Urminder Kaur

Koushik Saha

Subhash Bairagi

Abhishek Das

Thierry Roisnel

Tapan Kanti Paine

Sundargopal Ghosh
email: sghosh@iitm.ac.in

Structural and electronic analysis of bimetallic thiolate complexes of group-5 transition metal ions

Keywords: Thiolate, Tantalum, Niobium, Vanadium, Paramagnetic

 A series of binuclear thiolate complexes of group-5 metals have been synthesised and the role of metal and associated thiolate ligands influencing the structural and electronic properties of these complexes has been described.

 The experimental results have been complemented and rationalized through DFT studies.

Introduction

The chemistry of early transition metal complexes incorporating thiolate ligands has received considerable attention over the years for several reasons. The close relevance of these transition metal thiolates to certain biological systems and industrial catalysts, 1,2 their potentiality in the chemistry related to S-C bond cleavage and desulfurization, 3 their efficacy in the synthesis of metal sulfide as well as organosulfur materials by MOCVD processes, 4a are some of the motivating facts which led to the development of their chemistry. The increasing interest in these complexes stems from their resemblance with the metalloproteins such as metallothioneins or nitrogenase. 2 Moreover, the long-known polymetallic thiolate clusters act as synthetic models for biologically active sites, whereas polyoxometalates have shown promising biological and biochemical effects recently with their antiviral, antitumoral, and antibiotic properties. 4b Although the diamagnetic transition metal thiolates are an enormously rich class of compounds, the chemistry of paramagnetic metal thiolates appears to be relatively less explored and thus becomes an exciting and active research field. The isolation of first homoleptic paramagnetic group-5 thiolate complex [V 2 (SCH 2 CH 2 S) 4 ] in 1983 spurred the chemistry of transition metal thiolates exhibiting paramagnetic behaviour and became the focus for many inorganic and organometallic chemists. 5 The detailed investigation on magnetic as well as the structural properties of these paramagnetic complexes is important for understanding the nature of exchange interactions between the paramagnetic metal ions. In particular, the contribution from direct exchange through M-M bonds and from super exchange via bridged ligands can be estimated. Therefore, a thorough investigation in this matter becomes of interest.

Our recent studies are focused on the syntheses of transition metal complexes containing metal centres bridged by thiolate/dithiolate ligands. [6][7][8][9] In our previous report, we have isolated and structurally characterised a classical [B 2 H 5 ] -ion, [(Cp*Ta) 2 (μ-S 2 CH 2 -κ 2 S:κ 2 S') 2 (μ-H)(μ,η 2 :η 2 -B 2 H 5 )], I. 9 Later, we developed a new methodology to synthesize different thiolate complexes of group-5 metals that exhibit novel coordination modes of sulfur-containing ligands with interesting electronic properties. 8 For example, the room temperature reaction of [(CH 2 S 2 ) 4 B]Na 5 10 with [Cp*VCl 2 ] 3 generated diamagnetic as well as paramagnetic thiolate complexes. In contrast, niobium and tantalum yielded only diamagnetic species. Thus, we were interested to know the role of transition metal ions as well as thiolate ligand that determined the nature of the thiolate complexes. Thus, we have revisited this system with different reaction conditions. This report provides a detailed account of the chemistry of group-5 metal precursors with [(CH 2 S 2 ) 4 B]Na 5 . In addition, the density functional theoretical calculations were carried out to provide a clear picture of the bonding, electronic structures and transitions of these thiolate complexes.

Results and discussion

Reactivity of [(CH 2 S 2 ) 4 B]Na 5 with [Cp*VCl 2 ] 3

The reaction of [(CH 2 S 2 ) 4 B]Na 5 with [Cp*VCl 2 ] 3 at 60 °C over 6 h allowed the formation of [(Cp*V) 2 (μ-CS 3 -κ 2 S,S')(μ-H 2 CS 2 -κ 2 S'',S''')], 1, and [{S(CH 2 S) 2 }OVCp*] 8 , 2, in 11 and 32% yield, respectively (Scheme 1). The structural characterisation of 1 was carried out by single crystal X-ray diffraction study and its identity is described below.

(Scheme 1 near here)

To our surprise, the 1 H NMR spectrum of 1 did not show any meaningful chemical shifts in the diamagnetic region. The mass spectrometric data of 1 displayed an ion peak at m/z = 559.0039 [M+H] + with the isotope distribution pattern corresponding to the molecular formula C 22 H 32 S 5 V 2 (Fig. S3). This spectrometric data was not sufficient to propose the structure of 1.

The slow evaporation of CH 2 Cl 2 /hexane (30:70) solution of 1 at 5 °C produced the single crystals appropriate for X-ray diffraction.

As shown in Fig. 1, the solid-state X-ray structure of 1 displays that the methanedithiolate (CH 2 S 2 ) and trithiocarbonate (CS 3 ) ligands symmetrically coordinated to two {Cp*V} units in {μ,κ 2 } fashion. The mirror plane is passing through the molecule which contains both methanedithiolate and trithiocarbonate ligands. Both the metal centres in 1 adopted a pseudooctahedral coordination geometry. The trithiocarbonate ligand in 1 shows two types of C-S bond distances, 1.747(4) (C21-S1) and 1.622(4) Å (C21-S3), corresponding to single and double bond distances, respectively. 12 The V-V single bond length of 2.5548(7) Å is shorter as compared to those of reported bimetallic vanadaborane complexes, such as

[(CpV) 2 (μ,η 2 :η 2 -B 2 H 6 ) 2 ] 11 (2.787(2) Å) and [(Cp*V) 2 (μ,η 2 :η 2 -BH 3 S)(μ-SPh) 2 ], 12a (2.6816(12) Å). The C22-S4 (1.835(4) Å)
and C22-S5 (1.833(4) Å) bond distances are in the single bond range and similar to tungsten methanedithiolate complex, [(Cp*W) 2 (μ-S 2 CH 2 -κ 2 S,S')(μ-S) 2 ] 13 (c.a. 1.85 Å). In addition, the Cp* ligands are in eclipsed positions with respect to the V1-V2 bond. The V-S-V angles in the range of 62.77-63.61° are comparable to other bimetallic vanadium thiolate complexes. 13 The importance of complex 1 can be correlated to [(CpMo) 2 (μ-S 2 CH 2 -κ 2 S,S')(μ-S) 2 ], which participated in the hydrogenation of olefins. 14 Therefore, 1 is a unique entry to the family of bimetallic thiolate complexes.

(Fig. 1 near here)

Density functional theory (DFT) calculations at the BP86/def2-TZVP level were performed to understand the electronic and bonding situation in 1. The optimized geometry of 1 matches well with the solid-state structure. An energy gap of 0.85 eV between its HOMO and LUMO is observed in the molecular orbital (MO) analysis of 1 and that is consistent with its thermodynamic stability. Based on MO analysis, the α-SOMO-2 of 1, discloses the overlap of the d z 2 orbitals of both the vanadium centres indicating a strong bonding interaction between the two vanadium centres (Fig. 2a). This is further confirmed by the Wiberg bond index (WBI) of V-V bond (0.91) and the contour line diagram of 1 (Fig. 2d). As shown in Fig. 2b, the α-SOMO-9 of 1 shows the localization of electron density on all the sulfur atoms except S3 atom, in which the electron density is delocalized over S3 and C21 atoms by the side-to-side overlap of the atomic orbitals i.e., π-bonding and confirms the presence of C=S double bond. The α-SOMO-13

of 1 shows the d-p orbital interaction between vanadium and sulfur. Furthermore, the Laplacian electron density plot of 1 shows the bonding scenario of the plane consisting CS 3 and CH 2 S 2 ligands, which is perpendicular to the V-V bond (Fig. 2e). A negative natural charge on carbon and a positive natural charge on sulfur atoms of the methanedithiolate and trithiocarbonate ligands are observed by performing the natural bond orbital (NBO) analysis of 1 (Table S1).

(Fig. 2 near here)

Vanadium is found in several oxidation states that vary from (-1) to (+5), and among them vanadium ions in +2, +3 and +4 oxidation states are paramagnetic. 15 Vanadium(III) is generally found in marine invertebrates, cofactor of nitrogenase, nitrogen-fixing bacteria and other species.

Recent investigation suggested that vanadium(III) thiolate complexes are stable in physiological conditions. 16 Therefore, the electronic properties of vanadium thiolate complexes are of interest.

To get information in this regard, complex 1 was subjected to EPR spectroscopy.

Vanadium(III) complexes are generally EPR silent in conventional conditions due to their large zero-field splitting and integer spin number (S = 1). For such systems, high-frequency or highfield electron paramagnetic resonance (HFEPR) techniques are required to understand the magnetic properties of vanadium(III) species. 17 However, conventional X-band EPR spectroscopy is often useful in understanding the electronic structure of half-integer spin vanadium (II) (S = 3 / 2 ) or vanadium (IV) (S = 1 / 2 ) complexes. Interestingly, the X-band EPR spectrum of the solid sample of 1 at 77 K displays the eight-line pattern for vanadium ( 51 V; I = 7 / 2 ) with anisotropic g and A values (Fig. 3). In addition, each vanadium hyperfine exhibits eightline superhyperfine splitting. These unique splitting patterns in the EPR spectrum do not arise from a monomeric complex, rather two vanadium centres are connected to each other. In this case, the nuclear spins of one vanadium centre interact with the electron spin on the other vanadium centre. Thus, the spectral data confirms the existence of a strong bonding interaction between the two vanadium centres, as observed in the single crystal X-ray diffraction data and DFT calculations.

(Fig. 3 near here)

The experimental spectrum was simulated taking the above-mentioned electronic scenario into consideration. Simulation of the experimental spectrum yields the parameters g x = 1.990, g y = 1.995, g z = 1.95, and the hyperfine coupling constants A x = 50 x 10 -4 cm -1 , A y = 60 x 10 -4 cm -1 , and A z = 113 x 10 -4 cm -1 . The super-hyperfine coupling constants are calculated to be A x = 13.3

x 10 -4 cm -1 , A y = 20 x 10 -4 cm -1 , and A z = 10 x 10 (Scheme 2 near here)

Complex 3 was isolated as a green solid with 16% yield. The 1 H NMR spectrum of 3 shows resonance at δ = 2.17 ppm corresponding to the methyl protons of Cp* units which is further supported by the 13 C{ 1 H} NMR spectrum (Figs S4 andS5). The 11 B{ 1 H} NMR spectrum showed no resonance indicating the absence of boron. The mass spectrometric analysis showed a molecular ion peak at m/z 628.9130 consistent with the molecular formula of C 21 H 31 Nb 2 S 5 ([M+H] + ) (Fig. S6). The green crystals of 3 were obtained by slow evaporation of the CH 2 Cl 2 /hexane (30:70) solution of the complex at 5 °C.

