Claude Paul Malvy
email: claude.malvy@igr.fr

PhD Christine Vauthier Christine Vauthier

ANTISENSE STRATEGIES: DELIVERY OF OLIGONUCLEITIDES BY NON-VIRAL NANOPARTICLES

come

Introduction

Antisense oligonucleotides (AS-ODN) and small interfering RNA (siRNA) are very promising molecules for the development of specific treatment against cancer [START_REF] Burnett | RNA-based therapeutics: current progress and future prospects[END_REF][START_REF] Gavrilov | Therapeutic siRNA: principles, challenges, and strategies[END_REF][START_REF] Miele | Nanoparticle-based delivery of small interfering RNA: challenges for cancer therapy[END_REF][START_REF] Sen | Nucleic acid-based approaches to STAT inhibition[END_REF]. However, their systemic in vivo delivery is challenging. The active molecules need to be protected against degradation by nucleases while they also need to be helped to go across the many biological barriers found on their way between the site of administration and the cell cytoplasm where the target molecules are located [START_REF] Gavrilov | Therapeutic siRNA: principles, challenges, and strategies[END_REF]. Chemical modifications of AS-ODN and siRNA are known to improve their stability in vivo but this is often at the expense of a loss of their activity [START_REF] Gavrilov | Therapeutic siRNA: principles, challenges, and strategies[END_REF][START_REF] Watts | Chemically modified siRNA: tools and applications[END_REF]. Chemical modifications on their own are not sufficient to let the molecule to go across all barriers and to penetrate into cells. Alternative methods consist in associating the natural molecules which corresponds to the phosphodiester oligonucleotide with a carrier that will serve as transporter and transfection agent. Nucleic acids as anticancer drugs offer many possibilities. According to the properties of nucleic acids most of their applications are either using the coding properties of nucleic acids or the Watson Crick hybridization properties of nucleic acids. Coding properties fall in the Gene Therapy category and hybridization properties in the oligonucleotide (siRNA, AS-ODN) category. In cancer therapy the gene delivery in cancer cells inside a tumor requires a very high transfection efficiency which can be obtained only by using modified viruses to deliver genes (foreign genes have to be expressed in all cells of the tumor). Oligonucleotide with much shorter sizes than genes can be delivered at thousands copies in these same cells by using chemical vectors with lower transfection efficiency but which do not present the risks endogenous to modified viruses.

The number of proposed systems is impressive in relation to the youth of this area of research [START_REF] Toub | Innovative nanotechnologies for the delivery of oligonucleotides and siRNA[END_REF][START_REF] Morille | Progress in developing cationic vectors for non-viral systemic gene therapy against cancer[END_REF][START_REF] Wang | The design of vectors for RNAi delivery system[END_REF][START_REF] De Martimprey | Polymer nanocarriers for the delivery of small fragments of nucleic acids: oligonucleotides and siRNA[END_REF][START_REF] Howard | Delivery of RNA interference therpapeutics using polycation-based nanoparticles[END_REF][START_REF] Rudzinski | Chitosan as a carrier for targeted delivery of small interfering RNA[END_REF][START_REF] Zhou | Nanoparticle-Based Delivery of RNAi Therapeutics: Progress and Challenges[END_REF][START_REF] Shu | Thermodynamically stable RNA three-way junction for constructing multifunctionnal nanoparticles for delivery of therapeutics[END_REF]. In general, they achieved the first challenge which is to protect the nucleic acid fragments against the degradation by nucleases and other provided data are a rather rough description of their physicochemical characteristics. Only few systems were evaluated ahead to demonstrate their potential to deliver siRNA or AS-ODN producing an anticancer effect in vivo using appropriate animal models. The occurrence of a reduction of tumor growth was related to the expected antisense activity of the antisense agent associated with the carrier.

The results were extremely promising motivating the rapid development of clinical tests in human with the few formulations that have passed all the preclinical evaluations stages [START_REF] Zhou | Nanoparticle-Based Delivery of RNAi Therapeutics: Progress and Challenges[END_REF][START_REF] Bruno | Using drug-exipient interactions for siRNA delivery[END_REF][START_REF] Lee | Recent developments in nanoparticle-based siRNA delivery for cancer therapy[END_REF]. It remains a very hot topic that is developing very fast through the identification of new potential targets and improving the delivery methods [START_REF] Maier | Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics[END_REF]. In this review, the first part we will give a brief description of the different systems that are now in clinics in human or close to clinical testing. The second part described the biological activity of only few systems that are not yet in clinics but showed very promising effect on cancers caused by junction oncogenes.

Delivery systems for siRNA and AS-ODN by the systemic route that have reached advanced stages of development.

