 respectively) indicates that only five LecA tetramers are effectively interacting with glycoclusters 4a and 8. SPR experiments using LecA bound to the chip resulted in typical curves when circulating the multivalent ligand, with a fast association phase and slow dissociation, especially for high valency ligands (see ESI). Nevertheless, the dissociation constant obtained from the kinetic data (K D cin) is in the same range as the one obtained at equilibrium (K D th). The obtained affinities are more accurate than those obtained with ITC. SPR appears to be a more suitable method in this particular case, which may be due to the immobilization of the lectin on the chips avoiding the formation of aggregates, a common problem for solution experiments.

LecB Binding studies.

The fucosylated glycoclusters 4b-f were evaluated as potential LecB ligands (Table 2). Unfortunately, SPR experiments could not be performed due to alteration of the lectin's binding properties when covalently immobilized on the sensor chip surface or to high aggregation when the lectin was used in solution. Therefore, the binding properties of 4b-f were by ELLA and ITC which could be performed with concentration low enough to avoid strong aggregation. ELLA experiments revealed that the decavalent ligands displayed nanomolar IC 50 values with moderate improvement ( values) in comparison to -FucOMe (methyl -L-fucopyranoside) used as a reference monovalent ligand. However, increasing the length of the linker arm improved the binding to LecB in the decavalent glycocluster series going from 4b to 4c and then 4d. Increasing both the linker arm and valency in the icosavalent series gave the best ligand 4e however increasing the length of the spacer further 4f proved detrimental. This tendency was not observed in the ITC measurements which gave micromolar K D values to LecB that were not a significant improvement compared to the monosaccharide ligand methyl -L-fucopyranoside. Compound 4b appears even as a weaker ligand, presumably the short spacer preventing the fucose to be located in the most efficient way in the binding site. As a typical example, the ITC data obtained from ligand 4e are shown in Figure 3. The stoichiometry (N) of the lectin-glycocluster complexes varied from seven or eight LecB monomers bound to the decavalent ligand 4c-d (N = 0.13) to a maximum of ten for the icosavalent glycoclusters 4e-f (N = 0.09). The poor performance of the clusters towards LecB in solution may be explained by the topology of the lectin which presents its four binding sites far from each other and with different orientations at the tips of a tetrahedron. The multivalent ligands could therefore only induce aggregation, with no effective clustering of neighbouring binding sites on the lectin surface. BambL Binding studies. The binding properties towards BambL were then investigated for the six fucosylated glycoclusters (Table 3). ELLA experiments provided spectacular results and a  value of nearly 36 million was observed for the icosavalent glycocluster 4f with the longest linker arm composed of two triethyleneglycols. The IC 50 value was therefore in the picomolar range (IC 50 = 70 pM), which is the best result for a ligand reported to date for this lectin. The affinity was also highly improved for the shorter icosavalent ligand 4e ( ~ 300 000) reaching a nanomolar IC 50 value. Further investigations by ITC moderated the ELLA observations since the  values were below 60 for these fucosylated glycoclusters. The best ELLA ligands 4e and 4f were shown to have a K D value around 20 nM and an interaction with high enthalpic and entropic contributions. The very strong ELLA activity of 4f was not comparable with its affinity in solution, and was most likely due to very strong aggregative properties. Indeed, the ITC data in Table 3 and Figure 2 were obtained at very low concentration to obtain a reasonable stoichiometry value (N = 0.06, compatible with 20 fucose). When higher concentrations were used, very low values of N were obtained (N = 0.013) with much higher affinity (7 nM) that are more representative of a large aggregative network being formed in solution.

Similarly the affinities obtained by SPR were very high, ranging from 50 to 6 nM when measured at equilibrium (K D th). The values obtained by the kinetic approach (K D cin) were very different, but the dissociation of the compounds was very slow which introduces large errors in the estimation of k off . The two highest affinity ligands, 4e and 4f could not be evaluated with the kinetic approach because of their flat dissociation curves (see ESI).

Conclusions

The potential therapeutic applications of multivalent glycoclusters targeting bacterial adhesion lectins call for the design of core scaffolds presenting various valencies and topologies in order to understand the specificities of binding. Pillar [5]arenes are ideal candidates in this endeavor since they provide decavalent scaffolds a minimal molecular architecture leading to dense presentation of carbohydrate epitopes on their periphery. The synthesis of pillar [5]arenebased glycoclusters was readily achieved by CuAAC conjugations from the azido-and alkyne-functionalized precursors. The binding properties of the glycosylated multivalent ligands were studied for three lectins and by at least two complementary techniques in order to provide a good overview. The most important data were obtained from ITC and SPR experiments. Three lectins were selected from bacterial pathogens based on their potential therapeutic applications as anti-adhesive targets. Generally, multivalency improved the binding to lectins and the length of the spacer arm usually improved the affinity. The two galactosylated glycoclusters studied herein are strong ligands of LecA and an influence of both valency and linker arm for binding was observed. However, these ligands displayed binding properties similar or slightly poorer than previously reported glycoclusters. [29] In contrast, the fucosylated glycoclusters are amongst the most potent ligands for both LecB and Bambl. These results show clearly the potential of pillar [5]arene scaffolds for the design of bioactive multivalent nanomolecules. One important problem to solve is the stereochemistry of the pillar [5]arene core and the development of optically pure pillar [5]arene derivatives is an important challenge for future biological applications. On the other hand, the possibility to prepare rotaxanes with glycopillar [5]arene as the macrocyclic component offers the possibility of producing heteroglycoclusters targeting several lectins [59] or fluorescent derivatives for confocal microscopy studies. Therefore, the present results pave the way towards the development of a new generation of glycoclusters for various biological applications.