The single crystal X-ray diffraction studies revealed the identity of 3 as [(Cp*Nb) 2 (μ-SCS 2κ 1 S:κ 3 C,S',S'')(μ-S) 2 ] (Fig. 5). Note that, here we have discussed the data of one amongst the two independent molecules present in the asymmetric unit of 3. The structure of 3 clearly displays that a trithiolate (S 3 C) ligand is coordinated to two {Cp*Nb} units through one carbon and three sulfur atoms in a unique binding mode which can be described as {μ,κ 1 S:κ 3 C,S',S''}. (Fig. 5 near here)

In addition, the average Nb-S distance for Nb2 is 2.323 Å, which is relatively smaller compared to that for Nb1 (2.4865 Å). This may be due to the unsymmetrical bonding of the trithiolate ligand towards the metal centres in 3. The Nb-S-Nb angles Nb1-S3-Nb2 (75.81(2)°)

and Nb1-S4-Nb2 (76.36(2)°) are comparable with the sulfur bridged niobium complex [(Cp*Nb) 2 (CH 2 Si-Me 3 ) 2 (μ-S) 2 ] 18 78.8(1)°. One of the interesting aspects of complex 3 is the unique binding mode adopted by {CS 3 } moiety of trithiolate ligand which differs significantly from those found in the previously reported complexes such as [(OC) 5 (Cp*Mo) 2 (μ-η 2 :η 1 -CS 3 )] 13c , and [{(η 5 -C 5 H 3 ) 2 (SiMe 2 )(CMe 2 )Mo(CO) 2 } 2 (μ-η 1 :η 2 -CS 3 )] 20 and complex 1 (Table 1). 21

The different types of coordination modes known for the {CS 3 } ligand in bimetallic systems are listed in Table 1. The bonding mode of trithiolate ligand present in 3 belongs to the type D where one carbon and two sulfur atoms are directly bonded to one metal centre while the third sulfur atom is attached to the other metal centre. Whereas, complex 1 resembles to the type C where the trithiocarbonate ligand binds with two metals through two sulfur donors in {κ 2 -S,S'} fashion.

(Table 1 near here)

The MO analysis of 3 disclosed that the HOMO-LUMO gap of 3 (1.32 eV) is greater than that of 1. The Nb-Nb bonding interaction is depicted in HOMO-1 of 3, where the d orbitals of both the Nb centres are delocalized to form the Nb-Nb bond (Fig. 6a). This Nb-Nb bonding is also verified by WBI of 0.45. The HOMO-8 of 3 reveals the bonding interaction (d-p) of Nb1-C31

and anti-bonding interaction of Nb2-C31 (Fig. 6b). The considerable WBI for the Nb1-C31 bond (0.535) indicated a meaningful bonding interaction between Nb1 and C31, which is also supported by the contour line diagram along Nb2-C31-Nb1 plane (Fig. 6d). The HOMO-25 of 3

showed the end-to-end p-p orbital overlap between C and S that indicated the presence of σ-(C-S) bonds of CS 3 moiety (Fig. 6c). In addition, the computed C-S bond lengths (C31-S1, C31-S2 and C31-S5) of 1.757, 1.762 and 1.787 Å are comparable to those observed in 3. The Laplacian electron density plot along the S1-S2-S5 plane displayed a high amount of charge concentrations and the BCPs between C31-S1, C31-S2 and C31-S5 bonds specified strong bonding interactions (Fig. 6e).

(Fig. 6 near here)

Complexes 4 and 5 were characterised by multinuclear spectroscopy, mass spectrometry as well as single crystal X-ray diffraction analysis. The 11 B{ 1 H} NMR spectra of both 4 and 5

showed no resonances which suggested the formation of non-boron species. Notably, the coordination of (dimercaptomethyl)methanedithiolate unit towards the tantalum centres is exclusive as it coordinates to both the tantalum centres with three sulfur atoms. The unsymmetrical Ta-S bond distances (2.5641( 14) vs 2.4470( 13 (Fig. 7 near here)

In contrast, the solid-state X-ray structure of 5 consists of two {Cp*Ta} units which are bridged by methanedithiolate (S 2 CH 2 ) ligands in two different coordination modes (Fig. 7 (right)). One of the methanedithiolate moiety bridged two Ta centres through two sulfur atoms in {μ,κ 2 S,S'} fashion, whereas the other one is bridged in {μ,κ 2 S'':κ 1 S'''} fashion. Interestingly, this difference in the coordination of methanedithiolate units towards the tantalum centres is unique.

Note that, the structure of 5 is disordered over two components with a site occupancy ratio of 0.64:0.36 (Fig. S1). The bridged sulfur atom (S4) of the S 2 CH 2 ligand in 5 also makes an acute bridge (∠Ta1-S4-Ta2 = 75.00( 5 (2.441 Å). 22 The computational analyses of 4 and 5 revealed that the structural parameters of their optimized geometries are in good agreement with the experimentally observed values. The MO analyses revealed a larger HOMO-LUMO gap for 5 (1.91 eV) than that of 4 (1.70 eV). The HOMO-11 of 4 shows the Ta1-H36-Ta2 interaction (Fig. S16a) which is also confirmed by the Contour-line diagram of the Laplacian of the electron density along the Ta1-H36-Ta2 plane (Fig. S16b). The HOMO-18 of 5 shows end-to-end Cl1-Ta2 (p z -) overlap of atomic orbitals (or the presence of Cl1-Ta2 covalent bond) (Fig. S17a) which is also supported by the contour line diagram along the Ta1-Ta2-Cl1 plane (Fig. S17b).

Synthesis of tantalum thiolate complex, 6

Isolation of classical [B 2 H 5 ] -ion, I was achieved from the reaction of LiBH S13) and further supported by the 13 C{ 1 H} NMR spectrum (Fig. S14). The absence of 11 B{ 1 H} signal for 6 suggested a non-boron species. The mass spectrum of 6 displayed a molecular ion peak at m/z 874.9845 [M+Na] + (Fig. S15). Several attempts to obtain good crystals of 6 for single crystal Xray diffraction studies were unsuccessful. However, all the spectroscopic and mass spectrometric data along with the X-ray diffraction studies of a weakly diffracted crystal provided the core structure of 6 as [(Cp*Ta) 2 (μ-S)(μ-S 2 CH 2 -κ 2 S,S')(μ-H)(μ-CHS 3 -κ 2 S'':κ 2 S''',S'''')] (Fig. S2).

Complex 6 consists of methanetrithiolate (HCS 3 ) and methanedithiolate (H 2 CS 2 ) ligands symmetrically bridged between two {Cp*Ta} units in {μ,κ 2 S,S'} and {μ,κ 2 S'':κ 2 S''',S''''} fashion.

It is interesting to note that complexes 4, 5 and 6 are analogous to each other containing {S 4 C 2 H 3 }, {S 2 CH 2 } and {S 3 CH} ligands, respectively, in addition to the presence of a common methanedithiolate unit.

(Scheme 3 near here)

In order to understand the bonding situation in 4, 5 and 6, the computational studies were performed at the BP86/def2-TZVP level of theory. The MO analyses revealed that the HOMOs of these complexes are localized over the S atoms (p orbital) whereas the LUMOs are mostly centred on both the tantalum atoms (d-orbital) along with a little contribution from the ligand.

The WBI values of 0.32, 0.31 and 0.32 in 4, 5 and 6, respectively revealed a weak Ta-Ta bonding interaction. Further, the MO analysis showed that the HOMO-LUMO energy gap increases in the order 4 < 5 < 6 (Fig. 8). The NBO analyses revealed that the natural charge on both the Ta atoms is negative and quantitatively similar in 4 and 6. Whereas, in the case of 5, although the natural charge on Ta1 is negative as usual, it is positive on Ta2. This may be due to the direct Ta2-Cl bonding in which Cl atom acted as an electron withdrawing group. The natural charges on all the S atoms are positive due to charge transfer to metal centre except the bridging sulfur which is also reflected by the higher natural valence population as compared to other sulfur atoms.

(Fig. 8 near here)

Having a series of binuclear metal thiolate complexes in hand, we were interested to examine their structural parameters with other transition metal thiolate species, listed in Table 2. 8,23 Amongst the listed complexes, it is observed that as the metal centre varies, there is a change in M-M, M-S bond lengths as well as the M-S-M bond angle. For example, on moving from Vcomplex 1 to Ta-complex 5, there is an increase in the M-M and avg. M-S bond distances from 2.5548 Å to 3.1483 Å and 2.439 Å to 2.478 Å, respectively, which is further supported by calculated values (Table S1). The M-S-M bond angle also increases from 62.77° to 81.43°. This may be attributed to the increase in the atomic radius of the transition metal on moving from vanadium to tantalum, which results in the elongation of M-M and M-S bonds and causes the M-S-M bond angles to become less acute. 24 In addition, the 2 J H-H coupling constant for the methanedithiolate protons in the listed tantalum thiolate complexes were found to be in the range of 10-13 Hz.

(Table 2 near here) 1, 3, 4, 5 and6 The UV-vis absorption spectroscopy helped to measure the optical properties of complexes 1, 3, 4, 5 and 6. Their UV-Vis spectra reveal multiple bands around 280-600 nm (Fig. 9). The highenergy absorption bands around 280-290 nm are for the π-π* transition of Cp* ligands and that is characteristic of most of the Cp* based-metal complexes. 25 The low-energy bands around 340-550 nm are assigned to charge transfer bands. 8,26 (Fig. 9 near here)

UV-vis absorption studies of complexes

To get information about the electronic transitions, time dependent DFT calculations were carried out. These calculations suggest that the possible numbers of transitions are more in complex 1 compared to those of 3, 4, 5 and 6 (Table S2-S6, Fig. S18-S22). This accounts for the paramagnetic behaviour observed in 1, showing α and β subspaces, whereas complexes 3, 4, 5

and 6 are diamagnetic in nature. The molecular orbitals related to the most intense electronic transitions for 1, 3, 4, 5 and 6 are shown in Fig. S23-S27. In complex 1, the absorption near 341 nm may be assigned to the electronic transition corresponding to HOMO-1(α)LUMO(α), HOMO-1(α)LUMO+2(α), which may be considered as an intramolecular MLCT transition.

The low intensity absorptions near 463 and 543 nm could be the intramolecular LMCT transitions corresponding to HOMO(α)LUMO(α) and HOMO(α) LUMO+5(α), respectively.

The absorption band at 347 nm for complex 3 corresponds to MLCT transitions, whereas the absorptions near 458 and 533 nm seem to be intramolecular LMCT transitions. Similarly, the absorptions at ~ 370 nm for complexes 4, 5 and 6 appear due to MLCT transitions, whereas those in 432-511 nm region are mainly due to intramolecular LMCT transitions. A comparison among the low-intensity absorption bands ranged from 432-543 nm reveals a blue shift from V to Nb to Ta, which is also in accord with their HOMO-LUMO energy gaps. In contrast, a red shift is observed in case of high intensity absorption bands ranged from 341-371 nm.