Only few synthetic delivery systems have reached an advanced stage of development. The table 1 and 2 give a description of their composition and their structure is described in the figure 1. As indicated in Table 1 and2, several were already included in clinical trials in human for the delivery of siRNA and AS-ODN by the systemic route. It is noteworthy that the different proposed systems displayed a large variety of composition and structures that matched different groups of nanotechnologies developed as nanomedicine. Those made of polymers including nanospheres and nanocapsules were designed by polymerization of alkylcyanoacrylates used as monomers [START_REF] Maksimenko | In vivo potentialities of EWS-FLI-1 targeted antisense oligonucleotides-nanospheres complexes[END_REF][START_REF] Toub | Efficacy of siRNA-nanocapsules targeted against the EWS-FLI-1 oncogene in Ewing Sarcoma[END_REF][START_REF] De Martimprey | New core-shell nanoparticules for the intravenous delivery of siRNA to experimental thyroid papillary carcinoma[END_REF] and by methods based on the precipitation of an hydrophobic polymer in very well defined conditions including emulsification-followed by solvent evaporation [START_REF] Nafee | Chitosan-coated PLGA nanoparticles for DNA/RNA delivery: effect of the formulation parameters on complexation and transfection of antisense oligonucleotides[END_REF][START_REF] Tahara | Oral nuclear factor-kB decoy oligonucleotides delivery system with chitosan modified poly(D,L-lactide-co-glycolide) nanospheres for inflammatory bowel disease[END_REF]. It is noteworthy that many systems form thanks to the self assembly of their basic components. This can lead to the formation of particles having well defined structures and even, in some cases, internal crystal-like structures (Figure 1). proposed in the literature. A: AtuPLEX [START_REF] Strumberg | Phase I clinical development of Atu027, a siRNA formulation targeting PKN3 in patients with advanced solid tumors[END_REF], B: Stable Nucleic Acid Lipid Nanoparticles (LNP) [START_REF]RNAitherapeutic web site[END_REF], C: Atecollagen-siRNA complexes [START_REF] Takeshita | Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo[END_REF] , D: Poly(alkylcyanoacrylate) (PACA) nanocapsules [START_REF] Toub | Efficacy of siRNA-nanocapsules targeted against the EWS-FLI-1 oncogene in Ewing Sarcoma[END_REF][START_REF] Toub | Antisense oligonucleotides nanocapsules efficiently inhibit EWS-Fli-1 expression in a Ewing's sarcoma model[END_REF], E: PACA nanospheres type I [START_REF] Maksimenko | In vivo potentialities of EWS-FLI-1 targeted antisense oligonucleotides-nanospheres complexes[END_REF][START_REF] Zandanel | Poly(isobutylcyanoacrylate) Nanoparticles Decorated with Chitosan: Effect of Conformation of Chitosan Chains at the Surface on Complement Activation Properties[END_REF], F: PACA nanospheres type II [START_REF] De Martimprey | New core-shell nanoparticules for the intravenous delivery of siRNA to experimental thyroid papillary carcinoma[END_REF][START_REF] Ramon | siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma[END_REF][START_REF] Zandanel | Poly(isobutylcyanoacrylate) Nanoparticles Decorated with Chitosan: Effect of Conformation of Chitosan Chains at the Surface on Complement Activation Properties[END_REF], G: PACA nanospheres type II targeted [START_REF] Ramon | siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma[END_REF], H: Nanospheres with cyclodextrines [START_REF] Davis | Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles[END_REF], I: Nanospheres with dendrimers [START_REF] Taratula | Surface-engineered targeted PPI dendrimer for efficient intracellular and intratumoral siRNA delivery[END_REF]. DPhyPE: 1,2-diphytanoyl-sn-glycero-3phosphoethanolamine [START_REF] Patil | Internally cationic polyamidoamine PAMAM-OH dendrimers for siRNA delivery: effect of the degree of quaternization and cancer targeting[END_REF][START_REF] Taratula | Surface-engineered targeted PPI dendrimer for efficient intracellular and intratumoral siRNA delivery[END_REF] * NP : not published yet

The association of the nucleic acid is achieved by promoting ionic interactions between their negative charged given by the phosphate group and positively charged components of the carrier [START_REF] Howard | Delivery of RNA interference therpapeutics using polycation-based nanoparticles[END_REF][START_REF] Garcia-Fuentes | Chitosan-based drug nanocarriers: where do we stand?[END_REF][START_REF] Al-Qadi | Chitosan and its derivatives as nanocarriers for siRNA delivery[END_REF][START_REF] Vauthier | Chitosan-based nanoparticles for in vivo delivery of interfering agents including siRNA[END_REF] . The whole structure is also assembled by strong hydrophobic interactions between components which include fatty acid chains of lipids [START_REF] Bruno | Using drug-exipient interactions for siRNA delivery[END_REF], the squalene motive of the squalenoylated siRNA [START_REF] Ali | Effects of silencing RET/PTC1 oncogene in papillary thyroid carcinoma by siRNA-squalene nanoparticles with and without fusogenic companion GALA-Cholesterol[END_REF] and the use of cyclodextrines [START_REF] Davis | Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles[END_REF].

To be active in vivo, nanomedicines designed to deliver gene silencing effectors need to remain stable in biological fluids during transportation from the site of administration down to the target site while it should dissociate to release the nucleic acid once it has reached the interior of the cell. The stability of systems needs to be tuned very precisely to achieve the right balance between these contradictory properties. For instance, over 50 different amino lipids were tested in the formulation of the nucleic acid lipid nanoparticles (LNP) which are developed as a delivery platform for siRNA to find the optimal compromise [START_REF] Jayaraman | Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo[END_REF]. This system is now under evaluation in clinics for various siRNA targets to treat diseases in the liver [START_REF] Haussecker | The Business of RNAi Therapeutics in 2012[END_REF].

In the case of the ATUPlex, the second advanced delivery platform which is also based on a lipid self assembly SiRNA are inserted into bilayers of cationic lipids. The whole complex which occurs as a very small particle is stabilized by chains of poly(ethylene glycol) (PEG).

Although used to insure the colloidal stability of the nanoparticles, PEG was used to decorate the surface of many types of carriers to confer them stealth properties. It is a method for camouflaging nanomedicine surfaces to hamper recognition of by the immune system of the host organism. In this way, the delivery system circulates in the blood compartment and reaches tumors thanks to the enhanced permeation and retention effect or it can be used to target vascular endothelial cells. Recently it was shown that PEG is not the only hydrophilic polymer that can be used to camouflage the surface of nanomedicines. Polysaccharides can be used too to design stealth nanoparticles provided that the chains adopt a hair configuration on the surface of the nanomedicine and that the grafting density is high enough [START_REF] De Martimprey | New core-shell nanoparticules for the intravenous delivery of siRNA to experimental thyroid papillary carcinoma[END_REF][START_REF] Alhareth | Pharmacokinetics and tissue biodistribution in rats of doxorubicin loaded Poly(isobutylcyanoacrylate) nanoparticles prepared by redox radical emulsion polymerization[END_REF][START_REF] Ramon | siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma[END_REF].