Experimental Section

Synthesis.

General. All reagents were used as purchased from commercial sources without further purification. Compounds 1, [56] 2a, [24] 2b-f, [60] 5 [44] and 6 [START_REF] Cecioni | Synthesis of Azido-Functionalized Carbohydrates for the Design of Glycoconjugates[END_REF] were prepared according to previously reported procedures. Solvents were distilled over CaH2 (CH2Cl2), Mg/I2 (MeOH), Na/benzophenone (THF) or purchased dry. Reactions under microwave activation were performed on a Biotage Initiator system. Thin Layer Chromatography (TLC) was carried out on aluminium sheets coated with silica gel 60 F254 (Merck). TLC plates were inspected by UV light (λ = 254 nm, 365 nm) and developed by treatment with a mixture of 10% H2SO4 in EtOH/H2O (95:5 v/v) followed by heating. Silica gel 60 (230-400 mesh, 0.040-0.063 mm) for column chromatography was purchased from E. Merck. Optical rotation was measured using a Perkin Elmer polarimeter. NMR spectra were recorded on a Bruker AC 300 or AC 400 spectrometer with solvent peaks as reference. The following abbreviations were used to define the multiplicities: s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet, and their combinations. Complete signal assignments were based on 1D and 2D NMR correlations COSY and HSQC. High resolution (HR-ESI-QToF) mass spectra were recorded using a Bruker MicroTOF-Q II XL spectrometer and MALDI-TOF-LD+ were recorded using a Waters QTOF1 spectrometer. Infrared spectra were recorded on a Perkin-Elmer Spectrum One spectrometer. Elemental analyses were performed by the analytical service of the Chemistry Department of the University of Strasbourg (France).

Compound 3c.

As described for 3b, this compound was prepared from 1 (117 mg, 0.09 mmol), 2c (412 mg, 0.99 mmol), CuSO4•5H2O (2.2 mg, 0.009 mmol) and sodium ascorbate (5.0 mg, 0.027 mmol) in CH2Cl2/H2O (1:1, 10 mL). Column chromatography (SiO2, CH2Cl2 containing 2.8% of methanol) followed by gel permeation chromatography (Biobeads SX-1, CH2Cl2) gave 3c as a 1:1 mixture of two diastereoisomers (389 mg, 80%). Colourless glassy product. IR (neat): 1740 (C=O) cm -1 . 1

Compound 3d.

As described for 3b, this compound was prepared from 1 (97 mg, 0.075 mmol), 2d (382 mg, 0.83 mmol), CuSO4•5H2O mg, 0.008 mmol) and sodium ascorbate (4.5 mg, 0.023 mmol) in CH2Cl2/H2O (1:1, 8 mL). Column chromatography (SiO2, CH2Cl2 containing 3.0% of methanol) followed by gel permeation chromatography (Biobeads SX-1, CH2Cl2) gave 3d as a 1:1 mixture of two diastereoisomers (320 mg, 72%). Colourless glassy product. IR (neat): 1740 (C=O) cm -1 . 1

Compound 3f.

As described for 3b, this compound was prepared from 1 (40 mg, 0.031 mmol), 2f (400 mg, 0.34 mmol), CuSO4•5H2O (0.8 mg, 0.003 mmol) and sodium ascorbate (2.0 mg, 0.009 mmol) in CH2Cl2/H2O (1:1, 8 mL). Column chromatography (SiO2, CH2Cl2 containing 5.5% of methanol) followed by gel permeation chromatography (Biobeads SX-1, CH2Cl2) gave 3f as a 1:1 mixture of two diastereoisomers (289 mg, 71%). Colourless solid. IR (neat): 1744 (C=O) cm -1 . 1 General procedure for the Zemplén deacetylations. MeONa (10 eq.) was added to a solution of acetylated glycocluster (1 eq.) in distilled MeOH. The mixture was stirred at r.t. for 16 h, then filtered over a short column of Dowex TM 50WX8-200 or Amberlite IR 120 (H + resin form). The resin was washed with water. The obtained filtrate was concentrated to afford the corresponding deprotected glycocluster.