Conclusions

In this report, we have demonstrated a series of binuclear thiolate complexes of group-5 metals and the studies show that the metal as well as the associated ligands play important roles in tuning the structural and electronic properties of these complexes. The V-V bond in the binuclear vanadium complex 1 influences the electronic properties of the complex. Furthermore, the vanadium thiolate complex provides structural model systems, which might be useful for the identification and understanding of vanadium-sulfur centres in metallobiomolecules. In the latter context, we have discussed the niobium complex 3, which shows a unique binding mode of trithiolate ligand. In addition to this, the tantalum complexes 4, 5 and 6 display a variety of thiolate ligands, such as (dimercaptomethyl)methanedithiolate, methanedithiolate and methanetrithiolate, coordinated to the tantalum centres. These complexes show diamagnetic behaviour in contrast to the vanadium complex 1.

Experimental details

General procedures and instrumentation

All the syntheses were performed under argon atmosphere using standard Schlenk line and glove box techniques and all the solvents were purified by distillation method in presence of proper drying agents. CDCl 3 was degassed through three freeze-pump-thaw cycles. instrument was used for the measurement of the electron paramagnetic resonance spectrum. UVvis spectra were recorded on a JASCO V-650 spectrometer.

Synthesis of compound 1

A freshly prepared solution of [(CH 2 S 2 ) 4 B]Na 5 (0.013 mmol in 10 mL tetrahydrofuran, 0.0013 mol/L) was added dropwise in the toluene (10 mL) solution of [Cp*VCl 2 ] 3 , (0.05g, 0.065 mmol), taken in a flame dried Schlenk tube, over 15 min at -78 °C. The reaction mixture was stirred vigorously and kept at 60 °C for 6 hours. Then the solvent was dried using vacuum and the solid deposit was extracted using the hexane/THF (90:10 v/v) solution mixture and ran through a G3 sintered frit partially filled with cotton. Then the volatiles were removed under vacuum and the chromatographic workup was carried out on silica gel TLC plates using the solid residue. The hexane/toluene (80:20 v/v) solution mixture was used as eluent and that allowed us to isolate yellow 1 (0.003 g, 11%) and 2 8 (0.007 g, 32%).

1: MS (ESI + ) calcd for C 22 H 33 V 2 S 5 + [M+H] + m/z 559.0064, found 559.0039; UV-Vis [CH 2 Cl 2 , λ, nm]: 341, 463, 543. Well-grounded solid powder of complex 1 (~7 mg) was loaded into a sample holder under argon atmosphere and the X-band EPR spectrum was recorded at 77 K using the following parameters. Microwave frequency = 9136.87 MHz, microwave power = 0.99800 mW, modulation frequency = 100.00 kHz, modulation amplitude CH1= 100.0, CH2 = 2.0, field centre = 400 mT, time constant CH1 = 0.1 s, CH2 = 0.03 s and sweep time = 2.0 min.

Synthesis of compound 3

A freshly prepared solution of [(CH 2 S 2 ) 4 B]Na 5 (0.027 mmol in 10 mL tetrahydrofuran, 0.0027 mol/L) was added dropwise in the toluene (10 mL) solution of [Cp*NbCl 4 ], (0.05g, 0.136 mmol), taken in a flame dried Schlenk tube, over 15 min at -78 °C. The reaction mixture was stirred vigorously and kept at 60 °C for 6 hours. Then the solvent was dried using vacuum and the solid deposit was extracted using the hexane/CH 2 Cl 2 (60:40 v/v) solution mixture and ran through a G3 sintered frit partially filled with cotton. Then the volatiles were removed under vacuum and the chromatographic workup was carried out on silica gel TLC plates using the solid residue.

The hexane/CH 2 Cl 2 (60:40 v/v) solution mixture was used as eluent and that allowed us to isolate green 3 (0.005 g, 16%), and [(Cp*Nb) 2 (μ,η 2 :η 2 -SH 3 B)(μ-S)(μ-H 2 CS 2 -κ 2 S,S')] 8

(0.005 g, 11%).

3: MS (ESI

Synthesis of compounds 4 and 5

A freshly prepared solution of [(CH 2 S 2 ) 4 B]Na 5 (0.022 mmol in 10 mL tetrahydrofuran, 0.0022 mol/L) was added dropwise in the toluene (10 mL) solution of [Cp*TaCl 4 ], (0.05g, 0.110 mmol), taken in a flame dried Schlenk tube, over 15 min at -78 °C. The reaction mixture was stirred vigorously and kept at 60 °C for 6 hours. Then the solvent was dried using vacuum and the solid deposit was extracted using the hexane/CH 2 Cl 2 (40:60 v/v) solution mixture and ran through a G3 sintered frit partially filled with cotton. Then the volatiles were removed under vacuum and the chromatographic workup was carried out on silica gel TLC plates using the solid residue.

The hexane/CH 2 Cl 2 (40:60 v/v) solution mixture was used as eluent and that allowed us to isolate purple 4 (0.009 g, 28%) and orange 5 (0.005 g, 15%) along with [(Cp*Ta) 2 (μ-S){μ-(BH 2 S)(CH 2 S) 2 (BS 3 )-κ 2 B:κ 2 S:κ 4 S',S'',S''', S''''}] 8 (0.012 g, 24%).

4: MS (ESI+

Synthesis of compound 6

A 2.0 M LiBH 4 .THF solution (0.7 mL) was added dropwise in the toluene (10 mL) solution of [Cp*TaCl 4 ], (0.100 g, 0.22 mmol), taken in a flame dried Schlenk tube, over 15 min at -78 °C. The mixture was allowed to stir for 1 hour, then the freshly prepared solution of excess S 2 C•PPh 3 (0.075 g of PPh 3 dissolved in 6 mL of CS 2 ) was added over 5 min and the reaction mixture was stirred vigorously and kept at 60 °C for 6 hours. The reaction mixture color changed from yellow to brown. Then, the solvent was dried using vacuum and the solid deposit was extracted using the hexane/CH 2 Cl 2 (40:60 v/v) solution mixture and ran through a G3 sintered frit partially filled with cotton. Then the volatiles were removed under vacuum and the chromatographic workup was carried out on silica gel TLC plates using the solid residue.

X-ray structure determination

The crystal data of 1 and 6 were collected and integrated using Bruker APEX3

diffractometer with graphite monochromated Mo-Kα (λ = 0.71073 Å) radiation at 296(2) K. The crystal data of 3 and 4 were collected and integrated using D8 VENTURE Bruker AXS diffractometer with graphite monochromated Mo-Kα (λ = 0.71073 Å) radiation at 150(2) K. The crystal data of 5 was collected and integrated using Bruker APEX2 diffractometer with graphite monochromated Mo-Kα (λ = 0.71073 Å) radiation at 296(2) K. The molecular structure of 1 was solved by heavy atom methods using SHELXT-2014/5 and refined using SHELXL-2018/3. [START_REF] Sheldrick | [END_REF] The structures of 3 and 4 were solved by heavy atom methods using SHELXT-2014/4 and refined using SHELXL-2017/1. [START_REF] Sheldrick | [END_REF] The structures of 5 and 6 was solved by heavy atom methods using SHELXS-97 and refined using SHELXL-2018/3. [START_REF] Sheldrick | [END_REF] Olex 2 software was used to draw all the molecular structures. 32 The non-hydrogen atoms were refined with anisotropic displacement parameters. All hydrogens could not be located in the difference Fourier map for compound 4.

(Table 3 near here)

Computational details

Geometry optimizations and electronic structure calculations were carried out on the Gaussian 09 program [START_REF] Gaussian | Gaussian Inc[END_REF] analyses were carried out for all structures, and the absence of any imaginary frequency confirmed that all structures represent minima on the potential energy hypersurface. The gauge including atomic orbital (GIAO) 36 method was employed to calculate the NMR chemical shifts of the BP86/def2-TZVP optimized geometries using the hybrid Becke-Lee-Yang-Parr (B3LYP) 37 functional and aforementioned basis set. For computing 1 H NMR chemical shift, TMS(SiMe 4 ) has been taken as the internal standard (shielding constant 31.92 ppm). Natural bonding analyses were performed with the NBO partitioning scheme, as implemented in the Gaussian 09 suite of programs. 38 Wiberg bond indexes (WBI) were obtained on NBO analysis. [START_REF] Wiberg | [END_REF] All the optimized structures and orbital plots were generated by using visualization programs, such as Gaussview 40 and Chemcraft 41 . In order to understand the nature of bonding of the synthesized molecules in greater detail, the topological properties of the resultant electron density, ρ, obtained from the wave functions of all of the optimized structures were analyzed with the quantum theory of atoms in molecules (QTAIM). 42 QTAIM analysis was carried out utilizing the Multiwfn, V.3.6, package 43 whereas the wave functions were generated with Gaussian 09 at the same level of theory as that used for geometry.

Scheme 1

Syntheses of vanadium thiolate complexes.

Scheme 2.

Syntheses of thiolate complexes of niobium and tantalum.

Scheme 3. Synthesis of thiolate complex of tantalum 6. 14), Ta1-S4 2.4470( 13),

Fig

Ta1-S6 2.5641( 14), Ta2-S5 2.4419( 14), Ta1-Ta2 3.1156(3), S1-C21 1.826(6), S4-C22 1.826(6), S6-C23 1.838(6), S7-C23 1.792(6), S7-C22 1.821(6); C21-S1-Ta1 106.7(2), Ta2-S3-Ta1 80.72(4), C22-S4-Ta1 113.1(2). 5: Ta1-S3 2.411(4), Ta1-S1 2.392(6), Ta1-S4 2.600(2), Ta1-S5 2.544( 16), Ta1-Ta2 3.1483( 8), Ta2-Cl1 2.463( 14), Ta2-S4 2.590(17), S1-C21 1.850(2), S4-C22 1.900(2); C21-S1-Ta1 108.7(7), Ta2-S3-Ta1 81.43 (13), C22-S4-Ta1 93.00 (9).

Fig. 9.

Combined UV-vis spectra of 1, 3, 4, 5 and 6 in CH 2 Cl 2 .

Table 1.

Examples of transition metal complexes with unique coordination modes of bridging {μ-CS 3 } ligand.

Table 2.

Selected structural and spectroscopic parameters of binuclear metal thiolate complexes.

Table 3.

Crystal data and structure refinement details for compounds 1, 3, 4 and 5.