To improve delivery into cells, several systems were including a ligand to promote transfection by fusion with the cell membrane. For instance, the fusion peptide GALA was inserted in the structure of the squalenoylated delivery platform using a cholesterol moiety grafted on this peptide [START_REF] Ali | Effects of silencing RET/PTC1 oncogene in papillary thyroid carcinoma by siRNA-squalene nanoparticles with and without fusogenic companion GALA-Cholesterol[END_REF]. In the AtuPLEX, the fusogenic molecule is the 1,2-diphytanoylsn-glycero-3-phosphoethanolamine (DPhyPE) [START_REF] Strumberg | Phase I clinical development of Atu027, a siRNA formulation targeting PKN3 in patients with advanced solid tumors[END_REF]. Another type of decoration of the particle surface includes the insertion of a targeting ligand. It was transferrin in the case of the cyclodextrin-based system [START_REF] Davis | Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles[END_REF] or a monoclonal antibody associated on the surface of poly(alkylcyanoacrylate) nanoparticles [START_REF] Ramon | siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma[END_REF]. The platforms were designed in such a way that the targeting moiety can be interchangeable. In the cyclodextrin based system, cyclodextrins are exhibited on the surface of the carrier and serve as anchor for the targeting ligand onto which an adamantane residue is grafted (Figure 2A). Attachment of the targeting ligand on the poly(alkylcyanoacrylate) nanoparticles was achieved using a biotin-avidin-biotin sandwich system [START_REF] Segura-Sánchez | Synthesis and characterization of functionalized poly(gamma-benzyl-L-glutamate) derivates and corresponding nanoparticles preparation and characterization[END_REF]. The platform consists in biotinylated nanoparticles and targeting ligands are biotynylated as well. The assembly between these two partners is achieved by simple incubation with avidin (Figure 2B). The number of targeting antibody molecules associating with a single nanoparticle was low, 3 to 5 units, but this low number was sufficient to increase the delivery of the cargo to target sites in vivo after intravenous injection and to improve treatment efficacy [START_REF] Ramon | New targeted approaches for the treatment of Ewing's sarcoma family of tumors[END_REF]. The low number of targeting ligand required is an advantage regarding the modification of surface properties of the carrier introduced by the addition of the targeting ligands. Indeed, it remained low enough to preserve the stealthiness of the nanoparticles as acknowledged by the low capacity to activate the complement system of both the targeted and the parent nanoparticles and by the increased delivery of the cargo to tumor with the targeted nanoparticles. Surface properties of carriers are determinant for the in vivo fate of the nanoparticles.

Figure 2: Mode of association of a targeting ligand to delivery systems for siRNA. A: Biotinavidin-biotin sandwich system [START_REF] Ramon | siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma[END_REF][START_REF] Segura-Sánchez | Synthesis and characterization of functionalized poly(gamma-benzyl-L-glutamate) derivates and corresponding nanoparticles preparation and characterization[END_REF], B: adamantane-cyclodextrin association [START_REF] Davis | Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles[END_REF], C: chemical coupling [START_REF] Taratula | Surface-engineered targeted PPI dendrimer for efficient intracellular and intratumoral siRNA delivery[END_REF]. Targeting ligands are easily interchangeable on the different delivery platfoms.

Although surface properties must be designed precisely to achieve the correct biodistribution of the cargo, results from an extensive study of the influence of surface properties on the cytotoxicity and genotoxicity of carriers have pointed out that they can compromise the safety of the carrier [START_REF] Shah | Genotoxicity of different nanocarriers: possible modifications for the delivery of nucleic acids[END_REF]. A clear correlation was highlighted between the surface charge of the particles and their cytotoxicity. The same observation was reported regarding the evaluation of the genotoxicity of the particles through the formation of micronuclei in cells in contact with non cytotoxic concentration of the particles. It was suggested that newly designed carriers should display neutral or negatively charged surface to reduce such a risk of genotoxicity.

Examples of biological activity

All the nanoparticles-siRNA or antisense oligonucleotide listed in the table have been used

sucessfuly in biological systems either in cell culture or in vivo or both. Taking into account the diversity of their chemical nature it is very difficult to describe a unified common mechanism of action in vivo. However, in the cancer field one common point could be the Enhancement of Permeability Retention effect (EPR effect) which facilitates the uptake of nanoparticles by tumors taking into account the fenestrated nature of the tumor vascular endothelium. As examples we describe here some of the biological results obtained with squalenes, poly(alkylcyanoacrylate) nanoparticles and lipoplexes A siRNA targeting the fusion oncogene RET/PTC1, which is the most prevalent type of gene rearrangement found in papillary thyroid carcinoma, at the level of the mRNA junction of RET and PTC1 was coupled to squalene. It formed 200 nm nanoparticles as already shown [START_REF] Raouane | Synthesis, characterization, and in vivo delivery of siRNA-squalene nanoparticles targeting fusion oncogene in papillary thyroid carcinoma[END_REF]. Ali et al. [START_REF] Ali | Effects of silencing RET/PTC1 oncogene in papillary thyroid carcinoma by siRNA-squalene nanoparticles with and without fusogenic companion GALA-Cholesterol[END_REF] found that these nanoparticles carrying the siRNA inhibit in vivo with specificity RET/PTC1 at the level of mRNA and protein as well as tumor growth. The in vivo model used was a mouse xenograft of human BHP 10-3 SC mice cells expressing the RET/PTC1 oncogene. The total dose of siRNA delivered intravenously was 0.5 mg/Kg. At the same dose the same siRNA without squalene has no effect on tumor growth and expression of RET/PTC1.

Nanospheres of PIBCA coated with chitosan and carrying a siRNA targeting RET/PTC1 were used by De Martimprey to treat mice having received a xenograft of tumorigenic murine cells stably expressing the human RET/PTC1. Either by intratumor [START_REF] De Martimprey | siRNA nanoformulation against the ret/PTC1 junction oncogene is efficient in an in vivo model of papillary thyroid carcinoma[END_REF] or by intravenous delivery [START_REF] De Martimprey | New core-shell nanoparticules for the intravenous delivery of siRNA to experimental thyroid papillary carcinoma[END_REF] inhibition of RET/PTC1 expression and of tumor growth was obtained at a low dose of siRNA. Similar nanospheres have been used to carry either antisense oligonucleotide [START_REF] Maksimenko | In vivo potentialities of EWS-FLI-1 targeted antisense oligonucleotides-nanospheres complexes[END_REF] or a siRNA [START_REF] Ramon | siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma[END_REF] anti EWS/Fli-1 in preclinical models of Ewing sarcoma. With antisense oligonucleotides tumor (NIH3T3 cells expressing EWS/Fli-1) inhibition takes place after intratumor injection (5 mg/Kg total oligonucleotide dose) [START_REF] Maksimenko | In vivo potentialities of EWS-FLI-1 targeted antisense oligonucleotides-nanospheres complexes[END_REF] or after intravenous injection (30 mg/Kg total oligonucleotide dose) [START_REF] Elhamess | Antitumor vectorized oligonucleotides in a model of Ewing Sarcoma : Unexpected role of nanoparticles[END_REF]. With siRNA a 40 % inhibition of EWS/Fli1 in the tumor (A673 human Ewing cells) is obtained with the nanospheres but is increased to near 80 % when the nanospheres are coupled to an antibody against CD99, a biomarker of Ewing cells (100 µg total dose siRNA by IV injection). Interestingly, results from the group of Malvy [START_REF] Elhamess | Antitumor vectorized oligonucleotides in a model of Ewing Sarcoma : Unexpected role of nanoparticles[END_REF] show that, in addition to delivery, nanoparticles can sometimes erase unspecific polyanionic effects of nucleic acids.

