Jiang-Cheng Shen

Motoko Unoki

Damien Ythier

Alain Duperray

Lyuba Varticovski

Remy Pedeux

Curtis C Harris
email: curtis_harris@nih.gov.

ING4 Suppresses Cell Spreading and Cell Migration by Interacting with a Novel Binding Partner, Liprin α α α α1

Keywords: ING4, liprin α1, PPFIA1, tumor suppressor, migration

ING4 is a candidate tumor suppressor that plays a major role in gene regulation, cell cycle control, apoptosis, and angiogenesis. ING4 expression is downregulated in glioblastoma cells, and head and neck squamous cell carcinoma. Here, we identified liprin α1/PPFIA1, a cytoplasmic protein necessary for focal adhesion formation and axon guidance, as a novel interacting protein with ING4. ING4 and liprin α1 colocalized at lamellipodia in the vicinity of vinculin. Overexpressed ING4 suppressed cell spreading and cell migration. In contrast, overexpressed liprin α1 enhanced cell spreading and cell migration. Knockdown of endogenous ING4 with RNA interference induced cell motility, whereas knockdown of endogenous liprin α1 suppressed cell motility. ING4 also suppressed cell motility that was enhanced by liprin α1. However, ING4 did not further suppress cell motility when liprin α1

was suppressed with RNA interference, suggesting a functional and mechanistic interdependence between these proteins. In addition to its nuclear functions, cytoplasmic ING4 interacts with liprin α1 to regulate cell migration, and with its known anti-angiogenic function, may prevent invasion and metastasis.

Introduction

Inhibitor of growth (ING) family is comprised of six members in humans, including ING1-5 and INGx [START_REF] He | Phylogenetic analysis of the ING family of PHD finger proteins[END_REF][START_REF] Russell | Grow-ING, Age-ING and Die-ING: ING proteins link cancer, senescence and apoptosis[END_REF]. The first ING gene (ING1) was identified by using a strategy for tumor suppressor gene isolation [START_REF] Garkavtsev | Suppression of the novel growth inhibitor p33ING1 promotes neoplastic transformation[END_REF]. ING4 and other family members were subsequently identified by a sequence homology search [START_REF] Nagashima | DNA damage-inducible gene p33ING2 negatively regulates cell proliferation through acetylation of p53[END_REF][START_REF] Nagashima | A novel PHD-finger motif protein, p47ING3, modulates p53-mediated transcription, cell cycle control, and apoptosis[END_REF][START_REF] Shimada | Cloning of a novel gene (ING1L) homologous to ING1, a candidate tumor suppressor[END_REF][START_REF] Shiseki | p29ING4 and p28ING5 bind to p53 and p300, and enhance p53 activity[END_REF]. As a candidate tumor suppressor, ING4 is associated with a loss of heterozygocity on chromosomes in head and neck cancers [START_REF] Gunduz | Frequent deletion and down-regulation of ING4, a candidate tumor suppressor gene at 12p13, in head and neck squamous cell carcinomas[END_REF]. The ING genes negatively regulate cell proliferation [START_REF] Russell | Grow-ING, Age-ING and Die-ING: ING proteins link cancer, senescence and apoptosis[END_REF][START_REF] Campos | Biological functions of the ING family tumor suppressors[END_REF][START_REF] Shi | The fellowships of the INGs[END_REF]. ING4 binds to p53 and acetyltransferase p300, and thus, facilitates p53 acetylation by p300, especially on Lys382 [START_REF] Shiseki | p29ING4 and p28ING5 bind to p53 and p300, and enhance p53 activity[END_REF]. The ING4 protein also associates with the HBO1 HAT (histone acetyltransferase) complex [START_REF] Doyon | ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation[END_REF] and interacts with the methylated lysine 4 of histone H3 [START_REF] Pena | Molecular mechanism of histone H3K4me3 recognition by plant homeodomain of ING2[END_REF][START_REF] Shi | ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression[END_REF], suggesting a role in chromatin remodeling and transcription regulation. [START_REF] Shiseki | p29ING4 and p28ING5 bind to p53 and p300, and enhance p53 activity[END_REF]. In addition, ING4 plays a major role in cellular responses to hypoxia, repressing HIF (hypoxia inducible factor) activation by interacting with HIF prolyl hydroxylase [START_REF] Ozer | The candidate tumor suppressor ING4 represses activation of the hypoxia inducible factor (HIF)[END_REF]. ING4 regulates angiogenic growth induced by human glioblastoma cells in SCID mice, by interfering with the NF-κB pathway [START_REF] Garkavtse | The candidate tumour suppressor protein ING4 regulates brain tumour growth and angiogenesis[END_REF]. ING4 was also reported to suppress loss of contact inhibition [START_REF] Kim | A screen for genes that suppress loss of contact inhibition: identification of ING4 as a candidate tumor suppressor gene in human cancer[END_REF]. ING4 overexpression in breast cancer cells showed the attenuation of anchorage-independent cell growth in soft agar [START_REF] Kim | A screen for genes that suppress loss of contact inhibition: identification of ING4 as a candidate tumor suppressor gene in human cancer[END_REF].

ING4 overexpression induces apoptosis in cells

The conversion of a cancer cell into an invasive metastatic type is a hallmark of tumor progression, as well as the deregulation of cell proliferation [START_REF] Hanahan | The hallmarks of cancer[END_REF]. During this switch, cancer cells lose control of several morphological features such as cell polarization, filopodia and lamellipodia formation, cell spreading, and cell migration [START_REF] Ridley | Cell migration: integrating signals from front to back[END_REF], which are key regulating steps between the static and metastatic transition of a cancer cell. The morphological transition towards a migrating cell involves multiple regulatory components at different steps [START_REF] Tamura | Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN[END_REF].