  The mirror plane is passing through the molecule, which contains both the Nb atoms along with C31 and S5 atoms of {CS 3 } unit. The {CS 3 } moiety of trithiolate ligand is nearly planar with the sum of S-C-S bond angles of 359.64° (S1-C31-S2 112.26(14)°, S1-C31-S5123.46(16)° and S2-C31-S5 123.92(15)°). The Nb-Nb bond length of 2.9705(3) Å is in accord with that of other binuclear niobium complexes, for example, [(Cp*Nb) 2 (CH 2 SiMe 3 ) 2 (μ-S) 2 ] (2.978 Å) and [(Cp*Nb) 2 (C 3 H 5 ) 2 (μ-S) 2 ] (3.141 Å). 18 The C-S bond distances (C31-S1 1.756(3) Å, C31-S2 1.746(3) Å, C31-S5 1.776(3) Å) of trithiolate ligand in 3 are fall within single C-S bond distance observed in various metal thiolate complexes. 12b-c, 19

  ) Å) clearly indicate that the bridged sulfur atom is more loosely bonded with metal centres as compared to the terminal ones of the C 2 H 3 S 4 ligand . The bridged sulfur atom (S6) of this ligand forms an acute bridge (∠Ta1-S6-Ta2 = 73.49(4)°) with metal centres. The geometry around the sp 3 hybridized carbon atoms (C22 and C23) of this ligand are slightly deviated from the regular tetrahedron (∠S4-C22-S5 = 120.2(3)° and (∠S6-C23-S7 =118.7(3)°). The average C-S bond distance (1.820 Å) of the C 2 H 3 S 4 unit is in the range of single bond length. The decrease in the Ta-Ta bond distance in 4(3.1156(3) Å), as compared to that of [(Cp*Ta) 2 (μ-S 2 CH-κ 2 C:κ 2 S,S')(μ-S)(μ-CHS 3κ 2 S'':κ 2 S''',S'''')] (3.171 Å)8 , is the resultant of decrease in the corresponding Ta-S-Ta angle. On the other hand, the sp 3 hybridized carbon atom of methanedithiolate ligand lies in a distorted tetrahedral configuration (∠S1-C21-S2 = 115.9(3)°).

  )°) with the metals as observed in 4. This increase in Ta-S-Ta angle results in a longer Ta-Ta bond distance in 5 (3.1483(8) Å). All the four C-S bond lengths in methane-dithiolate ligands (C21-S1 = 1.85(2) Å, C21-S2 = 1.77(2) Å, C22-S4 = 1.90(2) Å and C22-S5 = 1.81(3) Å) are in range of a typical C-S single bond distance. 21 In addition to the two methanedithiolate ligands, a chlorine atom is directly bonded to one of the tantalum centres with a Ta1-Cl1 bond distance of 2.463(14) Å which is similar to that observed in complexes [(Cp*TaCl) 2 (B 5 H 11 )] (2.465 Å), [Ta 2 (PMe 3 ) 4 (Cl) 4 (H) 2 ] (2.418 Å), and [(Cp*TaCl) 2 (B 5 H 10 Cl)]

  package using the BP86 functional34 and the triple-ζ quality basis set def2-TZVP35 . In compound 1 both the vanadium centers are in (+III) oxidation state i.e. d 2 electronic configuration. So, there are possibilities of both, low spin diamagnetic singlet state (multiplicity = 1) configuration and high spin triplet state (multiplicity = 3) configuration. And calculation shows that the triplet spin state (E = -4737.74600201 a.u.) is more stable than diamagnetic singlet spin state (E = -4737.73137247 a.u.) by 0.01462954 a.u. In case of compound 3, 4, 5 and 6 both the metal centers are in (+V) oxidation state i.e. d 0 electronic configuration. So, there is the only possibility of the low spin diamagnetic singlet state configuration. Therefore, for the optimization and other theoretical analyses of compound 1, multiplicity = 3 and for other molecules, multiplicity = 1 has been considered. Vibrational

Fig. 2 .

 2 Fig. 2. (a) α-SOMO-2, (b) α-SOMO-9 and (c) α-SOMO-13 of 1. Contour values are ±0.04 (e/bohr 3 ) 1/2 . (d) and (e) Contour line diagram of the Laplacian of the electron density (∇ 2 ρ(r)) in V1-S1-V2 plane and the perpendicular plane with respect to V-V bond. The dashed black line displays the area of charge depletion (∇ 2 ρ(r) > 0) and the solid red line shows the area of charge concentration (∇ 2 ρ(r) < 0). Deep Brown line indicates the bond path whereas blue dot depicts the bond critical point (BCP).

Fig. 3 .

 3 Fig. 3.X-band EPR spectrum of the solid sample of complex 1 recorded at 77 K with microwave frequency of 9.136 GHz.

Fig. 4 .

 4 Fig. 4. Molecular orbitals showing α and β subspaces for the antiferromagnetically coupled vanadium centres in 1. Contour values are ±0.05 (e/bohr 3 ) 1/2 .

Fig. 5 .

 5 Fig. 5. Molecular structure and labelling diagram of and 3. The protons of Cp* rings are not shown for clarity. Selected bond lengths (Å) and angles ( ο ) are: Nb1-S1 2.4557(7), Nb1-S2 2.4412(7), Nb1-Nb2 2.9705(3), C31-S1 1.756(3), C31-S2 1.746(3), C31-S5 1.776(3), Nb2-S5 2.3977(7), Nb1-C31 2.259(2); S1-C31-S2

Fig. 6 .

 6 Fig. 6. (a) HOMO-1, (b) HOMO-8 and (c) HOMO-25 of 3. Contour values are ±0.04 (e/bohr 3 ) 1/2 . (d) and (e) Contour line diagram of Laplacian of the electron density ∇ 2 ρ(r) in Nb2-Nb1-C31 and S1-S2-S5 plane, respectively. The dashed black line displays the area of charge depletion (∇ 2 ρ(r) > 0) and the solid red line shows the area of charge concentration (∇ 2 ρ(r) < 0). Deep Brown line indicates the bond path whereas blue dot depicts the bond critical point (BCP).

Fig. 7 .

 7 Fig. 7. Molecular structure and labelling diagram of 4 (left) and 5 (right). The bridging hydrogen atom and the protons of Cp* rings are not shown for clarity. Selected bond lengths (Å) and angles (°) are: 4: Ta1-S1 2.4389(14), Ta1-S4 2.4470(13),

Fig. 8 .

 8 Fig. 8.Comparison of frontier molecular orbitals of 4, 5 and 6; respectively. Contour values are ±0.04 (e/bohr 3 ) 1/2 .

  Scheme 1

Fig. 2

 2 Fig. 2

Fig. 3

 3 Fig. 3

Fig. 4

 4 Fig. 4

Fig. 5

 5 Fig. 5

Fig. 6

 6 Fig. 6

Fig. 7 Fig. 8

 78 Fig. 7

Fig. 9

 9 Fig. 9

8 a

 8 = for methylene or methine protons, avg = average, V = Cp*V, Nb = Cp*Nb and Ta = Cp*Ta

  -4 cm -1 . The EPR parameters with all g values <2 and large A z value indicate that the unpaired electron resides in the d xy orbital of vanadium.

	2.2 Reactivity of [(CH 2 S 2 ) 4 B]Na 5 with [Cp*MCl 4 ] (M = Nb and Ta)
	Mild thermolysis of [Cp*NbCl 4 ] with [(CH 2 S 2 ) 4 B]Na 5 in toluene yielded [(Cp*Nb) 2 (μ-SCS 2 -
	κ 1 S:κ 3 C,S',S'')(μ-S) 2 ], 3 and [(Cp*Nb) 2 -(μ,η 2 :η 2 -SH 3 B)(μ-S)(μ-H 2 CS 2 -κ 2 S,S')] 8 (Scheme 2).
	Whereas In case of tantalum, the reaction of [(CH 2 S 2 ) 4 B]Na 5 with [Cp*TaCl 4 ] at room
	temperature yielded [(Cp*Ta) 2 (μ-S)(μ-S 2 CH 2 CHS 2 -κ 2 S:κ 2 S',S'')(μ-H)(μ-S 2 CH 2 -κ 2 S''',S'''')], 4,
	and [Cl(Cp*Ta) 2 (μ-H)(μ-S 2 CH 2 -κ 2 S,S')(μ-S)(μ-S 2 CH 2 -κ 2 S'':κ 1 S''')], 5, in 28 and 15% yields,
	respectively (Scheme 2). The reaction also yielded [(Cp*Ta) 2 (μ-S){μ-(BH 2 S)(CH 2 S) 2 (BS 3 )-
	The energy gap between the d xy and	orbitals is larger compared to that between d xz /d yz
	and d xy orbitals of vanadium. An increased covalency in the equatorial plane likely responsible
	for low g z value in the complex. As shown in Fig. 4, the molecular orbitals housing the two
	unpaired electrons indicate non-orthogonality of the d orbitals of vanadium in the α and β
	subspaces. Therefore, it is expected that the electrons on two vanadium centres would interact
	with each other. While the electrons in the	orbitals are involved in V-V bonding, the d xy
	orbitals of vanadium do not overlap appreciably due to the small V-S-V angle (vide supra). As a
	result, the electron in the d xy orbital of each vanadium centre of the weakly interacting symmetric
	dimer gives rise to the EPR signals.
	(Fig. 4 near here)

κ 2 B:κ 2 S:κ 4 S',S'',S''',S''''}]

8

. The spectroscopic and structural characterisations of complexes 3-5 are discussed below in detail.

  The molecular ion peak at m/z 898.9876 ([M+H] + ) and 821.0310 ([M-Cl] + ) were observed corresponding to the molecular formula of C 23 H 37 Ta 2 S 7 (for 4) and

	C 22 H 35 Ta 2 S 5 (for 5), respectively (Figs S10 and S13). Storage of concentrated hexane/CH 2 Cl 2
	(40:60) solutions at 5°C allowed the isolation of diffraction quality purple and orange crystals of
	4 and 5, respectively.
	The solid-state X-ray structure of 4, shown in Fig. 7 (left), consists of two (Cp*Ta) units
	which are bridged by a sulfur, a methanedithiolate (S 2 CH 2 ) and a (dimercaptomethyl)methane-
	dithiolate (S 4 C 2 H
	The 1 H chemical
	shifts at δ = 2.32, 2.26 ppm (for 4) and 2.31, 2.24 ppm (for 5) were observed and that correspond
	to the methyl protons of two chemically and magnetically inequivalent Cp* ligands (Figs S9 and
	S12). The presence of Cp* units in complexes 4 and 5 is further corroborated by their respective
	13 C{ 1 H} NMR spectrum (Figs S8 and S11). Along with the Cp* ligands, the 1 H chemical shifts
	in the range of δ = 7.47-2.90 ppm (for 4) and δ = 7.43-4.15 ppm (for 5); and the 13 C{ 1 H}

chemical shifts at δ = 55.7-47.8 ppm (for 4) and δ = 56.7-43.3 ppm (for 5) confirmed the presence of methylene units. 3 ) unit. The methanedithiolate ligand is bridged via two sulfur atoms in {μ,κ 2 S''',S''''} fashion, whereas the other C 2 H 3 S 4 moiety is bridged in {μ,κ 2 S:κ 2 S',S''} fashion.