PIBCA nanoscapsules were used with antisense oligonucleotides targeting EWS/Fli-1 in cell culture (NIH3T3 cells expressing EWS/Fli-1). Nanocapsules greatly increased the cellular uptake of oligonucleotides allowing the inhibition of EWS/Fli-1 and of the cell proliferation [START_REF] Toub | Antisense oligonucleotides nanocapsules efficiently inhibit EWS-Fli-1 expression in a Ewing's sarcoma model[END_REF]. The same nanocapsules were used to deliver siRNA targeting EWS/Fli-1 directly in the xenografted tumor [START_REF] Toub | Efficacy of siRNA-nanocapsules targeted against the EWS-FLI-1 oncogene in Ewing Sarcoma[END_REF]. At 1 mg/Kg total siRNA dose inhibition of EWS/Fli-1 expression as well as inhibition of tumor growth were observed.

Aleku et al. have used a siRNA targeting the protein kinase N3 in endothelial cells. This siRNA is associated to a lipoplex and the corresponding siRNA nanoparticle is called Atu027.

Antimetastic activity in different types of cancers in preclinical models has been shown by Aleku et al. [START_REF] Aleku | Atu027, a liposomal small interfering RNA formulation targeting protein kinase N3, inhibits cancer progression[END_REF] and Santel et al. [START_REF] Santel | Atu027 prevents pulmonary metastasis in experimental and spontaneous mouse metastasis models[END_REF].

Conclusion

Gene silencing approaches have now demonstrated their promise to develop efficient treatments of diseases caused by the abnormal over expression of a known gene. In vivo delivery of gene silencing mediating agents that include antisense oligonucleotides and siRNA remains the bottleneck for the success of treatments, but solutions are emerging thanks to the extremely active research aiming to find suitable delivery methods. As pointed out in this paper, several types of carriers have been identified and their clinical development is already on the way. Although tremendous progresses were made in a very short time, several challenges remain as we are still in the infant age developing therapeutic strategies based on gene silencing approaches. For instance, mechanisms by which the gene silencing activity is achieved in vivo need to be elucidated. It is also not understood so far why in vitro effect did not always correlate well with the observed in vivo effect and can even be contradictory in the sense that activity was only observed in vivo. We also need to improve our understanding of the biodistribution of nanocarriers that are designed to deliver antisense oligonucleotides and siRNA and their interactions with the immune system of the host. Another bottleneck can be the finding of relevant gene silencing mediators improving their specificity on the expression of the target gene and avoiding off target effects. In this regard, antisense oligonucleotides and siRNA targeting junction oncogenes that are unique in the diseased cells and not present in the healthy cells are probably advantageously targeted for the development of therapeutic approached based on this method. Today, many hopes are based on research conducted in this area. For sure nanomedicines will contribute further to the development of these compounds and their success proposing new treatments to patients.

Key terms

Antisense strategy: Classical drugs are protein inhibitors interacting directly with the active site of a protein. Antisense strategies aim at inhibiting with specificity the synthesis of a given protein. It can be triple helices at the level of DNA or double helices at the level of mRNA. Usually, these double helices lead to the mRNA cleavage and degradation through either RNaseH or RISC complex

Oligonucleotides: Short fragments of nucleic acids occurring as oligodesoxynucleotides and ribonucleic acids. These fragments can interfere with the expression of a specific gene thanks the complementarity of their sequence with the target gene or the target mRNA. In the cells, they are part of physiologic mechanisms occurring for the regulation of gene expression.

Antisense Oligonucleotide (AS-ODN): Short fragment of a single chain of oligodesoxynucleoride having a sequence complementary to a target gene or a target mRNA. They can be used to block the expression of the gene in the nucleus by formation of a triple helix on the complementary sequence of DNA found on the corresponding chromosome. They can compromise the production of a protein by blocking the translation of the mRNA forming a complex with the complementary sequence found on the target mRNA. In this case, the translation of the mRNA into the protein is compromised either because the mRNA is then degraded by the RNAseH or because the AS-ODN-mRNA complex formed hindered the association or the progression of the ribosome which is required for the synthesis of the protein.

Small interfering RNA (siRNA): Short fragment of double strands ribonucleic acid. One of the strand, the antisense strand, has a complementary sequence to that of the target mRNA. This strand associates with a group of proteins to form the RISC complex while the second strand, the sense strand, is degraded. Once formed, the RISC complex displays an RNAse activity against the mRNA complementary to the antisense strand of the siRNA. This leads to the degradation of the mRNA targeted by the siRNA hence to the inhibition of the production of the corresponding protein.

Non-viral nanoparticles:

In contrast with drug carriers designed from viruses, non-viral nanoparticles are drug carriers obtained by designing artificial nanoparticles from a drug and eventually other materials that include in general either lipids or polymers.

Chapter summary

-Gene silencing approaches have demonstrated high promise for the development of new treatments of diseases caused by the abnormal expression of a known gene. -Non-viral nanoparticles including lipid and polymer-based nanoparticles were identified to make possible in vivo delivery of biologically active gene silencing agents. -Clinical developments have started with several systems considering development of treatments against various cancers, viral infections and hypercholesterolemia. -The development of therapeutic strategies based on gene silencing approaches is still in its infant age and many challenges remain

Figure 1 :