Compound 4a. Obtained from 3a (150 mg, 0.029 mmol) as a pale yellow foam (100 mg, quantitatively). 1 Compound 4f. Obtained from 3f (50 mg, 3.79 µmol) as a colorless foam (31 mg, 72%). 1 H NMR (400 MHz, D2O): δ = 7.99 (s, 20H), 7.96 (s, 10H), 6.39 (s, 10H), 4.63-4.24 (m, 130H), 3.98 (q, J = 6.6 Hz, 20H), 3.81-3.51 (m, 100H), 3.36-3.30 (m, 90H), 1.12 (d, J = 6.6 Hz, 60H) ppm. 13

Hemagglutination Inhibition Assays (HIA). Determination of lectin concentration:

Hemagglutination inhibition assays (HIA) were performed in U-shaped 96-well microtitre plates. Rabbit erythrocytes were purchased from Biomérieux and used without further washing. Erythrocytes were diluted to a 4% solution in NaCl (150 mM). Lectin solutions of 2 mg/mL were prepared in TRIS-HCl 20 mM, NaCl 100 mM and CaCl2 100 μM. The hemagglutination unit (HU) was first obtained by the addition of 25 μL of the 4% erythrocyte solution to 25 μL aliquots of sequential (two-fold) lectin dilutions. The mixture was incubated at 25°C for 60 minutes. The HU was measured as the minimum lectin concentration required for hemagglutination. Lectin concentrations of 4 HU were used for the lectin inhibition assays. For LecA, this concentration was found to be 8 μg/mL. Determination of minimum inhibitory concentration (MIC): Subsequent inhibition assays were then carried out by the addition of 12.5 μL lectin solution (at the required concentration) to 25 μL of sequential dilutions of glycoclusters, monomer molecules and controls. These solutions were then incubated at 25°C for 2 h then 12.5 μL of 4% erythrocyte solution was added followed by an additional incubation at 25°C for 30 minutes. The minimum inhibitory concentration (MIC) for each molecule was determined for each duplicate.

Enzyme-Linked Lectin Assays (ELLA). Determination of lectin concentration: 96-Well microtitre plates (Nunc Maxisorb) were coated with -PAA-Gal for LecA and -PAA-Fuc for BambL: 100 μL of 5 μg/mL in carbonate buffer, pH 9.6 for 1h at 37°C then blocking at 37°C for 1h with 100 μL per well of 3% (w/v) BSA in PBS. Lectin solution (100 μL) were diluted (1/2) starting from 30 μg/mL. After 1h incubation at 37°C and 3 washings with T-PBS (PBS containing 0,05% Tween 20), 100 μL of horseradish streptavidin-peroxidase (HRP) conjugate (dilution 2:5000; Boehringer-Mannheim) was added and left for 1h at 37°C. Colouration was developed using 100 μL per well of 0.05 M phosphate/citrate buffer containing o-phenylenediamine dihydrochloride (0.4 mg/mL) and urea hydrogen peroxide (0.4 mg/mL) (OPD kit, Sigma-Aldrich) for 15 minutes and stopped with 50 μL of 30% sulfuric acid. Absorbance at 490 nm was then read using a microtiter plate reader (BioRad 680). Biotinylated lectins concentration were determined by plotting the relative absorbance versus lectin concentration. The concentration which leads to the highest response in the linear area was selected as the standard lectin concentration for the subsequent inhibition experiments. Determination of inhibition potency (IC50): ELLAs were conducted in the same conditions as above. Inhibitor solutions (50 μL) were submitted to serial dilutions (1/3) with PBS-BSA 0.3% (w/v). Then, 50 μL of biotinylated lectin solution (0.12 μg/mL for LecA and 3 ng/mL for BambL) were added in each well and the plates were incubated for 1h at 37°C. Plots of inhibition percentage versus inhibitor concentration and sigmoidal fitting provided IC50 determination.