Elucidating the regulatory networks among the known tumor suppressors and identifying new candidate tumor suppressor genes may provide valuable therapeutic strategies for preventing tumor progression and metastasis.

To reveal the molecular basis of ING4 function, we examined ING4-interacting proteins by a protein pull-down assay. We identified several ING4-associated proteins. We show here that ING4 interacts and colocalizes in lamellipodia with liprin α1/PPFIA1 [protein tyrosine phosphatase, receptor type f polypeptide (PTPRF), interacting protein (liprin), alpha 1]. Liprin α1 was identified as a binding protein of leukocyte common antigen-related (LAR) family receptor tyrosine phosphatases and colocalized with LAR at focal adhesions (LAR-RPTP) [START_REF] Serra-Pages | The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR-interacting protein co-localize at focal adhesions[END_REF].

Liprin α1 is required for the trafficking of synaptic vesicles [START_REF] Miller | Direct observation demonstrates that Liprin-alpha is required for trafficking of synaptic vesicles[END_REF] and is involved in the development and maintenance of excitatory synapses [START_REF] Dunah | LAR receptor protein tyrosine phosphatases in the development and maintenance of excitatory synapses[END_REF]. We further show that ING4 overexpression leads to the suppression of cell spreading and cell migration, and liprin α1 is required for these functions. We propose that ING4 regulates cell motility by interacting with liprin α1.

Materials and Methods

Cell lines and culture. RKO, HEK-293, and U-87 MG cells were purchased from American Type Culture Collection (ATCC; Rockville, MD) and cultured under recommended conditions.

Transient transfections of plasmids were performed with Lipofectamine and Plus reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA).

Plasmid construction. pFLAG-CMV2-ING4, pFLAG-CMV6-ING2 and

pFLAG-CMV2-ING5 were constructed previously [START_REF] Shiseki | p29ING4 and p28ING5 bind to p53 and p300, and enhance p53 activity[END_REF][START_REF] Pedeux | ING2 regulates the onset of replicative senescence by induction of p300-dependent p53 acetylation[END_REF]. The liprin α1 expression plasmid for future experiments. The precipitate that contains perinuclear and nuclear extracts was resuspended in lysis buffer B (same as lysis buffer A but with 500 mM NaCl), kept on ice for 30 min, and centrifuged at 13,000 x g for 15 min at 4 o C. The supernatant was incubated with FLAG M2-conjugated resins (Sigma-Aldrich) for 4 h at 4 o C (salt concentration is 250 mM), centrifuged, and washed three times with wash buffer 1 (20 mM HEPES, pH 7.9, 150 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 0.1% NP-40, 10% glycerol, 1 mM DTT, and the protease inhibitor cocktail), followed by three times with wash buffer 2 (same as wash buffer 1 but with 300 mM KCl). The pull-down proteins were eluted by elution buffer (20 mM Tris-HCl, pH 8.0, 150 mM KCl, 1 mM DTT, and 5% glycerol) containing 0.2 mg/ml of FLAG peptide at 4 o C for 4 h.

Coimmunoprecipitation. RKO cells transfected with FLAG-ING4 expression plasmids

were harvested after 48 h transfection, resuspended in extraction buffer A (50 mM Tris-HCl, pH 8.0, 500 mM KCl, 20% glycerol, 0.1% NP-40, 1 mM DTT, and the protease inhibitor cocktail), subjected to three rounds of freeze-thawing (liquid N2, 37 o C), and centrifuged at 13,000 x g for Membrane protrusion and spreading was observed by immunofluorescence microscopy based on the staining. The ratio of cells that had filopodia and/or lamellipodia (filo/lamellipodia) over cells with GFP expression was calculated by counting cells and is referred to as the percent of cell spreading. The average surface area per cell was measured by NIH ImageJ software. The statistical analysis was performed by Student's t-test with data from more than three independent fields in the experiment.

Modified Boyden chamber cell migration assay. Modified Boyden chamber cell migration assay was performed using a migration chamber containing 8 mM pore-size polystyrene 10 4 cells/cm 2 in a 100 mm dish and cultured for 16 h. Cells were transfected with 1 µg of plasmid and incubated for 16 h. The transfected cells were harvested, washed once with serum-free DMEM, and resuspended in serum-free DMEM at a concentration of 1 x 10 5 /ml.

For the FluoroBlok transmembrane migration assay, 0.5 ml of the cell suspension was transferred to the upper chamber of the assay and 0.75 ml of complete DMEM was laid in the lower chamber. The 24-well plate of the migration assay was immediately incubated at 37 o C for 22 h.

Quantitative cell migration data were obtained by fluorescence spectrometry (Victor II, PerkinElmer Life and Analytical Sciences, Boston, MA) after the transverse cells in the lower chamber were stained with calcein AM (Molecular Probes) at 37 o C for 1 h. Ras-GTPase activating protein, SH3 domain-binding protein 2a (G3BP2a), COP1-β', liprin α1, and several ribosomal proteins, were identified as ING4 interacting proteins by mass spectrometry (Fig. 1A). Among these pull-down proteins, we focused on liprin α1 in this report.

Results

ING4 interacts with liprin

The mass spectrometry results were further confirmed by coimmunoprecipitation of FLAG-ING4 with endogenous liprin α1, which was detected by anti-liprin α1 antibody in a

Western blot (Fig. 1B). This interaction was specific for ING4 among other ING family proteins; ING2 and ING5 did not coimmunoprecipitate with liprin α1 (Fig. 1B). We determined the ING4 motif responsible for the interaction using three C-terminal-truncated FLAG-ING4 constructs coexpressed with myc-tagged liprin α1. The peptide sequence between codons 135 and 180 was essential for ING4-liprin α1 interaction (Fig. 1C).