 1 Figure 1: Schemes of the structures of several systems for the delivery of siRNA as they areproposed in the literature. A: AtuPLEX[START_REF] Strumberg | Phase I clinical development of Atu027, a siRNA formulation targeting PKN3 in patients with advanced solid tumors[END_REF], B: Stable Nucleic Acid Lipid Nanoparticles (LNP)[START_REF]RNAitherapeutic web site[END_REF], C: Atecollagen-siRNA complexes[START_REF] Takeshita | Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo[END_REF] , D: Poly(alkylcyanoacrylate) (PACA) nanocapsules[START_REF] Toub | Efficacy of siRNA-nanocapsules targeted against the EWS-FLI-1 oncogene in Ewing Sarcoma[END_REF][START_REF] Toub | Antisense oligonucleotides nanocapsules efficiently inhibit EWS-Fli-1 expression in a Ewing's sarcoma model[END_REF], E: PACA nanospheres type I[START_REF] Maksimenko | In vivo potentialities of EWS-FLI-1 targeted antisense oligonucleotides-nanospheres complexes[END_REF][START_REF] Zandanel | Poly(isobutylcyanoacrylate) Nanoparticles Decorated with Chitosan: Effect of Conformation of Chitosan Chains at the Surface on Complement Activation Properties[END_REF], F: PACA nanospheres type II[START_REF] De Martimprey | New core-shell nanoparticules for the intravenous delivery of siRNA to experimental thyroid papillary carcinoma[END_REF][START_REF] Ramon | siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma[END_REF][START_REF] Zandanel | Poly(isobutylcyanoacrylate) Nanoparticles Decorated with Chitosan: Effect of Conformation of Chitosan Chains at the Surface on Complement Activation Properties[END_REF], G: PACA nanospheres type II targeted[START_REF] Ramon | siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma[END_REF], H: Nanospheres with cyclodextrines[START_REF] Davis | Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles[END_REF], I: Nanospheres with dendrimers[START_REF] Taratula | Surface-engineered targeted PPI dendrimer for efficient intracellular and intratumoral siRNA delivery[END_REF]. DPhyPE: 1,2-diphytanoyl-sn-glycero-3phosphoethanolamine

Table 1 : More advanced Lipid based carriers developped for in vivo delivery of siRNA and ODN

 1

	Type	of	Composition	Figure	Nucleic acid	Disease		References
	carrier/Name of				(Status of development)
	technology/
	Company
	Cationic lipoplexe/		Atu027 Atufect01: multivalent cationic	A	23mer siRNA against PKN3 Delivery to endothelial cells	Solid Cancer Clinical trials		[30]
	AtuPLEX		lipid
	lipoplex/		DPhyPE: fusogenic helper lipid,
	Silence		MPEG 2000-DSPE
	Therapeutics		Molar ratio: 50/49/1
	Neutral liposomes/ LNP	Amino lipid chosen among more than 50 molecules,	B	Already several targets located in the liver -siRNA targeting hepatic transthyretin (TTR) (ALN-	Transthyretin Amyloide (clinical	[26,34]
	liposomes/		DSPC,		TTR01)	trial)
	Alnylam Pharma	cholesterol		-siRNA targeting VEGF (vascular endothelial	Liver cancer (clinical trial)
	Tekmica Pharma	PEG-lipid		growth factor) and KSP (kinesin spindle protein)
			Molar ratio: 50/10/38.5/1.5		(ALN-VSP02)
					-siRNA targeting the polo-like kinase 1, (TKM-	Solid cancer and lymphoma
					PLK1)	(clinical trial)
					-siRNA targeting apolipoprotein B, (TKM-ApoB)	Hypercholesterolemia	(clinical
					-siRNA targeting PCSK9 (ANL-PCS02	trials)
					-Viral RNA, Ebola infections (TKM-EBOLA)	Viral infections (Clinical trial)
					-siRNA targeting the hepatits B virus HBV
	Lipidoids		-98N 12 -5(1): five-tailed isomer of triethylenetetramine-	NP**	Cationic lipoid nanoparticle siRNA against PD-L1 gene,	Antiviral immunity in kupffer cells Prostate cancer	[35-37]
			laurylaminopropionate with a		double stranded RNA (dsRNA) activating expression
			free internal amine		gene P21.
			-Cholesterol
			-mPEG 2000 -C14 Glyceride.
			Molar ratio: 42:48:10
	Squalenoylated particles		Nanoaggregate formed from the self-assembly of squalenoylated	NP**	siRNA against a junction oncogene Ret/PTC 1	Radio induced thyroid cancer	[38,39]
			siRNA (prodrug)

*DPhyPE: 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine ** NP : Not published yet

Table 2 : More advanced polymer-based carriers developped for in vivo delivery of siRNA and ODN

 2

	Type	of	Composition		Ref. to	Nucleic acid		Disease	References
	carrier/Name of			Figure 1			(Status of development)
	technology
	Atelocollagen-siRNA complex:	Complexes of atelocollagen (product obtained from the	C	21-23 mer siRNA, siRNA directed against the alpha p110 subunit of	metastatic tumors in bone tissue	[40]
			peptidolysis of collagen) with		human	phosphoinositide	3-
			siRNA			hydroxykinase;
	Miniature		Complex between siRNA and	NP*	SiRNA against K-RAS G12D,	Pancreatic cancer,	[41]
	biodegradable		LODER polymer, nature not				Intratumoral administration
	matrix		disclosed
	Silenseed Ltd
	polyplexe		Poly-electrolyte between chitosan or modified complex	NP*	Very active research on various targets. Various diseases	[10,21-23]
			chitosan and siRNA
	Nanocapsules PACA		Polymer Poly(alkylcyanoacrylate)	vesicles	D	AS-ODN against the junction oncogene EWS-Fli 1.	Ewing sarcoma (pediatric bone cancer)	[17]
			envelope entrapping AS-ODN				(Research)
	Nanospheres		Chitosan decorated nanoparticle made of poly(D,L-lactide-co-	NP*	decoy-ODN targeted nuclear factor kappa B (NF-κB) (proinflammatory	Ulcerative (inflammatory disease of the colitis	[20]
			glycolide) (PLGA). Chitosan		cytokine)		bowel) Oral administration
			was adsorbed on the PLGA core
			in the course of formation by
			nanoprecipitation.
	Nanospheres		Core-corona nanoparticles made	E,F	siRNA against the junction oncogene	Radio-induced thyroid cancer
			of chitosan copolymers Poly(alkylcyanoacrylate)-		Ret/PTC AS-ODN or siRNA against EWS-Fli1	(Research) Ewing sarcoma (pediatric	[16,18,29]
								bone cancer) (Research)
	Targeted nanospheres		Core decorated with antibodies corona nanoparticle	G	siRNA against the junction oncogene EWS-Fli 1.	Ewing sarcoma (pediatric bone cancer) (Research)	[29]
	Targeted nanospheres /		Self-assembly of 4 components siRNA,	H	siRNA reducing expression of RRM2 (previously denoted	Myeloid Leukemia clinical trial	registration	[25, 42]
	Calando Pharma		polycyclodextrine-including		siR2B15) (CALAA-01)	number NCT 00689065
			positively charged linkaged
			between cyclodextrins,
			PEG-adamantane, Transferrin-
			PEG-adamantane