Surface plasmon resonance (BambL). SPR inhibition experiments

were performed on a Biacore X100 instrument (GE Healthcare) at 25°C in HBS buffer (10 mM Hepes/NaOH, pH 7.5, 150 mM NaCl, 0.05% Tween 20) at a flow rate of 10 mL.min -1 . Streptavidin was immobilized on a research grade CM5 chip using standard procedures, and 200 resonance units of biotinylated polyacrylamide--L-fucopyranoside probe (Lectinity, 200 mg.mL -1 ) were captured on channel 2. Inhibition experiments were performed with the fucosylated channel 2, and plots represent the subtracted data (channel 2-channel 1). Inhibition studies consisted of the injection (association 180 s, dissociation 180 s) of incubated (30 min at room temperature) mixtures of BambL (0.2 mM) and various concentrations of inhibitor (3-fold cascade dilutions). For each inhibition assay, BambL was injected to observe the full adhesion of the lectin onto the sugar-coated surface (0% inhibition). The chip was fully regenerated by two successive injections of L-fucose (120 s, 1 M in running buffer). Binding was measured as resonance units over time after blank subtraction, and data were then evaluated by using the Biacore X100 evaluation software, version 2.0. For IC50 determination, the response was considered as the amount of lectin bound to the sugar surface at the end of injection. Inhibition curves were obtained by plotting the percentage of inhibition against the inhibitor concentration (logarithmic scale). Surface plasmon resonance. The studies were conducted using a Biacore X100 instrument (GE Healthcare) at 25°C with a CM5 sensor chip. A continuous flow of HEPES buffer (10 mM HEPES, 150 mM NaCl, 100 µM CaCl2, twin 20 0.05 %, pH 7.5) was maintained over the sensor surface at a flow rate of 10 μL.min -1 . The CM5 sensor chip was activated with an injection of a solution containing N-ethyl-N'-(3-diethylaminopropyl)carbodiimide (EDC) (0.2 M) and N-hydroxysuccinimide (NHS) (0.05 M) for 7 minutes. LecA (100 μg.mL -1 ) in NaOAc buffer (pH 4.2) was injected over the activated flow cell at flow rate of 10 μL.min -1 for 10 minutes to achieve a ~1000 RU. The same procedure was applied for BamblL achieving 200 RU capture The immobilization procedure was completed by an injection of ethanolamine hydrochloride (1 M) (70 μL), followed by a flow of the buffer (100 μL.min -1 ) in order to eliminate physically adsorbed compounds. Ethanol amine alone was used in channel 1 as a reference. For the LecA chip, Compounds 4a and 8 were injected using the single-cycle mode with 5 concentrations varying from 0.5 nm to 5 µM concentration. By the end of measures the sensor chip was regenerated with 100 seconds pulse of methyl β-Dgalactopyranoside (100 mM) followed by an injection of running buffer for 300 seconds.

Microcalorimetry. Recombinant lyophilized lectin was dissolved in buffer (LecA: 20 mM TRIS-HCl, 100 µM CaCl2, pH 7.5, NaCl 100 mM; LecB: 20 mM TRIS-HCl, 100 µM CaCl2, pH 7.5, NaCl 100 mM; BambL: 20 mM TRIS-HCl, pH 7.5, NaCl 150 mM) and degassed. The protein concentration was checked by measuring A280 by using a theoretical molar extinction coefficient (LecA: 28000, LecB: 6990, BambL: 40450). Carbohydrate ligands were dissolved in the same buffer, degassed, and loaded in the injection syringe. ITC was performed with a VP-ITC microcalorimeter (Microcal, GE Healthcare). The lectin solution was placed in 1.4478-mL sample cell at 25°C. Titration was performed with 10 µL injections of carbohydrate ligands every 400 s. Data were fitted with MicroCal Origin 7 software according to standard procedures. The fitted data yielded the stoichiometry (N), association constant (Ka), and enthalpy of binding (H). Other thermodynamic parameters (i.e. changes in free energy (G) and entropy (S)) were calculated from the equation G = H -TS = -RTlnKa, in which T is the absolute temperature and R = 8.314 J.mol -1 .K -1 . Two independent titrations were performed for each ligand tested.

Figure 1 .

 1 Figure 1. Crystal structures of three soluble bacterial lectins with peptide chains represented by ribbons with different colours, carbohydrate ligands represented as sticks, and calcium ions represented by green spheres. The following complexes are displayed from left to right: LecA/galactose (PDB code 1OKO), LecB/fucose (1GZT) and BambL/fucose (3ZZV).

Scheme 2 .

 2 Scheme 2. Synthesis of a galactosylated decavalent pillar[5]arene-based glycocluster using a deca-propargylated core scaffold. Reagents and conditions: (a) 1-azido-3,6-dioxa-octyl 2,3,4,6-tetra-O-acetyl--Dgalactopyranoside, CuSO4•5H2O, sodium ascorbate, CH2Cl2/H2O, 43%; (b) MeOH, MeONa, r.t., 16 h, >95%.

Figure 2 .

 2 Figure 2. Raw ITC data (top) obtained by injections of glycocluster 8 (190 µM) to a solution of LecA (79.5 µM) and the corresponding integrated titration curve (bottom).

  Minimum inhibitory concentration (MIC) required to inhibit the agglutination of erythrocytes. [b] Calculated as the ratio of the value obtained for the monovalent reference (methyl -D-galactopyranoside = -GalOMe) to the ligand's value. [c] Stoichiometry of binding defined as the number of glycoclusters per monomer of LecA.

Figure 3 .

 3 Figure 3. Raw ITC data (top) obtained by injections of glycocluster in a solution of lectin and the corresponding integrated titration curve (bottom).. Left : 4f (150 µM) in LecB (110 µM); right : 4f (30 µM) in BambL (30 µM)