  4 •THF with[Cp*TaCl 4 ] in presence of S 2 C•PPh 3 . Complex I was stabilized by binuclear tantalum template and methanedithiolate ligands.9 In order to isolate other diborane species, we reinvestigated this reaction under different reaction conditions. As a result, reaction of LiBH 4 •THF with [Cp*TaCl 4 ] followed by thermolysis in presence of S 2 C•PPh 3 was carried out. Although we did not able to isolate any diborane complexes, reaction yielded [(Cp*Ta) 2 (μ-S)(μ-S 2 CH 2 -κ 2 S,S')(μ-H)(μ-CHS 3κ 2 S'':κ 2 S''',S'''')], 6 along with I and [(Cp*Ta) 2 (μ,η 2 :η 2 -B 2 H 6 ) 2 ] 22 , II (Scheme 3). The 1 H chemical shift at δ = 2.23 ppm, corresponding to the Cp* protons in 6, are observed (Fig.

  The starting materials [Cp*VCl 2 ] 3 , 27 [Cp*NbCl 4 ], 28 [Cp*TaCl 4 ], 29 [S 2 C•PPh 3 ] 30 and [(CH 2 S 2 ) 4 B]Na 5 7,8 were synthesized following the corresponding literature. Thin-layer chromatography was performed using 250 mm aluminium supported silica gel TLC plates. Chemical shifts are referenced to residual solvent signals ( 1 H/ 13 C{ 1 H}; CDCl 3 : δ = 7.26/77.16 ppm). The 6545 Qtof LC/MS instrument was used for recording mass spectra of all compounds. JEOL Model JES FA200

  Me 5 ); UV-Vis [CH 2 Cl 2 , λ, nm]: 347, 458, 533.

	+ ) calcd for C 21 H 31 Nb 2 S 5	+ [M+H] + m/z 628.9157, found 628.9130; 1 H
	NMR (CDCl 3 , 500 MHz, 22 ºC): δ = 2.17 ppm (s, 30H; 2×Cp*); 13 C{ 1 H} NMR (CDCl 3 ,
	125 MHz, 22 ºC): δ = 12.8 ppm (s, C 5

  ) calcd for C 23 H 37 Ta 2 S 7 + [M+H] + m/z 898.9900, found 898.9876; 1 H NMR (CDCl 3 , 500 MHz, 22 °C): δ = 9.46 (s, 1H, Ta-H-Ta), 7.47 (s, 1H, CHS 3 ), ), 6.45(d, 2 J H-H = 10.2 Hz, 1H, CH 2 S 2 ), 4.28(d, 2 J H-H = 10.7 Hz, 1H, CH 2 S 2 ), 3.24(d, 2 J H-H = 7.7 Hz, 1H, CH 2 S 2 ), 2.90(d, 2 J H-H = 10.7 Hz, 1H, CH 2 S 2 ), 2.32, 2.26 ppm (s, 30H, 2×Cp*);13 C{ 1 H} NMR (CDCl 3 , 125 MHz, 22 °C): δ = 120.5, 120.0 (s, C 5 Me 5 ), 55.7 (s,

	CHS 3 ), 49.9, 47.8 (s, CH 2 S 2 ), 12.7, 12.6 ppm (s, C 5 Me 5 ); UV-Vis [CH 2 Cl 2 , λ, nm]: 371,
	511.
	5: MS (ESI+) calcd for C 22 H 35 Ta 2 S 5	+ [M-Cl] + m/z 821.0302, found 821.0310; 1 H
	NMR (CDCl 3 , 400 MHz, 22 °C): δ = 10.77 (s, 1H, Ta-H-Ta), 7.43 (d, 2 J H-H = 11.1 Hz,
	1H, CH 2 S 2 ), 5.95 (d, 2 J H-H = 10.6 Hz, 1H, CH 2 S 2 ), 4.55 (d, 2 J H-H = 10.6 Hz, 1H, CH 2 S 2 ),
	4.15 (d, 2 J H-H = 10.6 Hz, 1H, CH 2 S 2 ), 2.31, 2.24 ppm (s, 30H, 2×Cp*); 13 C{ 1 H} NMR
	(CDCl 3 , 100 MHz, 22 °C): δ = 120.9, 119.4 (s, C 5 Me 5 ), 56.7 (s, CH 2 S 2 ), 43.3 (s, CH 2 S 2 ),
	12.6, 12.3 ppm (s, C 5 Me 5 ); UV-Vis [CH 2 Cl 2 , λ, nm]: 369, 432, 504.

Table 2

 2 M-S-M (°) δ H (ppm, 2 J H-H (Hz)) a

	Complex	Structural parameters		Spectroscopic data	Ref.
		d(M-M) (Å)	d avg (M-S) (Å)
		2.698(3)	2.419	67.8	-	23
		2.5548(7)	2.439	62.77	-	this work
				63.47
		2.9705(3)	2.417	75.81	-	this work
				76.36
		3.173 (Nb)	2.441 (Nb)	77.90 (Nb)	6.93, 6.33	8
				88.04 (Nb)
		3.171 (Ta)	2.457 (Ta)	77.99 (Ta)	7.07, 5.55
				82.61 (Ta)
		3.1155(3)	2.472	73.49	6.45 (10.2), 4.28 (10.7),	this work
				80.71	3.24 (7.7), 2.90 (10.7)
		3.1483(8)	2.478	75.00	7.43 (11.1), 5.95 (10.6),	this work
				81.43	4.55 (10.6), 4.15 (10.6)
		-	-	-	7.34 (10.4), 6.02 (10.4)	this work
		2.9882(5)	2.490	70.39	6.68 (13.7), 6.24 (10.7),
					4.61 (13.7)

Acknowledgements

The support from Indo-French Center for the Promotion of Advanced Research (IFCPAR-CEFIPRA), Project No. 5905-1 is gratefully acknowledged. S.B. thanks IIT Madras and A.D.

thanks CSIR for research fellowships. The computational facility, IIT Madras, is thankfully acknowledged. The authors thank Prof. Chandan Mukherjee of IIT Guwahati, for his help with EPR simulation.

Declaration of Competing Interest

The authors declare no competing financial interest.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org


Dr Benoit Busser

Jean Luc

Amandine Hurbin
email: amandine.hurbin@ujf-grenoble.fr

J L Coll

Amandine Hurbin

vectorisation de drogues dans le cancer bronchique non-à petites cellules

Keywords: amphiregulin, non-small cell lung cancer, gefitinib, growth factor, apoptosis, drug resistance, EGFR, targeted therapy, tyrosine-kinase inhibitors

The increasing role of amphiregulin in non

The increasing role of amphiregulin in non-small cell lung cancer Le rôle grandissant de l'amphiréguline dans le cancer du poumon non-à petites cellules

Résumé

Les cancers bronchiques non-à petites cellules présentent une survie à 5 ans inférieure à 12%. Un tel pronostic s'explique par la capacité des cellules cancéreuses pulmonaires à échapper à l'apoptose et à résister aux traitements. L'amphiréguline est un facteur de croissance de la famille de l'Epidermal Growth Factor (EGF) sécrété par les cellules de cancer bronchique de façon autocrine/paracrine, afin de permettre une croissance tumorale autonome et la survie cellulaire. De plus, l'amphiréguline est impliquée dans la résistance du cancer bronchique non-à petites cellules aux inhibiteurs de la tyrosine kinase du récepteur de l'EGF. Cet éditorial met en évidence le rôle de plus en plus important de l'amphiréguline dans les cancers bronchiques non-à petites cellules, et son implication dans les mécanismes de résistance à l'apoptose et aux traitements. Ceci suggère une utilisation potentielle de l'amphiréguline comme marqueur de sensibilité au traitement et/ou comme marqueur pronostique dans les cancers bronchiques non-à petites cellules afin d'identifier les patients susceptibles de répondre à un traitement à base d'inhibiteurs de tyrosine kinase du récepteur à l'EGF.

Keywords : amphiregulin; non-small cell lung cancer; gefitinib; growth factor; apoptosis; drug resistance; EGFR, targeted therapy; tyrosine-kinase inhibitors Mots clés : amphiréguline; cancer bronchique non-à petites cellules; gefitinib; facteur de croissance; apoptose; résistance aux traitements; EGFR; thérapie ciblée, inhibiteurs de tyrosine kinase * 2) Manuscrit Amphiregulin (AREG) is an 84-aminoacid glycoprotein discovered in the late 80's by M. Shoyab et al. AREG was originally isolated from the conditioned medium of phorbol 12-myristate 13-acetate (PMA)-stimulated MCF-7 human breast carcinoma cell line. The human AREG gene (GeneID 374) is located on the q13-q21 region of chromosome 4, and is composed of 6 exons encoding the preprotein mRNA transcript.

AREG is thus synthesized as a 252-amino acid transmembrane precursor, named pro-AREG. Pro-AREG is subjected to sequential proteolytic cleavage at the plasma membrane within the ectodomain that leads to realeased N-glycosylated soluble AREG forms. This shedding is essentially mediated by tumor-necrosis factor-alpha converting enzyme (TACE), a member of the disintegrin and metalloproteinase (ADAM) family also known as ADAM-17.

The AREG gene expression profile indicates that AREG is constitutively expressed in various tissues, mainly in the genito-urinary and reproductive systems, as well as in the gastrointestinal tract, but also in bone marrow, liver and in the respiratory tract.

AREG is a member of the Epidermal Growth Factor (EGF) family of proteins, which include EGF itself, transforming growth factor alpha, heparin-binding EGF-like growth factor, betacellulin, and neuregulin. AREG binds to and activates the EGF receptor (EGFR or ErbB1) through autocrine, paracrine and juxtacrine mechanisms.

The EGFR is widely expressed on epithelial cell membranes. The binding of AREG to EGFR induces autophosphorylation of the EGFR intracellular tyrosine kinase domain, which in turn activates two major intracellular pathways, i.e. the MEK/ERK1/2 and the PI3K/AKT pathways. EGFR is for instance involved in cell proliferation and differentiation, cell metabolism, cell migration and survival, as well as in the control of cell cycle. Furthermore, the EGFR was the first tyrosine-kinase receptor to be directly linked to several human tumors. AREG possesses a mitogenic activity for cells and plays a role in several biological processes including nerve generation, bone formation, mammary duct formation, as well as the outgrowth and branching of several tissues such as lung, kidney and prostate. AREG is also expressed in several cancers, inducing cellular responses such as growth, proliferation, migration or invasiveness after the activation of EGFR.