Table 2 : More advanced polymer-based carriers developed for in vivo delivery of siRNA and ODN (Continued)

 2

	Type	of	Composition			Ref. to	Nucleic acid	Pathologies	References
	carrier/Name of				Figure 1
	technology
	Targeted		Complex of cationic dendrimers	I	siRNA against BCL2 mRNA	Cancer (Research)
	nanospheres		(poly(propyleneimine) (PPI) or
			Polyamidoamine (QPAMAM))
			with siRNA caged by surface
			crosslinking	with	disulfide
			bonds. Surface is neutral due to
			addition of PEG. A synthetic
			analogue	of	Luteinizing
			hormone releasing hormone
			(LHRH) confers specificity to
			cancer cells.


Funda Demirhan

Gülnur Taban

Miguel Baya

Chiara Dinoi

Jean-Claude Daran

Rinaldo Poli
email: poli@lcc-toulouse.fr

Prof Rinaldo Poli

Synthesis and structure of [{Cp*Mo(-SCH2COO

Keywords: Molybdenum, aqueous organometallic chemistry, alkylsulfido ligands, carboxilato ligands, C-S bond activation

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Aqueous organometallic chemistry is attracting growing interest because of its ''green'' impact [1][2][3] and because of its potential in catalytic [4,5] and biomedical applications [6]. Its development, however, has mostly been restricted to low-valent systems to which hydrophilic functionalities are added on the ligand periphery [7][8][9], in order to make the systems compatible with an aqueous environment. Whereas high oxidation organometallics, mostly supported by oxo ligands, are now a well established family of compounds [10,11], their systematic investigation in water has received little attention. Nevertheless, the metal bonded oxo functionality is related, through protonation equilibria, to hydroxo and aqua ligands, leading to the potential of aqueous solubility and to the generation of open coordination sites for substrate activation and catalysis. In addition, the greater metal electronegativity in the higher oxidation states confers a higher degree of covalency to the metal carbon bonds with odd-electron carbon ligands (i.e. alkyls, allyls, cyclopentadienyls, etc.), which consequently may become quite resistant toward hydrolytic decomposition. For a redox-active metal, reduction of a high oxidation state oxo complex should favour the generation of aqua ligands, opening the way to catalytic and electrocatalytic applications [12].

We have recently initiated a research program aimed at exploiting the chemistry of high oxidation state organometallic compounds in water and have focused our attention on the speciation [13] and electrochemical behaviour [14,15] of compound [Cp*2Mo2O5]. The Cp*-Mo bond is quite hydrolytically stable, being split only quite slowly at very low pH [16].

By chemical reduction with zinc in an acidic aqueous medium, novel oxo-supported complexes of Cp*Mo V and Cp*Mo IV have been obtained, the vacant positions left over by the removal of water ligands (from the expected protonation of oxo ligands) being filled by the anions of the acids used (acetic, trifluoroacetic) [17,18].

In this contribution, we report the reactivity of [Cp*2Mo2O5] with a thiol-containing organic acid, the thioglycolic acid, HSCH2COOH. This reaction is interesting in comparison with the well-studied reduction of MoO4 2-by the same reagent, of interest in bioinorganic chemistry as a model for the action of cysteine in molybdenum hydroxylase enzymes. [19][20][21][22][23] It will be shown that the substrate is capable to reduce the starting complex, leading to a thioglycolate complex of Cp*Mo IV . The abstraction of a sulfide ion also takes place during this transformation.

Experimental Section

General Methods

All preparations and manipulations were carried out with Schlenk techniques under an oxygen-free argon atmosphere. All glassware was oven-dried at 120 C. Solvents were dried by standard procedures and distilled under dinitrogen prior to use. G). Purification was attempted by chromatography through a silica gel column (ca. 1 cm diameter x 10 cm), using pentane/MeOH (7:3) as eluent. The title product was recovered as a red powder from a first, major red fraction, after evaporation to dryness. However, this was still contaminated by the EPR-active by-product. In addition, the compound is slowly decomposed by contact with the silica support.

A single crystal for the X-ray analysis was obtained by diffusion of a pentane layer into a red CH2Cl2 solution of the crude reaction product at room temperature. 1 H NMR (CDCl3): In a separate experiment, the reaction mixture was evaporated to dryness and the residue was washed with pentane. The extract was evaporated and the residue was dried and dissolved in DMSO-d6. This solution exhibited an NMR spectrum with a major peak at 

X-ray analysis

Single crystal was mounted under inert perfluoropolyether at the tip of glass fibre and cooled in the cryostream of the Oxford-Diffraction XCALIBUR CCD diffractometer.

The structures was solved by direct methods (SIR97) [26] and refined by least-squares procedures on F 2 using SHELXL-97 [START_REF] Sheldrick | SHELXL97. Program for Crystal Structure refinement[END_REF]. All H atoms attached to carbon were introduced in calculation in idealised positions and treated as riding models. The drawing of the molecules was realised with the help of ORTEP32 [START_REF] Farrugia | [END_REF]. The statistically distributed disulfide and dichloromethane molecules were freely refined. The crystal data and refinement parameters are shown in Table 1 and selected geometric parameters are collected in Table 2.

<Table 1 and Table 2> Crystallographic data (excluding structure factors) have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 283407.

Copies of the data can be obtained free of charge on application to the Director, CCDC, 12

Union Road, Cambridge CB2 1EZ, UK (fax: (+44) 1223-336-033; e-mail: deposit@ccdc.cam.ac.uk).

Results and Discussion

The title compound is produced by the direct interaction, without any reducing agent, of thioglycolic acid, HSCH2COOH, with [Cp*2Mo2O5], in an strongly acidic (CF3COOH) mixed MeOH/water solvent. As confirmed by the X-ray structure (vide infra), the product is a dinuclear complex in which the two metal atoms have the formal oxidation state IV.