  Compound 3a. A mixture of 1 (29 mg, 0.022 mmol), 2a (129 mg, 334 mmol), CuI (2 mg, 0.011 mmol) and DIPEA (55 L, 334 mmol) in DMF (3 mL) was heated at 110°C for 15 min under microwave irradiation. The crude mixture was concentrated and co-evaporated with toluene 6 times. Column chromatography (CH2Cl2/MeOH 99/1 to 95/5) gave 3a as a 1:1 mixture of two diastereoisomers (65 mg, 56%). Colourless glassy product. IR (neat): 1745 (C=O) cm -1 . 1 H NMR (400 MHz, CDCl3): δ = 7.80 and 7.77 (2 s, 10H), 6.56 (s, 10H), 5.34 (m, 10H), 5.14 (m, 10H), 5.02-4.85 (m, 20H), 4.81-4.56 (m, 40H), 4.30-4.00 (m, 40H), 3.98-3.83 (m, 10H), 3.25 and 3.23 (2 s, 10H), 2.11-1.85 (m, 120H) ppm. 13 C NMR (100 MHz, CDCl3): δ = 170.4, 170.2, 170.0, 169.6, 169.5, 149.5, 144.4, 144.3, 128.7, 123.4, 116.0, 100.3, 70.7, 68.7, 67.3, 67.0, 62.7, 62.6, 61.1, 61.0, 50.2, 29.6, 20.7 (2 peaks), 20.6, 20.5 ppm. HR-ESI-QTOF (positive mode): m/z 1721.5847 ([M+3H] 3+ , calcd. for C225H283N30O110: 1721.5824). Compound 3b. A mixture of 1 (95 mg, 0.073 mmol), 2b (263 mg, 0.80 mmol), CuSO4•5H2O (1.8 mg, 0.007 mmol) and sodium ascorbate (4.0 mg, 0.022 mmol) in CH2Cl2/H2O (1:1, 8 mL) was vigorously stirred at room temperature under Ar. After 72 h, H2O was added (15 mL) and the aqueous layer was extracted with CH2Cl2 (3 x). The combined organic layers were dried (MgSO4), filtered and concentrated. Column chromatography (SiO2, CH2Cl2 containing 1.9% of methanol) followed by gel permeation chromatography (Biobeads SX-1, CH2Cl2) gave 3b as a 1:1 mixture of two diastereoisomers (280 mg, 84%). Colourless glassy product. IR (neat): 1741 (C=O) cm -1 . 1 H NMR (300 MHz, CDCl3): δ = 8.07 and 7.88 (2 s, 10H), 6.60 and 6.45 (2 s, 10H), 5.30 (m, 10H), 5.20 (m, 20H), 5.12 (m, 10H), 4.74 (m, 40H), 4.31 (m, 5H), 4.15 (m, 20H), 3.98 (m, 5H), 3.36 and 3.26 (2 s, 10H), 2.16 (s, 30H), 2.01 and 1.99 (2 s, 30H), 1.97 and 1.96 (2 s, 30H), 1.08 and 0.96 (2 d, J = 6 Hz, 30H) ppm. 13 C NMR (100 MHz, CDCl3): δ = 170.8, 170.7, 170.5, 170.5, 170.1, 170 .1, 149.6, 149.5, 144.6, 144.3, 128.8, 128.6, 123.8, 123.7, 116.0, 115.7, 95.9, 95.9, 71.2, 71.2, 68.2, 68.1, 68.1, 68.0, 67.5, 67.3, 64.9, 64.8, 61.6, 61.4, 50.4, 29.4, 29.1, 20.9, 20.8, 20.8, 16.0, 15.9 ppm. ESI-TOF-MS (positive mode): m/z 1528.966 (100%, [M+3H] 3+ , calcd. for C205H263N30O90: 1528.23), 2291.937 (5%, [M+2H] 2+ , calcd. for C205H262N30O90: 2291.84).

  H NMR (400 MHz, CDCl3): δ = 7.85 (s, 10H), 6.61 (s, 10H), 5.34 (m, 10H), 5.29-5.25 (m, 10H), 5.10 (m, 20H), 4.78-4.65 (m, 40H), 4.30-4.14 (m, 30H), 3.77 (m, 10H), 3.64 (m, 60H), 3.52 (broad s, 10H), 3.25 (broad s, 10H), 2.15 (s, 30H), 2.05 (s, 30H), 1.97 (s, 30H), 1.05 (d, J = 4 Hz, 30H) ppm. 13 C NMR (100 MHz, CDCl3): δ = 170.8, 170.6, 170.2, 149.6, 145.3, 128.8, 123.3, 116.1, 96.4, 71.3, 70.7, 70.2, 69.9 (2x), 68.3, 68.2 (2x), 67.6, 64.7, 64.4, 51.0, 50.2, 29.5, 21.0, 20.9, 20.8, 15.9 ppm. MALDI-TOF-MS: m/z 5465.765 ([M] + , calcd. for C245H340N30O110: 5465.20).