Lung cancer is the leading cause of cancer mortality in the world, both in males and females. Non-Small Cell Lung Cancer (NSCLC) accounts for 80% of lung cancers.

Once diagnosed, NSCLC 5-year survival rate hardly reaches 12% despite different treatments such as chemotherapy, radiotherapy, and surgery. Such a poor prognosis is partially explained by NSCLC ability to resist apoptosis and treatments. In this editorial, we report the roles of AREG in such resistance mechanisms leading to NSCLC progression.

Implication of AREG in NSCLC apoptosis resistance

The ability to evade apoptosis is a hallmark of almost all types of cancers. Cancer cells can acquire resistance to apoptosis through a variety of strategies, including the inactivation of the well-known protein p53. Another way to circumvent programmed cell death is to transmit antiapoptotic survival signals, essentially through autonomous production of growth factors by the tumor. Cancer cells produce growth factors that can be secreted, thus binding to cell surface receptors and stimulating their own growth and apoptosis resistance in an autocrine manner. The autocrine roles of EGFlike growth factors in lung cancer pathogenesis and in apoptosis resistance have been extensively studied. The involvement of AREG in autocrine loops of activation is well described in several human malignancies and AREG antiapoptotic activity is already demonstrated in breast, lung and hepatocarcinomas. In NSCLC, AREG presence is correlated with tumor size, illustrating its functional activity as a growth factor enhancing cell proliferation. AREG is also an indicator of reduced overall survival for NSCLC, suggesting an interesting role as a prognostic marker [START_REF] Fontanini | Evaluation of epidermal growth factor-related growth factors and receptors and of neoangiogenesis in completely resected stage I-IIIA non-small-cell lung cancer: amphiregulin and microvessel count are independent prognostic indicators of survival[END_REF]. AREG, in collaboration with Insulin-like Growth Factor Type-1 (IGF-1), inhibits serum deprivation-induced apoptosis in NSCLC cell lines [START_REF] Hurbin | Inhibition of apoptosis by amphiregulin via an insulin-like growth factor-1 receptor-dependent pathway in non-small cell lung cancer cell lines[END_REF]. AREG is able to activate the IGF1-receptor independently of its own EGF receptor. IGF1-receptor activation is in turn inducing the secretion of AREG and IGF1 in an autocrine loop. The combination of AREG and IGF1 and the subsequent receptor activation stimulate an original protein kinase C-dependant MAPK and PI3K-independant pathway that leads to NSCLC apoptosis resistance [START_REF] Hurbin | Cooperation of amphiregulin and insulin-like growth factor-1 inhibits Bax-and Bad-mediated apoptosis via a protein kinase C-dependent pathway in non-small cell lung cancer cells[END_REF].

Implication of AREG in NSCLC resistance to treatment

Since autocrine-stimulated mitogenesis and apoptosis resistance contribute significantly to cancer progression, clinical development of anticancer treatments interrupting autocrine loops are under evaluation. In lung cancer, the use of EGFRtyrosine kinase inhibitors (EGFR-TKI) such as gefitinib and erlotinib can block such autocrine EGFR activation is to use. Gefitinib showed potent anti-tumor effects in clinical trials for NSCLC treatment after previous chemotherapy, but failed to improve overall survival benefit in an unselected population. Predictive markers of EGFR-TKI sensitivity have thus been investigated to identify patients likely to benefit from such a targeted therapy.

As a matter of fact, AREG is involved in NSCLC resistance to EGFR-TKI. Gefitinib responders and non-responders present a differential expression of AREG gene in biopsy samples obtained from gefitinib-treated NSCLC patients. These data are confirmed by RT-PCR, immunohistochemistry, and serologic ELISA assays : AREG is indeed significantly overexpressed in gefitinib non-responders but undetectable in responders. Moreover, NSCLC patients with the worst prognosis and a shorter survival time present high levels of seric AREG, which is correlated with gefitinib resistance [START_REF] Ishikawa | Increases of amphiregulin and transforming growth factor-alpha in serum as predictors of poor response to gefitinib among patients with advanced non-small cell lung cancers[END_REF]. This subset of gefitinib non responders patients contains principally males, smokers with non-adenocarcinomas NSCLC. Interestingly, independent studies showed that most of gefitinib sensitive patients are principally females, non-smokers with NSCLC adenocarcinomas [START_REF] Sharma | Epidermal growth factor receptor mutations in lung cancer[END_REF], and with EGFR mutations.

Altogether these data suggest that seric AREG might be an important predictor of gefitinib resistance among advanced NSCLC patients. In other malignancies, AREG is also responsible of resistance to conventional chemotherapeutic drug, such as cisplatin, and to hormonal antitumor treatments such as exemestane, an oral steroidal aromatase inhibitor.

Conclusion

The implication of EGF-related growth factors in cancer progression is indisputable.

AREG is already involved in various malignancies and we reported several oncogenic roles for AREG in NSCLC progression. AREG is secreted by tumor cells in an autocrine/paracrine manner, leading to present AREG as a prognostic marker in NSCLC. Furthermore, AREG allows resistance to apoptosis in NSCLC. Anti-AREG antibodies restored an apoptotic activity in NSCLC cells [START_REF] Hurbin | Inhibition of apoptosis by amphiregulin via an insulin-like growth factor-1 receptor-dependent pathway in non-small cell lung cancer cell lines[END_REF], suggesting that AREG might be an interesting target to overcome apoptosis resistance. In addition, AREG is involved in therapeutic resistance in breast cancer and NSCLC, thus suggesting the interest of AREG as a biological marker of sensitivity to both conventional and EGFRtargeted therapy. The use of AREG as a biomarker should be encouraged since AREG is correlated with a poor prognosis, resistance to treatments and to apoptosis.


Stanislas Dehaene
email: dehaene@shfj.cea.fr

A Jobert

L Naccache

P Ciuciu

J B Poline

D Le Bihan

L Cohen

Letter binding and invariant recognition of masked words: Behavioral and neuroimaging evidence

Fluent readers recognize visual words across changes in case and retinal location, while maintaining a high sensitivity to the arrangement of letters. To evaluate the automaticity and functional anatomy of invariant word recognition, we measured brain activity during subliminal masked priming. By preceding targets words with an unrelated prime, a repeated prime, or an anagram made of the same letters, we separated letterlevel and whole-word codes. By changing the case and the retinal location of primes and targets, we evaluated the invariance of those codes. Our results indicate that an invariant binding of letters into words is achieved unconsciously through a series of increasingly invariant stages in the left occipito-temporal pathway.

Considerable machinery lies behind the seemingly simple feat of word recognition (Posner & McCandliss, 1993). First, words must be recognized in spite of changes in font, size and retinal location. Second, letters must be bound together in a specific order, because different words can be written with the same letters. The present research had two aims: first, to clarify the cerebral stages of processing that lead to invariant word recognition, and second, to examine whether those stages can proceed in the absence of consciousness.

In literate adults, an extended strip of the left fusiform gyrus activates whenever visual words are presented [START_REF] Cohen | The visual word form area: Spatial and temporal characterization of an initial stage of reading in normal subjects and posterior split-brain patients[END_REF][START_REF] Cohen | Language-specific tuning of visual cortex? Functional properties of the Visual Word Form Area[END_REF][START_REF] Nobre | Word recognition in the human inferior temporal lobe[END_REF]. This region, which has been termed the Visual Word Form Area (VWFA), is responsive only to written words, not to spoken words [START_REF] Dehaene | The visual word form area: a prelexical representation of visual words in the fusiform gyrus[END_REF]. Its lesioning results in a severe word identification impairment, pure alexia, which is restricted to the visual modality [START_REF] Leff | The functional anatomy of single-word reading in patients with hemianopic and pure alexia[END_REF]. Thus, it is a plausible candidate for the neural basis of invariant visual word recognition.

To further specify the nature of word coding in the VWFA, we used the priming method [START_REF] Grill-Spector | fMR-adaptation: a tool for studying the functional properties of human cortical neurons[END_REF]Naccache & Dehaene, 2001). Using an fMRI version of the masked priming paradigm [START_REF] Forster | Repetition priming and frequency attenuation in lexical access[END_REF], we previously observed that, when the same visual word is presented twice, first as a subliminal prime and then as a visible target, less activation is observed in the VWFA than when two different words are presented [START_REF] Dehaene | Cerebral mechanisms of word masking and unconscious repetition priming[END_REF]. A possible interpretation of this finding, based on single-cell studies in monkeys [START_REF] Miller | A neural mechanism for working and recognition memory in inferior temporal cortex[END_REF], is that a population of neurons has been partially habituated by the presentation of the initial subliminal prime, and does not react as much when the same word appears as target.

By varying the degree of similarity between prime and target, one can then evaluate the selectivity of the coding of words. In our previous work, we observed that priming in the VWFA occurred even when there was a change of case between the prime and the target (e.g. prime RADIO, target radio). This suggests that this area achieves case invariance (Dehaene et al., 2001). Here, we extend this work by examining whether the VWFA abstracts away from retinal features of the visual stimulus, and responds to single letters or to larger units.

Experiment 1 searched for regions with genuine case-invariant recognition. Most letters have similar shapes in upper and lower case (e.g. O-o). Thus, cross-case repetition priming effect might be explained by a generic capacity for size-invariant shape recognition, without implying a specialization for word recognition. However, some letters, such as A and a, have so radically different shapes in upper and lowercase that their correspondence is essentially arbitrary [START_REF] Posner | Chronometric analysis of classification[END_REF]. Experiment 1 tested whether cross-case repetition priming occurred for words composed exclusively of such letters (e.g. RAGE-rage).

Only regions genuinely engaged in case-invariant word recognition should be able to detect the identity of upper and lowercase words written solely with visually dissimilar letters [START_REF] Bowers | Orthographic, phonological, and articulatory contributions to masked letter and word priming[END_REF][START_REF] Humphreys | Orthographic processing in visual word identification[END_REF].

In experiment 2, we then studied whether priming in those regions depends on the presence of individual letters or of larger constituents. This was achieved using "circular anagrams", pairs of words that can be transformed into each other by moving a single letter from front to back (e.g. range-anger; emit-mite). By priming a French word such as reflet ("mirror image") with its anagram TREFLE ("clubs"), we could repeat almost all of the same letters while presenting two unrelated words. By shifting the prime by one letter relative to the target, it was even possible to repeat almost all letters at the same retinal location, still without presenting the same word. If the VWFA is sensitive only to a word's component letters, it should show priming whenever letters are repeated, whether or not they compose a different word. This would fit with the hypothesis that only fragments of words can be extracted under conditions of subliminal presentation [START_REF] Abrams | Parts outweigh the whole (word) in unconscious analysis of meaning[END_REF]. If, however, a wholeword code can be extracted subliminally, then we would expect greater priming when a word is repeated than when it is preceded by its anagram. If such whole-word repetition effects survives a shift in the position of individual letters, it would further demonstrate that the neural code in the VWFA is capable of some degree of spatial invariance.