Therefore, a first role of the thioglycolic acid is as a reducing agent, taking the metal oxidation state from VI to IV. It is well known that thiols can act as one-electron reducing agents, being in turn oxidized to disulfide derivatives. The presence of compound HOOC-CH2-S-S-CH2-COOH in solution has been confirmed by 1 H NMR and mass spectrometry of the reaction mixture before separation and crystallization of the metal product. In addition, the S-S coupling product is trapped as an interstitial molecule in the crystal structure of the product (vide infra), thus unambiguously proving the reducing action of the sulfur reagent.

The isolated product contains one doubly deprotonated thioglycolic acid molecule as a ligand per each Mo atom. Thus, a second role of the thioglycolic acid is as a ligand, transferring its protons to the oxo ligands, with the elimination of water. Finally, the product contains a bridging sulfido ligand, which must necessarily derive from the thioglycolic acid reagent through a process of C-S bond cleavage.

It is interesting to remark that, contrary to the reaction reported here, no sulfur extraction from thioglycolic acid has been documented for the related reaction of the same thiol reagent with MoO4 2-. [19][20][21][22][23] The various products that have been isolated and characterized from that reaction only contain the doubly deprotonated thioglycolic acid as a ligand and terminal or bridging oxo ligands: 21,23]. Other mononuclear, EPR active intermediates have also been detected, but not isolated. In addition, a more reduced, air stable Mo IV product has been isolated but, to the best of our knowledge, its structure remains unknown. [20] All those products, like the compound reported here, were obtained working in aqueous media with commercially available thioglycolic acid (without further purification). The reagent used does not contain any H2S impurity (Ag(CN)2 -test). [START_REF] Charlot | Les Réactions chimiques en solution : l'analyse qualitative minérale[END_REF] On the other hand, a 1 H NMR investigation indicates that it is contaminated by a small amount (ca. 8%) of its methyl ester, HSCH2COOMe. This compound should in principle react similarly to the acid with respect to the sulfide abstraction process, thus the outcome of the reaction does not appear to be attributable to the presence of impurities in the reagent. It seems, therefore, that the sulfide extraction process is more favorable when using the organometallic reagent Cp*2Mo2O5, relative to the inorganic molybdate.

[{Mo V O(SCH2CO2)2}2(-O)] 4-[22], [{Mo V O(SCH2CO2)}2(-O)(-SCH2CO2)] 2-[22], and [{Mo V O(SCH2CO2)}(-SCH2CO2)2] 2- [
In previous mechanistic studies, it was established that the first step of the of the MoO4 O, and more susceptible to reduction.

It is also possible to conceive an alternative pathway: after coordination of the sulfur donor, the S-C bond could be reductively cleaved (the electrons being provided by the Mo center or by additional thioglycolic acid equivalents) under the influence of the Mo affinity for S. Other C-S bond breaking reactions on dinuclear cyclopentadienylmolybdenum complexes have been reported [START_REF] Dubois | [END_REF][32][33][34][35][36]. In this respect, the reaction would mimic the hydrodesulfurization process, which is in fact typically catalyzed by molybdenum-sulfur compounds [37,38]. Among the various sulfur-containing substrates that have been used for model studies [39,40], simple thiols are the easiest to desulfurize [41] and the assistance of the carboxylic function in thioglycolic acid may render such a process particularly favorable.

The NMR spectrum of the isolated product is fully consistent with the solid state molecular structure, since it shows an AB patters for the diastereotopic methylene protons of the SCH2CO2 ligands, and a single Cp* resonance, in a 1:1:15 ratio. The mass spectrum (chemical ionization with NH3) shows the molecular ion plus a captured proton as the most intense isotopic envelope (see Figure 1), the second most intense one being 17 mass units higher and corresponding to the molecular ion plus a captured ammonium cation. Other examples, however, are known for Mo III , e.g. [Cp2Mo2(-SMe)3(CO)2] + [36] and [44]. The most interesting metric parameter (see Table 2) It is interesting to compare the reductive reaction leading to the title compound with the oxidative addition of the methyl ester of thioglycolic acid, HSCH2COOCH3, to Cp2Mo2(CO)4 [48] (see Scheme 1). The two products are closely related to each other, because both are Sand thioglycolate-bridged dinuclear CpMo IV complexes. In the case of the oxidative process with the methyl ester in Scheme 1(a), the carboxylate functions are blocked by the Me substituents and do not coordinate the molybdenum center. The ideal replacement of the methyl groups with protons, followed by elimination of H2S, would lead to the product of the other reaction, Scheme 1(b). However, the two compounds have a totally different genesis, one deriving from an oxidative decarbonylation process (the presumed oxidizing agent being the SH protons, leading to H2), the other one being a reduction of a high oxidation state oxo compound. It may be predicted that a reaction of Cp2Mo2(CO)4 with the thioglycolic acid could lead to the same product of Scheme 1(b), and that the reaction of Cp2Mo2O5 with the methyl ester of thioglycolic acid may lead to the same product of Scheme 1(a).

[Cp2Mo2(-SMe)2(-SH)(CO)2] +
is the metal-metal distance [2.792(7) Å],

Conclusion

We have shown in this paper that the interaction of [Cp*2Mo2O5] with thioglycolic acid in a aqueous environment leads to a spontaneous reduction to Mo IV , to the incorporation of the thioglycolate ligand, and to sulfide extraction. The reaction occurs quite readily at room temperature. It will now be interesting to test whether this reaction can be generalized to other sulfur compounds and whether the resulting products lead to any interesting stoichiometric or catalytic transformations.

  Reduction of [Cp*2Mo2O5] by thioglycolic acid in an aqueous medium: Synthesis and structure of [Cp*Mo(µ-SCH2COO)2(µ-S)] Funda Demirhan, Gülnur Taban, Miguel Baya, Chiara Dinoi, Jean-Claude Daran, Rinaldo Poli To cite this version: Funda Demirhan, Gülnur Taban, Miguel Baya, Chiara Dinoi, Jean-Claude Daran, et al.. Reduction of [Cp*2Mo2O5] by thioglycolic acid in an aqueous medium: Synthesis and structure of [Cp*Mo(µ-SCH2COO)2(µ-S)]. Journal of Organometallic Chemistry, 2006, 691 (4), pp.648-654. 10.1016/j.jorganchem.2005.10.006. hal-03196152

2 .