Because liprin α1 is known to carry out its function mainly in the cytoplasm, and ING4 has been demonstrated to possess gene regulatory activities in the nucleus, we investigated whether ING4 was also present in the cytoplasm. By subcellular fractionation and Western blotting, we demonstrated that ING4 was present in both nuclear and cytoplasmic extracts (Fig. 1D). We also found that liprin α1 was present in both nuclear and cytoplasmic extracts, whereas the focal adhesion molecule, vinculin, was more strictly found in the cytoplasmic extract, and a nuclear marker, lamin A/C, was only detected in the nuclear extract (Fig. 1D).

ING4 colocalizes with liprin α α α α1 in protruding membranes. The subcellular localization of ING4 and liprin α1 was examined by immunofluorescence. We have observed that overexpressed ING4 (ING4_v1) mainly localized in the nucleus when cells were in contact with each other [START_REF] Unoki | Novel splice variants of ING4 and their possible roles in regulation of cell growth and motility[END_REF]. However, overexpressed ING4 had a tendency to be distributed in the cytoplasm under low cell density conditions in RKO cells (Fig. 2A). ING4 was widely spread out, but mostly in the cytoplasm, with elongated protruding membranes stimulated by serum in the cells (Fig. 2B). It is intriguing that ING4 proteins preferred to cluster around the perinuclear areas, and most remarkably, assembled into intensive foci along the protruding membrane.

Looking closely at the protruding membrane, we found that ING4 localized densely at the forefront of the leading edge, suggesting a role for ING4 in mechanisms of membrane protrusion, such as focal adhesion (Fig. 2B). Similar to ING4, endogenous liprin α1 resided all around the cytoplasm and formed puncta in the polar lamellipodium. The colocalization of ING4 and liprin α1 in the protruding membrane or polar lamellipodium was significant (Fig. 2B). The colocalization was further confirmed by confocal microscopy analyses in RKO cells cotransfected with liprin α1 and ING4 (Supplementary Fig. S1A). We also observed similar colocalization of ING4 and liprin α1 in protruding membranes or polar lamellipodia in HEK-293 cells and U-87 MG glioblastoma cells (Supplementary Fig. S1B andC).

Because ING4 and liprin α1 colocalized mainly at the protruding membranes, we surmised that ING4 may be involved in a focal adhesion complex. We examined whether ING4 colocalizes with the focal adhesion molecule, vinculin, in the cells. ING4 clustered at the tip of polar lamellipodia in front of vinculin or at the front edge of membrane ruffles that was surrounded by vinculin (Fig. 2C).

Effect of overexpressed ING4 and liprin α α α

α1 on cell motility. Liprin α1 plays a major role in neuron cell growth, particularly in axon guidance and in terminal branches during dendrite extension [START_REF] Dunah | LAR receptor protein tyrosine phosphatases in the development and maintenance of excitatory synapses[END_REF]. Because ING4 interacts with liprin α1, we were interested in understanding whether ING4 regulates cell spreading and cell motility. First, we performed a cell spreading assay. Cells were synchronized by serum starvation and stimulated by adding serum. The membrane morphology was observed by F-actin staining. The transfection efficiency, evaluated by a GFP expression vector, was high enough (80-90%) for the cells to be used for this experiment (Supplementary Fig. S2A). The surface area of ING4-overexpressed cells was

In addition to the previously reported nucleus-based functions, we provided evidence of ING4 cytoplasmic functions. We show here that ING4 binds to a cytoplasmic protein, liprin Recent studies in axon guidance indicated that liprin α1 is required for the trafficking of synaptic vesicles [START_REF] Miller | Direct observation demonstrates that Liprin-alpha is required for trafficking of synaptic vesicles[END_REF] and is involved in the development and maintenance of excitatory synapses [START_REF] Dunah | LAR receptor protein tyrosine phosphatases in the development and maintenance of excitatory synapses[END_REF].

Thus, liprin α1 could serve as a scaffold protein that is able to assemble protein complexes in a transportation vehicle destined to the presynapse. Interestingly, in our study, ING4 tends to cluster at the perinuclear area or at lamellipodium in RKO, U-87 MG, and HEK-293 cells, suggesting a transportation pathway from the nucleus to lamellipodium. In addition, the physical interaction of ING4 with COP1-β', another protein identified by the present experiment, may further support this hypothesis.

Cell migration is a highly coordinated process involving multiple components and steps that mirror the complexity of embryonic morphogenesis [START_REF] Ridley | Cell migration: integrating signals from front to back[END_REF]. A migration movement begins with cell polarization, which includes vesicle trafficking toward the leading edge, followed by membrane protrusion and adhesion. At the lagging end, adhesion disassembles to allow forward movement. Although liprin α1 has not been specifically studied for its role in the polarity of cell migration, its associated protein, GIT1, has been shown to play a major role in the regulation of membrane protrusive activity and cell migration [START_REF] Manabe | GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration[END_REF]. Here, we showed that ING4 suppressed membrane protrusion by delaying actin polymerization in the spreading cells.

This negative regulation of actin polymerization may play a major role in suppressing cell migration. By liprin α1 knockdown and ING4 overexpression simultaneously, we found that liprin α1 was required for ING4's capacity to suppress cell spreading and migration. These results suggest that appropriate subcellular localization and/or proper protein-protein interactions are critical for ING4 function in regulating the cell motility. Liprin α1 may play an essential role in the trafficking of ING4 to its destination and/or in assembling the required protein complex for ING4 activities.