  H NMR (400 MHz, CDCl3): δ = 7.84 (m, 10H), 6.59 (m, 10H), 5.35 (m, 10H), 5.29 (m, 10H), 5.12 (m, 5 H), 5.09 (m, 15H), 4.77 -4.65 (m, 40H), 4.30 -4.11 (m, 30H), 3.78 (m, 10H), 3.63 (m, 110H), 3.25 (broad s, 10H), 2.16 (s, 30H), 2.06 (s, 30H), 1.97 (s, 30H), 1.12 (d, J = 8 Hz, 30H) ppm. 13 C NMR (100 MHz, CDCl3): δ = 170.8, 170.6, 170.2, 149.6, 145.4, 128.8, 123.3, 116.1, 96.4, 71.3, 70.8, 70.7, 70.6, 70.2, 69.8, 68.3, 68.1, 67.6, 67.5, 64.6, 64.4, 50.3, 29.5, 20.9, 20.8, 20.8, 16.0 ppm. MALDI-TOF-MS: m/z 5905.73 ([M] + , calcd. for C265H380N30O120: 5905.4667). Anal. Calcd. for C265H380N30O120.2C6H12 (6074.31): C, 54.77; H, 6.71; N, 6.92. Found: C, 54.75; H, 6.41; N, 6.65. Compound 3e. As described for 3b, this compound was prepared from 1 (40 mg, 0.031 mmol), 2e (315 mg, 0.34 mmol), CuSO4•5H2O (0.8 mg, 0.003 mmol) and sodium ascorbate (2.0 mg, 0.009 mmol) in CH2Cl2/H2O (1:1, 8 mL). Column chromatography (SiO2, CH2Cl2 containing 5.0% of methanol) followed by gel permeation chromatography (Biobeads SX-1, CH2Cl2) gave 3e as a 1:1 mixture of two diastereoisomers (252 mg, 77%). Colourless glassy product. IR (neat): 1740 (C=O) cm -1 . 1 H NMR (400 MHz, CDCl3): δ = 7.97 (m, 30H), 6.61 (s, 10H), 5.30 (m, 20H), 5.25 (m, 30H), 5.15 (broad s, 20H), 5.04 (m, 20H), 4.78 (m, 30H), 4.62 (m, 40H), 4.49 (m, 20H), 4.33-4.12 (m, 70H), 3.65 (s, 20H), 3.58 (s, 40H), 3.51 (s, 20H), 3.21 (broad s, 10H), 2.13 (s 60H), 1.98 (s, 30H), 1.97 (s, 30H), 1.93 (s, 60H), 1.12 (s, 30H), 1.10 (s, 30H) ppm. 13 C NMR (100 MHz, CDCl3): δ = 170.7, 170.6, 170.6, 170.5, 170.1, 149.5, 145.0, 143.7, 143.6, 128.7, 125.4, 125.3, 123.4, 115.9, 115.9, 95.3, 95.0, 71.2, 70.7, 70.4, 70.1, 69.7, 68.2, 68.2, 67.9, 67.9, 67.5, 64.8, 64.8, 64.4, 64.4, 60.9, 60.8, 50.6, 50.6, 50.3, 50.3, 29.8, 20.9, 20.9, 20.8 (2x), 20.7 (2x), 16.0, 16.0 ppm. MALDI-TOF-MS: m/z 10547.46 ([M] + , calcd. for C455H620N90O200: 10550.13). Anal. Calcd. for C455H620N90O200.4CH2Cl2 (10890.02): C, 50.62; H, 5.81; N, 11.58. Found: C, 50.52; H, 5.60; N, 11.40.

Compound 7 .

 7 H NMR (400 MHz, CDCl3): δ = 7.76 (broad s, 30H), 6.62 (s, 10H), 5.34 (m, 10H), 5.32 (m, 10H), 5.26 (s, 30H), 5.10 and 5.09 (2 s, 10H), 5.07 and 5.04 (2 broad s, 30H), 4.75 -4.58 (m, 70H), 4.47 (m, 20H), 4.29-4.08 (m, 70H), 3.77 (m, 20H), 3.64 -3.48 (m, 300H), 3.22 (broad s, 10H), 2.14 (s 60H), 2.04 (s, 60H), 1.96 (s, 60H), 1.11 (s, 30H), 1.10 (s, 30H) ppm. 13 C NMR (100 MHz, CDCl3): δ = 170.7, 170.5, 170.1, 149.6, 145.0, 128.7, 128.7, 124.9, 123.5, 123.4, 116.0, 105.6, 96.4, 82.8, 81.8, 71.3, 70.8, 70.7, 70.6, 70.4, 70.2, 70.1, 69.8, 69.7, 68.8, 68.3, 68.1, 67.6, 66.8, 64.6, 64.4, 50.7, 50.3, 27.0, 20.9, 20.9, 20.9, 20.8, 20.8, 16.3, 16.0 ppm. MALDI-TOF-MS: m/z 13193.35 ([M+H] + , calcd. for C575H861N90O260: 13193.71). As described for 3b, this compound was prepared from 5 (20 mg, 0.020 mmol), 6 (133 mg, 0.263 mmol), CuSO4•5H2O (25 mg, 0.100 mmol) and sodium ascorbate (40 mg, 0.200 mmol,) in CH2Cl2/H2O (1:1, 4 mL). Column chromatography (SiO2, CH2Cl2 containing 9% of methanol) gave 7 as a 1:1 mixture of two diastereoisomers (52 mg, 43%). Colourless glassy product. IR (neat): 1746 (C=O) cm -1 . 1 H NMR (400 MHz, CDCl3): δ = 7.90 (s, 10H), 6.90 (s, 10H), 5.35 (d, J = 2.3 Hz, 10H), 5.15 (dd, J = 9.9, 8.7 Hz, 10H), 4.99 (dd, J = 9.9, 2.3 Hz, 10H), 4.91-4.78 (m, 20H), 4.56-4.44 (m, 30H), 4.11 (m, 20H), 3.89 (m, 40H), 3.77-3.49 (m, 80H), 2.12, 2.01, 1.98 (3s, 120H) ppm. 13 C NMR (100 MHz, CDCl3): δ = 170.5, 170.4, 170.2, 169.6, 149.8, 144.3, 128.6, 124.2, 115.5, 101.4, 71.0, 70.7, 70.6, 70.5, 70.2, 69.5, 69.3, 69.2, 68.9, 67.2, 62.3, 61.3, 50.2, 29.6, 20.9, 20.8, 20.7 ppm. HR-ESI-QTOF (positive mode): m/z 3023.6128 (8%, [M+2H] 2+ , calcd. for C265H362N30O130: 3023.6365), 2015.0849 (89%, [M+3H] 3+ , calcd. for C265H363N30O130: 2015.0900), 1512.3273 (100%, [M+4H] 4+ , calcd. for C265H364N30O130: 1512.3222).