 2 09 ppm (s, 15H, Cp*), 2.94 (AB system, 4H, SCH2CO2,  = 56.0 Hz; 2 JHH = 16.0 Hz). MS (CI/NH3) : m/z 692 (M+NH4 + , 23), 674 (MH + , 100), 659 (MH + -O, 5.5), 617 (MH + -CH2CO2, 5.5), 591 (MH + -CH2CO2-CH2C, 7.5), 575 (MH + -CH2CO2-CH2CO, 22), 559 (MH + -2CH2CO2, 18).

3 .

 3 65, close to the position reported for the disulfide compound HOOCCH2SSCH2COOH (3.63) [25], and mass values in the EI MS (70 eV) at m/z 137 (M-COOH, 2.9%), 123 (M-CH2COOH, 2.4%), 119 (M-COOH-H2O, 7.9%), which are also reported for the same molecule [25].

Figure 1 .Figure 2 .

 12 Figure 1. Mass spectrum (CI/NH3) of compound [Cp*Mo(-SCH2COO)]2(-S). (a) Experimental. (b) Simulated for C24H34Mo2O4S3.

  which is rather close to those of the above mentioned dinuclear Mo IV compounds with analogous structure [2.720(8) Å in [Cp*2Mo2(-O)(-Cl)2Cl2] [42] and 2.866(2) Å in [Cp*2Mo2(-Cl)3Cl2] + ] [43]. The metal-metal distance is also quite similar for the related Mo III dinuclear complexes {2.785(2) Å in [Cp2Mo2(-SMe)3(CO)2 + ] [36] and 2.772(2) Å in [Cp2Mo2(-SMe)2(-SH)(CO)2] +[44]}. This is expected, since the two electrons that make the difference between the electronic structures of Mo III and Mo IV triply-bridged (CpMo)2-type compounds are located in essentially nonbonding orbitals[43]. The Mo-S distances to the bridging sulfido ligand are shorter [average 2.310(4) Å], as expected, than those to the S atom of the thioglycolato ligand [average 2.452(17) Å].

Figure 2 .

 2 Figure 2. An ORTEP view of the [Cp*Mo(-SCH2COO)]2(-S) molecule. The ellipsoids are drawn at the 40% probability level and the H atoms are omitted for clarity.

Figure 3 .

 3 Figure 3. ORTEP diagram showing the hydrogen bonding network between HOOCCH2S-SCH2COOH and the dimolybdenum unit, and the disorder of the interstitial disulfide and dichloromethane molecules around the inversion center (shown). Only one of the two symmetry-related positions for each molecules is shown for clarity.

  Scheme 1

  2-reaction is formation of the mononuclear [Mo VI O2(SCH2CO2)2] 2-complex, which is subsequently reduced by the excess thioglycolic acid to produce mononuclear species, followed by dimerization. A similar process may well occur here. It is interesting to remark that an EPR active species (presumably monomeric) is also formed during the Cp*2Mo2O5 reduction, see Experimental part. Our efforts to separate this species from the dinuclear Mo IV product, however, were unsuccessful. It is not clear at what point and by what precise mechanism the sulfur atom is abstracted during the formation of the title compound. The

formation of H2S from the thioglycolic acid reagent by exchange with the water solvent appears unlikely, since this reaction is not described in the literature. Rather, it is well known that C-O bonds exchange with functions containing the S-H bonds to yield products containing C-S and O-H bonds, even under strongly acidic conditions like those used in our reaction.

[START_REF] March | Advanced Organic Chemistry[END_REF]

It is possible, however, that the sulfide abstraction is favored by the greater affinity of Mo for S vs. O in the +IV oxidation state. Indeed, the presence of the Cp* ligand could render the Mo acidic center softer, therefore having a greater relative affinity for S vs.

Table 2

 2

), consistent with localized single and double bonds, respectively, for a molybdenum carboxylate unit. However, the C2-O12 bond, which is engaged in the hydrogen bond with the O42 proton, is slightly longer than the C4-O22 bond

[1.233(3)

vs. 1.217(3) Å], which is not involved in hydrogen bonding. Conversely, C2-O11 is not as long as C4-O21

[1.289(3)

vs. 1.310(3) Å].

Table 1 .

 1 Crystal data and refinement parameters for [Cp*2Mo2(-SCH2COO)2(-S)]•1.5(CH2Cl2)•0.5(HOOCCH2SSCH2COOH)

	Empirical formula	C24 H34 Mo2 O4 S3, (CH2Cl2)3/2,(C4H6O4S2)1/2
	Formula weight	893.07
	Temperature	180(2) K
	Wavelength	0.71073 Å
	Crystal system	Triclinic
	Space group	P -1
	Unit cell dimensions	a = 12.0469(11) Å	= 104.398(7)°.
		b = 12.4252(12) Å	= 93.885(7)°.
		c = 13.7051(10) Å	 = 115.745(9)°.
	Volume	1753.1(3) Å 3
	Z	4
	Density (calculated)	1.692 Mg/m 3
	Absorption coefficient	1.221 mm -1
	F(000)	904
	Crystal size	0.37 x 0.34 x 0.19 mm 3
	Theta range for data collection	3.27 to 26.31°.
	Index ranges	-13<=h<=15, -15<=k<=14, -17<=l<=17
	Reflections collected	13162
	Independent reflections	7136 [R(int) = 0.0225]
	Completeness to theta = 26.31°	99.7 %
	Absorption correction	Semi-empirical from equivalents
	Max. and min. transmission	0.7236 and 0.6514
	Refinement method	Full-matrix least-squares on F 2
	Data / restraints / parameters	7136 / 0 / 454
	Goodness-of-fit on F 2	1.055
	Final R indices [I>2sigma(I)]	R1 = 0.0301, wR2 = 0.0743
	R indices (all data)	R1 = 0.0375, wR2 = 0.0777
	Largest diff. peak and hole	0.934 and -0.704 e.Å -3

Acknowledgements

This research was supported by the CNRS in France and by TUBITAK in Turkey. Supplemental travel support by a bilateral CNRS-TUBITAK (TBAG-U/62(102T211) program is also gratefully acknowledged. In addition, RP acknowledges support from the European Commission through the Research Training Network "AQUACHEM" (contract No. MRTN-CT-2003-503864) and the COST D29 program (working group 0009-03). FD thanks the University of Celal Bayar (FEF 2002/101, FEF 2005/023) for research grants.