We and others have previously shown that liprin α1 and ING4 are expressed in both normal tissue and multiple cancer types [START_REF] Unoki | Novel splice variants of ING4 and their possible roles in regulation of cell growth and motility[END_REF]. Recently, ING4 was identified in a screening assay designed to search for genes that suppress loss of contact inhibition among cells. Ectopic expression of ING4 attenuated anchorage-independent cell growth and suppressed loss of contact inhibition ( 16). Garkavtsev et al [START_REF] Garkavtse | The candidate tumour suppressor protein ING4 regulates brain tumour growth and angiogenesis[END_REF] reported that an interaction between ING4 and NF-κB plays an important role during angiogenesis. The interaction between ING4 and liprin α1 may also participate in endothelial cell migration and angiogenesis. In addition, development and function of the mammary gland is impaired in homozygous LAR-deficient mice [START_REF] Schaapveld | Impaired mammary gland development and function in mice lacking LAR receptor-like tyrosine phosphatase activity[END_REF]. Thus, the interaction of ING4 and liprin α1 may play a role during development. The role of ING4 interaction with liprin α1 in normal cell migration, e.g.) endothelial cells, and during development, warrants further investigation.

Previously, we reported that the endogenous ING4 expression level was induced by

10 min at 4 2 ( 5 ' 3 ( 5 '

 42535 o C. The cellular extracts (400 µg) were immunoprecipitated with FLAG M2 resin (salt concentration is 266 mM) for 4 h at 4 o C, centrifuged and washed with extraction buffer A four times. Subcellular fractionation. RKO cells transfected with FLAG-ING4 plasmid were grown for 48 h before being harvested. Cytoplasmic and nuclear fractions were separated by NE-PER kit (Pierce, Rockford, IL). Immunofluorescence microscopy. Cells were plated on glass coverslips at 4 x 10 4 /cm 2 and cultured for designated incubation times with designated cell treatments. Cells were fixed in 4% paraformaldehyde, permeated by 0.1% Triton X-100, and blocked by 1% BSA in PBS. Cells were incubated with a primary antibody at room temperature for 1 h and then treated with fluorophor-conjugated secondary antibody for another hour at room temperature. The cells were examined by fluorescence microscopy (Axioplan 2 imaging: Carl Zeiss, Thornwood, NY). siRNAs. Small interference RNA (siRNA) targeting ING4 and liprin α1 transcripts were designed and synthesized by QIAGEN (Valencia, CA). siRNA sequences for ING4 were as follows: ING4 siRNA-1 (5'-TCCCTTTGAATTACAGAGAAA-3'), ING4 siRNA--ATGCCTGTGGATCCCAACGAA-3'), ING4 siRNA--GAGGCTGATCTCAAGGAGAAA-3'), and ING4 siRNA-4 (5'-AAGGAGAAACAGATTGAGTCA-3'). siRNA sequences for liprin α1 were as follows: liprin α1 siRNA-1 (5'-CACGAGGTTGGTCATGAAAGA-3') and liprin α1 siRNA-2 (5'-CTGGTGTTTCCGAGACGGATA-3'). Lipofectamine 2000 (Invitrogen) was used for transfection of the siRNAs. Cell spreading assay. RKO cells transfected with ING4 expression plasmids were plated on glass coverslips in serum free medium and cultured overnight. The starved cells were stimulated by 5% fetal bovine serum (FBS) in DMEM without glutamine and antibiotics. At designated time points, cells were fixed and stained on F-actin by rhodamine-phalloidin.

α α α α1 .

 α1 We performed a pull-down assay using FLAG-tagged ING4 (ING4_v1) protein overexpressed in RKO cells to understand the network of ING4-interacting proteins. Several cytoplasmic proteins, Ubc4/5, Calcium binding protein 1 (CaBP1),

α1,

  and translocated to lamellipodia where it is colocalized with liprin α1, suggesting a dynamic change of ING4 subcellular localization under some conditions. Liprin α1 was originally identified as a binding protein of the LAR family receptor protein tyrosine phosphatases (LAR-RPTP) and was shown to be colocalized with LAR at focal adhesions, where liprin α1's location is proximal to the rear end of focal adhesion, which disassembles in migration during membrane protrusion (20). ING4 interaction with liprin α1 may explain the subcellular localization of ING4 being proximal to the focal adhesion molecule, vinculin. Although the exact function of liprin α1 in the formation and disassembly of focal adhesion is unknown, it has been suggested that liprin α1 may play a role in the regulation of focal adhesion disassembly.

  serum starvation and reduced by serum activation in normal fibroblast cells[START_REF] Unoki | Novel splice variants of ING4 and their possible roles in regulation of cell growth and motility[END_REF]. This observation is consistent with the present observation using siRNAs against ING4. Therefore, endogenous ING4 seems to suppress cell migration in normal cells, and the decreased ING4 expression in cancer cells[START_REF] Gunduz | Frequent deletion and down-regulation of ING4, a candidate tumor suppressor gene at 12p13, in head and neck squamous cell carcinomas[END_REF][START_REF] Garkavtse | The candidate tumour suppressor protein ING4 regulates brain tumour growth and angiogenesis[END_REF] could lead to cancer cell migration and metastasis. In the same report[START_REF] Unoki | Novel splice variants of ING4 and their possible roles in regulation of cell growth and motility[END_REF], we described four splice variants of ING4 (ING4_v1, v2, v3 and v4). In this report, we focused on the initially described ING4, ING4_v1, because of its strong binding affinity to liprin α1. Other splice variants have a partially missing NLS (deletion is between 128aa to 132aa) that corresponds to a flanking region that is critical for binding with liprin α1 (135aa-180aa), identified in this report. One of the four splice variants, ING4_v4, showed dominant-negative effects on the functions of ING4_v1. Therefore, ING4 may regulate multiple cellular functions by changing its subcellular localization, binding partners, and associations with the variants. In summary, we have identified an ING4-associated protein, liprin α1, that may function in directing ING4 to its cytoplasmic destination, where ING4 may function as a regulator of membrane dynamics, and consequently, cell motility. These findings shed a new light on our understanding of the biological role of the candidate tumor suppressor, ING4, in addition to its documented nuclear transcription regulation.