  H NMR (500 MHz, DMSO-d6 + D2O): δ = 8.31 (s, 10H), 6.73 and 6.68 (2s, 10H), 5.05-4.74 (m, 30H), 4.72-4.56 (m, 10H), 4.52-4.37 (m, 10H), 4.30-4.19 (m, 10H), 4.12 (bs, 10H), 3.66-3.60 (m, 10H), 3.57-3.50 (m, 20H), 3.45-3.22 (m, 30H), 3.21-3.08 (m, 10H) ppm. 13 C NMR (125 MHz, DMSO-d6 + D2O): δ = 148.8, 144.2, 144.0, 128.5, 124.3, 114.9, 102.8, 102.6, 75.4, 73.4, 70.6, 68.3, 67.6, 61.33, 60.6, 49.9, 28.3 ppm. HR-ESI-QTof (positive mode): m/z 1183.4278 ([M+3Na] 3+ , calcd. for C145H200N30Na3O70: 1183.4230). Compound 4b. Obtained from 3b (50 mg, 0.01 mmol) as a colorless foam (26 mg, 79%). 1 H NMR (400 MHz, D2O): δ = 8.13 and 8.05 (2 s, 10H), 6.30 and 6.26 (2 s, 10H), 4.94 (d, J = 3.4 Hz, 10H), 4.74-4.50 (m, 40H), 3.99 (s, 10H), 3.74-3.70 (m, 20H), 3.62 -3.43 (m, 30H), 3.25-3.18 (m, 10H), 0.94 and 0.90 (2 d, J = 6.2 Hz, 30H) ppm. 13 C NMR (100 MHz, D2O): δ = 149.7, 144.4, 144.2, 129.2, 129.0, 125.2, 115.7, 98.7, 98.5, 71.7 (2 peaks), 69.5, 68.1, 67.3, 67.2, 66.7, 60.8, 60.6, 50.0, 15.28 ppm. MALDI-TOF-MS-LD + : m/z 3344.3 ([M+Na] + , calcd. for C145H200N30O60Na: 3344.34), 3199.4 ([M-C6H11O4+Na] + , calcd. for C139H189N30O56Na: 3199.2). HR-ESI-QTof (positive mode): m/z 3344.3445 ([M+Na] + , calcd. for C145H200N30O60Na: 3344.34). Compound 4c. Obtained from 3c (50 mg, 9.14 µmol) as a colorless foam (34 mg, 89%). 1 H NMR (400 MHz, D2O): δ = 8.04 and 7.95 (2 s, 10H), 6.41 and 6.32 (2 s, 10H), 4.55-4.46 (m, 30H), 4.11-3.24 (m, 140H), 1.09 (s, 30H) ppm. 13 C NMR (100 MHz, D2O): δ = 149.9, 144.1, 129.0, 125.1, 116.0, 98.6, 71.8, 69.7, 69.6, 69.5, 68.8, 68.1, 67.5, 66.7, 66.5, 63.0, 50.2, 50.0, 15.4 ppm. MALDI-TOF-MS-LD + : m/z 4227.1 ([M+Na] + , calcd. for C185H280N30O80Na: 4227.4), 4081.0 [M-C6H11O4+Na] + , 4057.9 [M-C6H11O4] + , 3935.9 [M-C12H22O8+Na] + , 3910.9 [M-C12H22O8] + , 3789.8 [M-C18H33O12+Na] + , 3763.8 [M-C12H22O8] + . Compound 4d. Obtained from 3d (48 mg, 8.13 µmol) as a colorless foam (32 mg, 85%). 1 H NMR (400 MHz, D2O): δ = 8.05 (broad s, 10H), 6.37 (s, 10H), 4.54 (s, 30H), 4.04-3.48 (m, 180H), 1.10 (d, J = 6.6 Hz, 30H) ppm. 13 C NMR (100 MHz, D2O): δ = 149.8, 129.2, 125.2, 116.0, 98.6, 71.8, 69.7, 69.5, 68.9, 68.1, 67.66, 67.74, 66.6, 63.0, 50.2, 15.4 ppm. MALDI-TOF-MS-LD + : m/z 4667.3 ([M+Na] + , calcd. for C205H320N30O90Na: 4667.9), 4521.3 [M-C6H11O4+Na] + , 4373.2 [M-C12H22O8+Na] + , 4229.1 [M-C18H33O12+Na] + , 4206.1 [M-C18H33O12] + , 4084.1 [M-C24H44O16+Na] + , 4061.1 [M-C24H44O16] + , 3914.0 [M-C30H55O20] + , 3769.0 [M-C36H66O24] + , 3621.9 [M-C42H77O28] + , 3477.8 [M-C48H88O32] + , 3329.7 [M-C54H99O36+Na] + , 3178.6 [M-C60H110O40] + . Compound 4e. Obtained from 3e (56 mg, 5.31 µmol) as a colorless foam (36 mg, 84%). 1 H NMR (400 MHz, D2O): δ = 8.02 (s, 20H), 7.96 (s, 10H) 6.40 and 6.39 (2s, 10H), 4.93 (d, J = 3.0 Hz, 20H), (m, 30H), 4.02-3.69 (m, 72H), 3.55-3.28 (m, 110H), 1.06 (d, J = 6.4 Hz, 60H) ppm. 13 C NMR (100 MHz, D2O): δ = 149.8, 144.2, 144.0, 128.9, 126.0, 124.9, 115.7, 98.4, 76.7, 71.7, 69.7, 69.5, 69.4, 69.0, 67.9, 66.7, 63.1, 60.4, 50.9, 49.9, 15.3 ppm. MALDI-TOF-MS-LD + : m/z 8051.1 ([M+Na] + , calcd. for C335H500N90O140Na: 8051.1), 7904.8 ([M-C6H11O4+Na] + , 7758.7 ([M-C12H22O8+Na] + .