Method

Participants. 26 right-handed native French speakers participated (experiment 1: 2 men and 8 women, average 24 years; experiment 2: 5 men and 11 women, average 22 years). All subjects gave written informed consent. The study was approved by the regional ethical committee for biomedical research.

Procedure for experiment 1. Fourteen words were made of letters whose shape is similar in upper and lower case (Cc, Ff, Kk, Oo, Pp, Uu, Vv) and fourteen were made of dissimilar letters (Aa, Ee, Gg, Rr, Tt). They were matched in length (3-4 letters; means 3.36 and 3.43, t(26)=0.37) and frequency (means log 10 frequency 1.26 and 1.10, t(26)=0.39). All were monosyllabic. On each trial, a sequence of fixation point, mask, prime, mask and target was presented as in [START_REF] Dehaene | Cerebral mechanisms of word masking and unconscious repetition priming[END_REF] (figure 1). The prime and target words always appeared in a different case, randomized across trials. Because of the small number of available words, it was not possible to use a classical task such as semantic classification. Rather, participants classified targets into those containing the letters A or E, and those containing the letters O or U. On non-repeated trials, the prime was a word of the same length drawn randomly from the other set of words, ensuring that the prime and target did not share a single letter.

The event-related fMRI protocol comprised 5 randomly intermixed event types. Four trial types were defined by the 2 x 2 combination of two factors, prime-target relation (same or different words) and letter similarity (target from the "similar" or "dissimilar" set). In a fifth trial type (baseline), only the masks were presented, and participants were told to rest for a few seconds. Trials were presented at a 2.4 s interval in 4 runs of 150 trials (30 of each type).

Procedure for experiment 2. Twenty pairs of circular anagrams appeared as both primes and targets (e.g. the French words TREFLE and REFLET; 4-6 letters, mean 4.83; 1-3 syllables, mean 1.73; mean log 10 frequency 0.66). This restricted set of target words precluded using the same task as in experiment 1. Rather, participants classified the target words into bisyllabic words versus other words (with 1 or 3 syllables).

The fMRI protocol comprised 6 randomly intermixed event types defined by factors of prime-target relation and relative location (plus the same baseline condition as in experiment 1). First, primes and targets could be the same word (SW), or two anagrams (AN), or two different words (DW). Second, primes and targets could be presented at the same location (SL) or shifted by one letter (different location or DL; see figure 2). In each of 4 fMRI runs, 180 trials were presented, defining 6 conditions for fMRI analysis (C1 = 30 SW-SL words, C2= 30 SW-DL, C3 = 30 AN-SL, C4 = 30 AN-DL, C5 = 15 DW-SL + 15 DW-DL, and C6 = 30 baseline trials).

Assessment of prime visibility. After scanning, while still in the scanner, participants were told of the presence of primes and performed a 2-alternative forced-choice identification task.

On each trial, a stimulus sequence identical to the main experiment was followed by a choice between two horizontally presented words. One word was identical to the prime, the other was unrelated and did not share any letter with the prime. Performance was at chance level (experiment 1: 53.8% correct, p=0.14; experiment 2: 51.4% correct, p=0.40), and participants denied seeing any primes.

Functional magnetic resonance imaging. We used a 3-Tesla whole body system (Bruker, Germany) and a gradient-echo echo-planar imaging sequence sensitive to brain oxygen-level dependent (BOLD) contrast (26 contiguous axial slices, 4.5 mm thickness; TR = 2.4 seconds). Data processing, performed with SPM99 software, included corrections for EPI distortion, slice acquisition time and motion; normalization; smoothing (8 mm); fitting with a linear combination of functions derived by convolving a canonical hemodynamic response function and its time derivative with the known time series of the stimulus types; and randomeffect group analysis. Effects of subliminal priming on BOLD activation were assessed within small regions of interest rather than using a whole-brain search. We first identified voxels involved in word processing in experiment 1 by contrasting word-present trials with baseline trials (voxel p<0.01, cluster-extent p=0.05 corrected across the brain volume). This initial contrast isolated 1155 voxels (bilateral occipital, supramarginal, central, precentral and cerebellar cortices as well as left fusiform and supplementary motor regions). Using the small volume correction of SPM, those voxels were then searched for significant priming effects.

We examined the main effect of repetition (less activation to repeated than to unrepeated trials), the simple main effects of repetition restricted to similar or to dissimilar sets, and the conjunction of those two independent contrasts. In turn, the voxels that showed case-invariant priming in experiment 1 were used as a mask to constrain the search for priming effects in experiment 2. Because different subjects were used, this mask was smoothed at 8 mm. It was also symmetrized to avoid biasing the search towards the left hemisphere. In the end, 256 bilateral fusiform and parietal voxels derived from experiment 1 were searched for priming in experiment 2.

All comparisons reported in the table were tested at voxel p<0.02 and cluster-extent p<0.05 corrected for multiple comparisons across the small volume. Conjunctions were tested at voxel p<0.01 and were masked by the corresponding contrasts at p<0.02. Note that, given enough statistical power, subliminal priming effects reach significance in a whole-brain search and with the high thresholds conventionally used in fMRI. For instance, contrasting the different-case same-word and different-word conditions that were replicated in the present experiments 1 and 2 and in our earlier report [START_REF] Dehaene | Cerebral mechanisms of word masking and unconscious repetition priming[END_REF], for a total of 36 subjects, subliminal priming was observed in a left fusiform cluster of 27 voxels (voxel p<0.001, cluster-extent p=0.002, corrected for whole-brain search; peaks at -48, -48, -12, Z=4.55, and -44, -60, -8, Z=4.24).

Activations curves were obtained using a non-parametric regularized least square estimate of the event-related time course (Ciuciu et al., submitted). Percent signal change was then calculated by subtracting the mean peak activation, observed at 4.8 seconds following the stimulus, and the mean baseline activation value measured during a 4.8 second interval prior to stimulus presentation. Error bars indicate the between-subject standard error of the mean of each condition relative to the subject's grand mean.

Results

Invariance for case. Experiment 1 examined cross-case repetition priming for words made of letters with similar or different shape in upper and lower case (figure 1). Behaviorally, response times (RTs) were 23 ms faster for repeated words than for non-repeated words, F(1,9)=33.9, p=0.0003. There was no main effect of similarity (p>0.93) and no interaction of similarity and repetition (p>0.14). The repetition effect was significant with similar words (20 ms, F(1,9)=18.4, p=0.002) and with dissimilar words (26 ms, F(1,9)=38.9, p=0.0002). This replicates earlier results [START_REF] Bowers | Orthographic, phonological, and articulatory contributions to masked letter and word priming[END_REF][START_REF] Humphreys | Orthographic processing in visual word identification[END_REF], and indicates that a case-invariant representation, which has learned the arbitrary mapping between upper and lower case, was activated by the subliminal words.

In fMRI, reduced activation on repeated trials was seen in the left fusiform gyrus and, to a lesser extent, the right fusiform gyrus and the left parietal region (table 1). The large left fusiform cluster (antero-posterior coordinates -76 to -44 mm), most of which fell within the occipito-temporal sulcus at the border of the fusiform gyrus proper, replicated closely our earlier finding of case-independent priming [START_REF] Dehaene | Cerebral mechanisms of word masking and unconscious repetition priming[END_REF]. Conjunction analysis showed that the left fusiform, but not the right fusiform, showed priming on both the similar and the dissimilar conditions (figure 1). In separate contrasts, priming with similar letters was significant in the left fusiform gyrus (67 voxels) and the left parietal cortex (21 voxels), and fell short of significance in the right fusiform gyrus (8 voxels, p=0.066). With dissimilar letters, priming reached significance in the left fusiform gyrus only (coordinates -44, -56, -12; 10 voxels). The interaction of word repetition with letter similarity never reached significance, however, even in the right fusiform gyrus (t=0.93, n.s.). Thus, the results indicate case invariance in the left fusiform gyrus, and merely suggest, but do not prove, that the right fusiform may be sensitive to visual similarity rather than to genuine case invariance.

Letter binding and invariance for letter position. Experiment 2 used a 3x2 design with prime type (same word, anagram, or different word), and retinal location (same or different) as factors (figure 2). An ANOVA on median RTs revealed a main effect of prime type (F(2,30)=5.67, p=0.008). The same-word condition was 9 ms faster than the anagram condition (F(1,15)=6.82, p=0.020) and 11 ms faster than the different-word condition (F(1,15)=10.4, p=0.006). The latter conditions did not differ (F<1). There was no effect or interaction involving location (all Fs<1). In particular, repetition priming remained significant when the retinal locations of the prime and target were shifted by one letter (12 ms effect, F(1,15)=5.26, p=0.037). Conversely, there was no priming between a word and its anagram, even when most letters were presented at the same retinal location (4 ms effect, F<1). Thus, behavioral priming depended exclusively on a location-invariant representation of the whole word. FMRI, however, revealed a subdivision of the VWFA into at least two subareas (figure 2,table 1). First, in the left and right posterior fusiform, activation was reduced only in two conditions: when the same word was presented at the same location, or when an anagram was presented at a location shifted by one letter position. Those are the only cases in which the same letters were repeated at the same retinal location. Thus, those regions may comprise "letter detectors" tuned the presence of a given letter at a specific retinal location [START_REF] Peressotti | Letter-position coding in random constant arrays[END_REF].

Second, more anteriorily in the middle fusiform gyrus, activation was reduced whenever the same word was presented twice, even when shifted by one letter location. This suggested that location-invariance was achieved in this region. Location-invariant priming was observed mainly in the left fusiform gyrus, while the right homolog region showed a mixture of location-specific and location-invariant responses within the same voxel (figure 2C, right column).

To confirm that the left posterior and middle fusiform were functionally different, we extracted their peak activation relative to baseline and submitted them to an ANOVA with factors of region and conditions (5 levels; see fMRI Methods). There was indeed a significant condition X region interaction (F(4,60)=3.15, p=0.020). Targeted contrasts revealed locationspecific letter priming in the posterior (F(1,15)=8.00, p=0.013) but not the middle fusiform (F<1), and location-invariant word priming in the anterior (F(1,15)=6.32, p=0.024) but not the posterior fusiform (F<1).