Figure 1 .

 1 Figure 1. ING4 interacts with liprin α1. A, Cell extracts from RKO cells transfected with

Figure 2 .

 2 Figure 2. ING4 colocalizes with liprin α1 at lamellipodia in the vicinity of vinculin. A,

Figure 3 .

 3 Figure 3. Effect of overexpressed ING4 and liprin α1 on cell spreading and migration. A,

Figure 4 .

 4 Figure 4. Downregulation of ING4 enhances cell motility. A, The downregulation of ING4

Figure 5 .

 5 Figure 5. Interaction of liprin α1 and ING4 on regulating cell motility. A, Endogenous liprin

Acknowledgments

This work was supported by the Intramural Research Program of Center for Cancer Research, National Cancer Institute, National Institute of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore, be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We are grateful to the IAB microscopy facility for the production of cell migration videos. We thank Drs. Paul Randazzo and Kensuke Kumamoto for scientific advice, Damien Nissou and Alexei Grichine for technique assistance, and Dr. Tom Holroyd and Dorothea Dudek-Creaven for editorial assistance.

significantly smaller than that of the control cells (Fig. 2A left and Supplementary Fig. S2B).

The ratio of cells that had filopodia and/or lamellipodia (filo/lamellipodia) over cells with GFP expression was calculated and is referred to as the percent of cell spreading. The results showed that 1 h after serum stimulation, ING4 significantly delayed cell spreading by nearly 4-fold relative to the vector control (Fig. 3A right and Supplementary Fig. S2A). The mechanism by which ING4 suppresses cell spreading may also assist in cell migration regulation, particularly to the polarized protrusion membranes during migration. Thus, we next performed a modified Boyden chamber assay to measure transmembrane cell migration by fluorescence scoring. Overexpressed ING4 also significantly suppressed cell migration (Fig. 3B). This result was further confirmed by the in vitro scratch assay (Supplementary Fig. S3A,B, and Video 1). Time-lapsed motion pictures that monitored cell migration taken every 5 min over a 24-h period showed a marked suppression of cell migration when ING4 was overexpressed in RKO cells.

Next, we examined the effect of overexpressed liprin α1 on cell motility. After 45 min serum stimulation, overexpressed liprin α1 enhanced cell spreading (Fig. 3C and Supplementary Fig. S4). Coexpression of ING4 with liprin α1 suppressed the cell spreading enhanced by liprin α1 (Fig. 3C). Overexpression of liprin α1 also induced cell migration, but this induction was suppressed by ING4 (Fig. 3D).

Effect of endogenous ING4 on cell motility.

To understand the role of endogenous ING4, we reduced endogenous ING4 by four small interference RNAs (siRNAs). All four siRNAs reduced protein levels more than 50%, and siRNA-3 almost completely eliminated ING4 protein expression detected by anti-ING4 antibody (Fig. 4A). For the spreading assay, F-actin in the cells transfected with the siRNAs was stained after 30 min serum stimulation. Cells with control siRNA did not spread out much in 30 min, while the ING4 siRNA-transfected cells were swiftly extending their membranes in the form of filo/lamellipodia (Supplementary Fig. S5, Fig. 4B andC). siRNA-3 showed statistical significance in results determined either by scoring the percentage of cells with the formation of filo/lamellipodia (Fig. 4B) or by measuring the average cell surface area (Fig. 4C) consistent with ING4 protein levels (Fig. 4A). Next, we performed the modified Boyden chamber assay. The result showed that siRNA knockdown of ING4 also enhanced cell migration (Fig. 4D). Again, siRNA-3 enhanced cell migration most effectively consistent with the protein level (Fig. 4A).

Regulation of cell migration by ING4 and liprin


Yangguang Gao

Xianran He

Fei Ding

Yongmin Zhang
email: _yongmin.zhang@upmc.fr

Recent progress in chemical syntheses of sphingosines and phytosphingosines

Keywords: sphingosine, phytospingosine, synthesis, chiral pool, auxiliary, epoxidation

Sphingolipids and their derivatives such as glycosphingolipids and sphingomyelins exist ubiquitously in biomembrane of all eukaryotic cells, which play pivotal roles in cell proliferation, recognition, adhesion, and signal transduction. Sphingosine is shown to be the important lipid moiety in the large majority of glycosphingolipids and sphingomyelins, while phytosphingosine is one of the major long-chain moieties of glycosphingolipids. Due to the significance of the two bioactive lipids, tremendous efforts have been made to synthesize sphingosine or phytosphingosine using chiral pool approaches, chiral auxiliary and asymmetric reactions to construct the continuous stereogenic centers in them.

Introduction

Sphingolipids and glycosphingolipids are expressed on the surface of cell membrane and distributed throughout all eukaryotic cells, which are of physiological importance for cell growth, recognition, adhesion, neuronal repair, and signal transduction. 1 Sphingonsine, an amino alcohol, assigned as [(2S, 3R, 4E)-2-amino-3-hydroxyoctadec-4-en-1-ol)](Scheme 1), which is major lipid moiety of various sphingolipids, has itself displayed potent inhibitory activity against protein kinase C and plays key roles in cell signaling. 2 Study on sphingosine has underwent a long time since its first isolation by Thudichum from human brain in 1884, 3 the correct relative structural confirmation of the key functional groups by Carter in 1947, [START_REF] Carter | [END_REF] and first total synthesis by Shapro and Segal in 1954. 5 Phytosphingosine, one of the major backbone of glycosphingolipids found in higher plants, protozoa, yeast and fungi, [6][7][8][9] is a sphingoid base incorprating a long aliphatic chain and a polar 2-amino-1,3-diol group at head end. The fixed amino function and variation in hydroxyl stereogenic centers of phytosphingosine leads to four diastereomers, which exhibit different activities and metabolisms. Phytosphingosine is also a bioactive lipid, and its glycosylated derivatives display hopeful antitumor and antivirus activity. [10][11] For example, D-ribophytosphingosine can work as a cytotoxic agent against human leukemic cell lines. 12 In addition, D-ribo-phytosphingosine acts as hopeful heat stress signaling molecule in yeast. 13 To note, there is an underlying disorder related to D/L descriptors since two nomenclatures (i.e. amino-acid nomenclature and carbohydrate nomenclature) are employed for phytosphingosines. [START_REF]International Union of Biochemistry Biochemical Nomenclature and Related Documents[END_REF] For the sake of consistency, a traditional carbohydrate nomenclature in the amino-acid sense is used in this review unless otherwise stated.