  C NMR (100 MHz, D2O): δ = 149.9, 144.3, 144.0, 129.0, 126.1, 124.9, 115.8, 98. 7, 76.7, 71.8, 69.8, 69.6, 69.5, 69.1, 68.1, 67.4, 66.8, 66.6, 63.0, 51.0, 49.9, 15.4 ppm. MALDI-TOF-MS-LD + : m/z 10693.8 ([M+Na] + , calcd. for C455H740N90O200Na: 10694.1), 10546.6 ([M-C6H11O4+Na] + , calcd. for C449H729N90O196Na: 10546.0). Compound 8. Obtained from 7 (35 mg, 0.006 mmol) as a pale yellow foam (25 mg, quantitatively). 1 H NMR (400 MHz, DMSO-d6 + D2O): δ = 8.24 (s, 10H), 6.97 (s, 10H), 5.06 (d, J = 10.0 Hz, 10H), 4.77 (d, J = 10.0 Hz, 10H), 4.57-4.38 (m, 20H), 4.08 (d, J = 6.7 Hz, 10H), 3.86-3.74 (m, 30H), 3.63 (bs, 10H), 3.58-3.42 (m, 100H), 3.42-3.21 (m, 30H) ppm. 13 C NMR (100 MHz, DMSO-d6 + D2O): δ = 148.8, 143.2, 128.1, 124.5, 114.4, 103.6, 75.2, 73.5, 70.5, 69.72, 69.67, 69.5, 68.8, 68.1, 67.7, 61.5, 60.4, 49.4, 28.8 ppm. HR-ESI-QTof (positive mode): m/z 2204.9186 (8%, [M+2Na] 2+ , calcd. for C185H280N30Na2O90: 2204.9055), 1476.9385 (100%, [M+3Na] 3+ , calcd. for C185H280N30Na3O90: 1476.9311).

Table 2 .

 2 ELLA

	Ligand	ELLA	ITC
		IC50
		(nM)

and ITC measurements for binding of pillar

[5]

arene-based multivalent glycoclusters 4b-f to LecB.

Acknowledgements

The authors thank the Université Claude Bernard Lyon 1, the Université de Strasbourg, the International Center for Frontier Research in Chemistry (icFRC) and the CNRS for financial support. This project was also supported by COST Action CM-1102 MultiGlycoNano and the Agence Nationale de la Recherche (ANR, Programme Blanc 2011, Sweet60s). A.I. acknowledges support from GDR Pseudomonas and Labex ARCANE (ANR-11-LABX-003). Dr F. Albrieux, C. Duchamp, N. Henriques and J.-M. Strub are gratefully acknowledged for mass spectrometry analyses. N.G. thanks the Région Rhône-Alpes (Cluster de Recherche Chimie) for additional funding and the Royal Society for funding his internship undertaken in S.E.M.'s laboratory (JP090449).

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