Finally, there was some suggestion of another more anterior subdivision. Within the location-invariant region, the middle portion of the left fusiform gyrus (y=-56) showed identical priming in the same-word and anagram conditions. This suggests that only fragments of the words, common to the words and their anagrams, were coded at this level. However, the more anterior subpeak of this cluster showed more priming for same-words than for anagrams (F(1,15)=4.92, p=0.042). This paralleled response times and suggested that wholeword coding was beginning to emerge at that stage. This result should be interpreted cautiously, however, because the condition X region interaction failed to reach significance (F<1) when middle and anterior fusiform activations were entered into a common ANOVA.

Discussion

The present findings demonstrate the potential of joint behavioral and neuroimaging priming studies for the dissection of stages of processing. Our previous work [START_REF] Dehaene | Cerebral mechanisms of word masking and unconscious repetition priming[END_REF] dissociated two levels of word priming: the right occipital cortex was sensitive only to the repetition of the same physical stimuli, suggesting a coding of low-level visual features, while an extended strip of left ventral occipito-temporal cortex showed cross-case priming, suggesting a more abstract coding. The present results clarify the contribution of this region to word recognition. Experiment 1 indicates that this region is not merely coding for the visual similarity of upper and lowercase words. Rather, it encodes the arbitrary mapping between upper and lowercase letters. Furthermore, experiment 2 shows that this region can be decomposed into at least two smaller subareas. The posterior subpart, observed bilaterally, is not invariant for location: it shows priming only when the same letters are presented at the same retinal location. Thus, this region may contain the location-specific letter detectors postulated by some psychological models [START_REF] Peressotti | Letter-position coding in random constant arrays[END_REF]. Conversely, we found priming across retinal locations in the middle fusiform gyrus, mostly in the left hemisphere. The suggestion that this area holds a location-invariant representation of words fits with previous fMRI and ERP studies, which indicate that words presented to the left or right hemifield, although initially processed in distinct retinotopic visual areas, eventually converge towards the left occipito-temporal region [START_REF] Cohen | The visual word form area: Spatial and temporal characterization of an initial stage of reading in normal subjects and posterior split-brain patients[END_REF][START_REF] Cohen | Language-specific tuning of visual cortex? Functional properties of the Visual Word Form Area[END_REF][START_REF] Mccandliss | Repetition effects in processing visual words: a high density ERP study of lateralized stimuli[END_REF].

Here, we only tested invariance for a shift of one letter position. Nevertheless, computationally, this already raises an invariance problem, because it changes radically the pattern of brain activity in early visual areas. The logic of the priming method suggests that the left middle fusiform region may contain neurons that recognize the same word regardless of small position changes, and that invariance is attained in a series of stages, with a case-invariant but location-specific representation of letters preceding a more globally invariant code.

Our results tentatively suggest a further antero-posterior subdivision within this location-invariant region. Most of this region shows identical priming to repeated words and to anagrams, but the most anterior voxels showed more priming for words than for anagrams.

Unfortunately, severe signal drop-off due to susceptibility artifacts in fMRI prevented us from pursuing priming effects more anteriorily in the ventral temporal cortex. The behavioral results, however, clearly showed priming sensitive exclusively to whole words, not their component letters. This implies that a whole-word level of coding must exist and can be activated by subliminal masked primes. The anterior fusiform region, which responds crossmodally to words, is a plausible candidate for the source of this whole-word priming effect [START_REF] Büchel | A multimodal language region in the ventral visual pathway[END_REF][START_REF] Nobre | Word recognition in the human inferior temporal lobe[END_REF].

A possible interpretation of the observed posteror-to-anterior progression is that neurons in the fusiform region are tuned to progressively larger and more invariant units, from visual features in extrastriate cortex to broader units such as graphemes, syllables, morphemes or even entire words as one moves anteriorily in the fusiform gyrus. A similar posterior-toanterior gradient is observed in the human ventral pathway when scrambling objects into progressively smaller fragments [START_REF] Lerner | A hierarchical axis of object processing stages in the human visual cortex[END_REF].

Our use of the priming method [START_REF] Grill-Spector | fMR-adaptation: a tool for studying the functional properties of human cortical neurons[END_REF]Naccache & Dehaene, 2001) relied on several assumptions. We argued that priming in the left fusiform was due to repetition of the same visual word form, but the prime and target also had the same phonology and the same meaning. In the future, the contribution of phonological and semantic codes could be tested by replicating the present experiments with homonyms or synonyms.

Nevertheless, words are unlikely to be coded phonologically or semantically in the middle fusiform gyrus because this region responds only to visual words and is thought to be prelexical, as demonstrated by its identical response to words and to pronounceable pseusowords [START_REF] Dehaene | The visual word form area: a prelexical representation of visual words in the fusiform gyrus[END_REF]. Furthermore, lesion of this region leads to pure alexia, a selective deficit of visual word identification with no semantic or phonological impairment [START_REF] Leff | The functional anatomy of single-word reading in patients with hemianopic and pure alexia[END_REF].

The priming method also supposes that repetition effects in fMRI reflect the local code within a brain area [START_REF] Grill-Spector | fMR-adaptation: a tool for studying the functional properties of human cortical neurons[END_REF]Naccache & Dehaene, 2001). However, the fact that the prime and target words are identical might be recognized first in a distant area, perhaps using a phonological or semantic code, and only later retro-propagated to the fusiform gyrus, either activating a case-invariant representation or even several case-specific representations. Although this possibility requires investigation, we consider it unlikely because neurophysiological studies indicate that subliminal masking reduces stimulusinduced activity to a transient bottom-up and prevents the top-down re-entry of information [START_REF] Lamme | Why visual attention and awareness are different[END_REF][START_REF] Lamme | Masking interrupts figure-ground signals in V1[END_REF]. Furthermore, we and others [START_REF] Grill-Spector | fMR-adaptation: a tool for studying the functional properties of human cortical neurons[END_REF][START_REF] Vuilleumier | Multiple levels of visual object constancy revealed by event-related fMRI of repetition priming[END_REF] have observed distinct priming effects within a few centimeters in the fusiform gyrus, which is hard to explain if top-down effects were propagating throughout the visual system.

It is indeed noteworthy that a greater variety of forms of priming was observed in fMRI than in response times, which were solely sensitive to an invariant whole-word code.

Assuming serially organized stages of processing, response times should have reflected the sum of priming effects evoked at each stage, including a contribution from location-specific letter representations. Response time, being a composite measure of many successive processes, may be inherently less sensitive than fMRI. Alternatively, the processing stage that gets reflected in behavior may depend on task instructions. Indeed, when the task is changed to letter-nonletter classification, behavioral priming is found to depend on a locationdependent single-letter stage [START_REF] Peressotti | Letter-position coding in random constant arrays[END_REF]. Similarly, when subjects are submitted to a strong speed pressure in a word classification task, performance depends on word fragments rather than the whole-word level [START_REF] Abrams | Parts outweigh the whole (word) in unconscious analysis of meaning[END_REF]. This suggests that subjects can orient their attention towards a specific processing stage, resulting in an increased influence of that stage on response times. Such task effects may tentatively explain why we observed priming in the left parietal region in experiment 1, but not in experiment 2 nor in our previous work [START_REF] Dehaene | Cerebral mechanisms of word masking and unconscious repetition priming[END_REF]. The letter content judgment used in experiment 1 may have put particular emphasis on this region, which is known to be activated during letter and phoneme monitoring tasks (e.g. [START_REF] Simon | Topographical layout of hand, eye, calculation and language-related areas in the human parietal lobe[END_REF].

We conclude by considering the implications of our results for subliminal processing.

It is of course difficult to establish the complete absence of consciousness of the primes.

Nevertheless, we used a very short prime duration (29 ms) and powerful forward and backward geometrical masks. Participants did not report seeing the primes and performed randomly on a forced-choice test. Thus, our findings suggest that advanced processing along the ventral visual pathway is possible in the absence of consciousness. Ventral visual activation to unseen stimuli has now been reported in a variety of paradigms including masking [START_REF] Dehaene | Cerebral mechanisms of word masking and unconscious repetition priming[END_REF], dichoptic fusion [START_REF] Moutoussis | The relationship between cortical activation and perception investigated with invisible stimuli[END_REF], and extinction in neglect patients [START_REF] Rees | Unconscious activation of visual cortex in the damaged right hemisphere of a parietal patient with extinction[END_REF][START_REF] Vuilleumier | Neural response to emotional faces with and without awareness: event-related fMRI in a parietal patient with visual extinction and spatial neglect[END_REF]. Thus, contrary to some proposals (e.g. [START_REF] Bar | Cortical mechanisms specific to explicit visual object recognition[END_REF], fusiform activity is not necessarily associated with conscious awareness.

Finally, a new implication of our results is that the binding of letters into words can be performed subliminally, since behavioral repetition priming was highly sensitive to letter order. This fits with neurophysiological recordings, which reveal a high selectivity of inferotemporal neurons to specific combinations of object parts even in anesthetized monkeys [START_REF] Tsunoda | Complex objects are represented in macaque inferotemporal cortex by the combination of feature columns[END_REF]. [START_REF] Singer | Consciousness and the structure of neuronal representations[END_REF] distinguishes two forms of binding: a rigid binding, achieved by dedicated neurons that have learned to respond to particular combinations, and a transient creation of novel combinations, perhaps mediated by temporal synchrony and more tightly associated with conscious processing. Since words are highly overlearned, our results bear only on the first form of binding and thus contribute to refine, but are not necessarily inconsistent with, the view that higher forms of binding require consciousness [START_REF] Crick | Toward a neurobiological theory of consciousness[END_REF].

Figure 1 .

 1 Figure 1. Behavioral and brain-imaging correlates of case-invariant word recognition. A, schematic representation of our masking paradigm. B, response times showed repetition priming independent of the visual similarity of the upper and lowercase letters. C, statistical

Figure 2 .

 2 Figure 2. Dissociation of location-dependent and location-invariant stages of word recognition. A, Experimental design. Using anagrams, we dissociated letter and word

Table 1 . Localization of significant fMRI repetition priming effects

 1

		x	y	z
	4.10	-44 -48 -12
	3.73	-52 -60 -8
	2.57	-44 -72 0
	3.21	-40 -44 60
	2.41	-36 -48 44

suppression for repeated words at same location (replicating experiment 1) conjunction of repetition effects for similar and dissimilar letters main effect of repetition suppression

	Area	# voxels in cluster	cluster-level p value (corrected)	Z value at local maximum
		EXPERIMENT 1
	left fusiform	65	< 0.00001
	left parietal	24	0.001
		EXPERIMENT 2
	conjunction

of repetition effects for repeated words at same or different location

	left fusiform	12	0.005
	left fusiform	13	0.004
	left fusiform	16

repetition suppression for same letters at same location

Acknowledgements

We thank Nancy Kanwisher and Sid Kouider for comments. Supported by INSERM, CEA, and the McDonnell Foundation.