Because of the diverse biological activies and novel structural characters of sphingosines and phytosphingosines mentioned above, much attention has been drawn from biological and chemical community, especially from chemical community due to the scarcity of the two lipids in nature. To date, several reviews have been present in literatures. 15 Herein, we would like to introduce some recent advance in chemical syntheses of sphingosines and phytosphingosines published from 2000 to 2015, the synthetic procedures would be discussed together based upon their structural similarity.

Chiral Approach

Chirality from sugar

Chirality from mannose or mannitol

Pandey has reported an enantioselective and concise synthesis of (2S,3R,4R)-D-xylo-phytosphingosine with 7 steps in 36% overall yield utilizing D-mannitol triacetonide as chiral pool (Scheme 2). 16 In his scheme, D-mannitol triacetonide was converted into βlactam 7 according to literature's procedure 17 to install all of the required stereogenic centers. Wittig olefination was employed for chain elongation followed by two-step reduction to give compound 8. Finally, full deprotection under acidic condition, then, peracetylation with acetic anhydride/pyridene gave N,O,O,O-tetraacetyl-D-xylo-phytosphingosine (10) in a good yield.

Mettu has accomplished the syntheses of tetraacetyl-D-ribophytosphingosine (16) and triacetyl-D-erythro-sphingosine (18) using a common intermediate 11 obtained from cyclohexylidene protected D-glyceraldehyde (12), which was readily prepared from D-mannitol (Scheme 3). 18a The key steps included high diastereoselective Sharpless asymmetric epoxidation, regioselective epoxide-opening reaction by azide nucleophile, and Wittig olefination. Panza also adopted compound 12 as chiral pool to synthesize 3-O-benzoyl azidosphingosine 22. 18b Nucleophilic addition of the Grignard reagent to D-glyceraldehyde gave the propargylic alcohol 19 with low diastereoselectivity (syn/anti = 4:6). The undesired anti addition product was recycled through deacetylation followed by Mitsunobu inversion. Different from triflate or mesylate as leaving group in literatures, chloromesylate Scheme 2 Synthesis of N,O,O,O-tetraacetyl-D-xylo-phytosphingosine (10) from D-mannitol triacetonide (6).

was utilized as leaving group which was subjected to displacement by azide nucleophile to afford azidosphingosine in a very satisfactory yield.

Scheme 3 Syntheses of tetraacetyl-D-ribo-phytosphingosine ( 16), triacetyl-D-erythro-sphingosine (18), and 3-O-benzoyl azidosphingosine (22) from cyclohexylidene protected D-glyceraldehyde (12).

Recently, Martinkov described the total synthesis of protected Lg-arabino-phytosphingosine (31), L-ribo-phytosphingosine (28) from 2,3:5,6-di-O-isopropylidene-D-mannofuranose (Scheme 4)(g means for glyceraldehyde). 19 The pivotal reactions involved [3,3]sigmatropic rearrangement to introduce the desired amino functionality, and chain elongation through Wittig olefination. Notably, thermal Overman rearrangement of 24 furnished inseperable rearranged products in a low yield and pool diastereoselectivity, while the use of microwave heating afforded high-yielding of rearranged products and greatly shortened reaction time. The protected L-ribo-phytosphingosine (28) could be accessible from rearranged product 25b through several manipulations. Utilizing the same procedure as described for the preparation of protected L-ribo-phytosphingosine (28), synthesis of compound 31 was achieved from compound 25a. An alternative route for preparation of 31 was commenced with allylic thiocyanate 29 according to the similar procedure for synthesis of compound 28. Though aza-Claisen rearrangement of 29 was carried out in modest yield, displayed better stereoselectivities than those observed for the Overman rearrangement of trichloroacetimidate 24.

Also from 2,3:5,6-di-O-isopropylidene-D-mannofuranose, Lin reported a concise and efficient synthesis of D-ribo-phytosphingosine (2), which employed eight-step conventional manipulation in 57% overall yield using Wittig olefination and azide nucleophilic replacement as key reactions (Scheme 4). 20

2017-01-23 page

Scheme 4 Syntheses of compound 28, 31 and 2 from compound 23.

Chirality from D-lyxose

A short and very efficient route for synthesis of D-ribophytosphingosine from D-lyxose was reported by Lin group in 2003 (Scheme 5). 21 In his work, D-ribo-phytosphingosine (2) was prepared from D-lyxose over 6 steps in 28% overall yield using Wittig olefination and substitution by tetramethylguanidinium azide (TMGA) as crucial steps. A similar synthesis of D-ribophytosphingosine was also achieved by Lin group, in which both Wittig olefination and olefin cross-metathesis (CM) were adequately employed to extend carbon chain. 22 The later strategy seemed not to be concise compared to the former; however, it afforded rapid access to syntheses of the phytosphingosine derivatives.

Scheme 5 Synthesis of D-ribo-phytosphingosine (2) from D-lyxose.

Chirality from D-xylose

Panza has reported the synthesis of 3-O-benzoylazidosphingosine (22) from 3,5-O-isopropylidene-D-xylofuranose using Peterson olefination and allylic displacement by Grignard reagent as key steps (Scheme 6). 23 The deficiency of the strategy was low trans selectivity (E/Z=2/1) in allylic displacement. Still from cheap D-xylose, Kocienski has reported a twelve-step synthesis of the Derythro-sphingosine utilizing a 1,2-metallate rearrangement as the key step. In addition, Brook rearrangement and substitution by diphenylphosphoryl azide (DPPA) via Mitsunobu reaction were equally indispensable for the synthesis. 24 This synthesis provided a novel strategy for effective construction of trans double bond.

Scheme 6 Synthesis of D-erythro-sphingosine (1) and its derivative compound 22 .

Chirality from D-glucose and its derivatives

In 2012, Rao has accomplished the synthesis of acetyl derivative of Lg-lyxo-phytosphingosine (48) commencing with known 1,2:5,6di-O-isopropylidene-D-glucofuranose derived from D-glucose (Scheme 7). 25 In this work, Z-selectivity Wittig olefination was employed as key step to elongate carbon chain though stereochemistry of the double bond was unnecessary because of the subsequent hydrogenation. As shown in Schme 7, the important intermediate 49 was also prepared from 1,2:5,6-di-Oisopropylidene-D-glucofuranose over 4 steps. Synthesis of Derythro-sphingosine (1) was achieved by Dhavale via E-selective olefin cross-metathesis between compound 49 and long-chain terminal alkene in 65.4% overall yield. 26 A direct synthesis of 16 from D-glucosamine hydrochloride via the D-allosamine derivative 50 as key intermediate has been reported by Hung in 2002. 27 Similar to the synthesis of acetyl derivative of Lg-lyxo-phytosphingosine (48), Z-selectivity Wittig olefination was employed as well for extension chain to synthesize tetraacetyl-D-ribo-phytosphingosine (16). Besides, amino-azido conversion and highly regioselective benzoylation were also essential to this work. Bundle has utilized selective iodination, zinc-mediated reductive ring-opening reaction and olefin cross-metathesis as crucial steps to prepare tetraacetyl-D-xylo-and D-ribo-phytosphingosine (10 and 16) starting from the methyl β-glycosides of D-GlcNAc (53a) and D-AllNAc (53b), respectively (Scheme 8). 28 The spotlight of the synthesis was excellent E/Z selectivity (E/Z = 19:1 for 55a, only E for 55b) of olefin cross-metathesis (OCM), and the OCM could be applied to synthesize phytosphingosine derivatives with desired alkyl chains length.

Chirality from D-galactose and its derivatives

Duclos Jr reported a concise and conventional synthesis of Derythro-sphingosine (1) starting from D-galactose over 7 steps in 2001 (Scheme 9), 29a which was based on the previous protocol adopted by Schmidt in 1988. 30 The key step was Wittig olefination between 1,3-O-benzylidene-D-threose (56) and n-tetradecyl ylide to exclusively form trans double bond. The same protocol was utilized by Demchenko to synthesize L-erythro-sphingosine (ent-1) from 1,3-O-benzylidene-L-threose (prepared from L-arabitol) in 2010. 29b In 2000, Schmidt also utilized 1,3-O-benzylidene-Dthreose for preparing D-ribo-azidophyto-sphingosine (59). 31 Highlight of this work involved stereoselective addition of 1,3-Obenzylidene-D-threose by n-tetradecyl magnesium chloride to exclusively produce chiral hydroxyl at C4 position, and regioselective mesylation at C2 position. In 2008, Ye group accomplished a facile synthesis of tetraacetyl-D-ribo-phytosphingosine ( 16) over 5 steps in 74% overall yield from 3,4,6-tri-O-benzyl-D-galactal (Scheme 10). 32 Spotlight of this synthesis included a high-yielding Wittig olefination of lactol 60 and one-step azide substitution via Mitsunobu reaction.

Chirality from D-ribose

Very recently, Martinková 33 accomplished the syntheses of tetraacetyl-D-arabino-phytosphingosine (66), tetraacetyl-D-ribophytosphingosine (16) starting from D-ribose on the basis of the same procedures as described for preparation of their enantiomers in 2011 (Scheme 11). 34 The key step involved aza-Claisen rearrangement of allylic thiocyanate 64 to afford rearranged products 65a, 65b in an approximate ratio of 3:1 with 50% yield, which was further used to synthesize compound 66 and 16, respectively. Unlike Martinková's protocol, Sutherland employed Overman rearrangement of allylic trichloroacetimidate to install the chiral amino group of compound 70a as a single diastereomer in a satisfactory yield (72%) (Scheme 12). 35 While the other two stereogenic centers of phytospingosines were also derived from D-ribose. Thus, a new access to D-ribophytosphingosine

(2) was achieved using Overman rearrangement, trans-selective CM reaction, and stereoselective reduction of unsaturated ketone via CBS-reduction as key steps. The synthesis of L-arabino-phytosphingosine (ent-3) was prepared from 69b according to the same preceduce as decribed for 2.

Scheme 11 Syntheses of tetraacetyl-D-arabino-phytosphingosine (66) and tetraacetyl-D-ribo-phytosphingosine ( 16) from D-ribose. Scheme 12 Synthesis of D-ribo-phytosphingosine (2) from D-ribose.

Chirality from D-fructose

In 2011, a new methodology for synthses of D-ribo-phytosphingosine (2) and L-arabino-phytosphingosine (77) from D-fructose was developed by Perali (Scheme 13). 36 In this scheme, zincmediated fragmentation of 71 was used as key step to give the cerresponding ketone. Interestingly, subsequent reduction by NaBH4 almost yielded 72b, corresponding to 72a as major product (72a : 72b = 7 : 3) via reduction by LiAlH(O-t Bu)3. Both 72a and 72b were subjected to crucial olefin cross-metathesis for chain extention, azido substitution, and other simple conversions to successfully afford 2. To note, the reason for retention of configuration at C2 position from 74b to 2 was that substitution of 74b by NaN3 underwent SN1 type substitution. To invert this configuration, Mitsunobu reaction was finally utilized, thus, Larabino-phytosphingosine (77) was obtained from compound 75.

Scheme 13 Syntheses of D-ribo-phytosphingosine (2) and L-arabino-phytosphingosine (77) starting from D-fructose.

Chirality from serine and its derivatives

Chirality from serine ester

In 2002, Chuang group presented a short and efficient route for synthesis of all four stereomers of sphingosine from serine (Scheme 14). 37 The authors employed HWE olefination to form trans double bond in high yield. Treatment of 3-ketosphingosine derivative 78 with NaBH4 in the presence of CeCl3•7H2O afforded syn reduction product, which leaded to further prepare L-threosphingosine (79) and its acetyl derivative (80). However, reduction of deprotection product of 78 by Zn(BH4)2 gave anti reduction product, which was subjected to further conversion to D-erythrosphingosine (1) and its acetyl derivative (18). According to the same procedure, L-erythro-sphingosine and D-threo-sphingosine were easily prepared from D-serine methyl ester HCl. The similar stereoselective reduction protocol was also employed by Katsumura and Bittman to synthesize D-erythro-sphingosine (1) from L-serine or N-Boc-L-serine methyl ester. 38 Olefin crossmetathesis and elimination of sulfoxide intermediate was respectively utilized by Katsumura and Bittman to construct trans double bond. Liebeskind reported a concise and very efficient synthesis of Derythro-sphingosine (1) from N-Boc-L-serine over 6 steps in 72% overall yield in 2007 (Scheme 15). 39 In this work, new methodology for cross-coupling between thiol ester 81 and vinyl boronic has been developed to construct the classic alkenyl ketone intermediate 82, which was further subjected to stereoselective reduction mentioned above to give the target compound 1.

Scheme 15 Synthesis of D-erythro-sphingosine (1) from N-Boc-L-serine.

An efficient, stereocontrolled synthetic method for the preparation of D-xylo-phytosphingosine (4) and D-arabinophytosphingsine (3) was reported by Ham et al in 2012 starting from L-serine methyl ester via chiral 1,3-oxazines intermediate (Scheme 16). 40 The crucial reactions involved stereoselective intramolecular oxazine formation catalyzed by palladium (0) and CM reaction for chain extention. Notably, both 85a and 85b could be separately obtained as major products by changing the reaction temperature. 85a leaded to give D-xylo-phytosphingosine (4), however, 85b leaded to give D-arabino-phytosphingosine (3) in the same manner.

Chirality from Garner's aldehyde

In 2002, Murakami 41a achieved efficient and high diastereoselective syntheses of N-Boc-D-erythro-sphingosine (88a) and N-Boc-L-threo-sphingosine (88b) using addition of 1-alkenyl nucleophiles to Garner's aldehyde as pivotal steps (Scheme 17). Notably, addition of 1-alkenylzirconocene chloride to Garner's aldehyde in the presence of ZnBr2 gave anti-isomer, conversely, addition of 1-alkenyl-ethyl-zinc to Garner's aldehyde afforded synisomer. An alternative synthesis of intermediate 87a was reported by Arenz through addition of Garner's aldehyde by vinylmagnesium bromide and subsequent CM reaction in 37% yield. 41b The similar addition of 1-alkenyl nucleophile to Garner's aldehyde to prepare D-erythro-sphingosine was also employed by Ferjančić in 2014 using alkenylchromium (III) reagent instead, however, with a lower diastereoselectivity (7:1) and yield (46%) compared with Murakami's protocol. 42a In contrast, Montgomery took advantage of nickel-catalyzed reductive coupling of Garner's aldehyde with silyl alkyne to give compound 87c with good yield (78%) and satisfactory diastereoselectivity (>95:5), which easily converted into 87a over 2 steps. 42b

Scheme 17 Syntheses of N-Boc-D-erythro-sphingosine (88a), N-Boc-L-threosphingosine (88b) and D-erythro-sphingosine (1) from Garner's aldehyde.

A short and convenient synthesis of D-erythro-sphingosine (1) was accomplished by Cárdennas's group over 4 steps in 33% overall yield from Garner's acid in 2013 (Scheme 18). 43 Key steps included efficient addition of terminal alkyne to benzotriazole esters (89a, 89b) and stereoselective reduction.

Scheme 18 Synthesis of D-erythro-sphingosine (1) from Garner's acid. Unlike Lomobardo's strategy, Kim et al employed OsO4-catalyzed dihydroxylation reactions of (Z)-allylic amines derived from (Z)selective Wittig olefination of Garner's aldehyde to set the stereochemistry at C3, C4 positions. 44 As shown in Scheme 19, dihydroxylation of (Z)-allylic amine with N,N-diBoc groups gave anti-selective isomer 92a, however, dihydroxylation of N-Boc-(Z)allylic amine (91) gave syn-selective isomer 92b. This can be explained by severe 1,2-allylic strain between the N,N-diBoc groups and the vinylic hydrogen atom. The stereocontrolled dihydroxylation of (Z)-allylic amines was better than Sharpless dihydroxylation to some degree, upon which syntheses of both tetraacetyl derivatives of D-ribo-phytosphingosine ( 16) and Larabino-phytosphingosine (93) were achieved.

Scheme 19 Syntheses of both tetraacetyl derivatives of D-ribo-phytosphingosine (16) and L-arabino-phytosphingosine (93) from Garner's aldehyde.

Chirality from D-ribo-phytosphingosine

Since D-ribo-phytosphingosine is easily accessible from a yeast fermentation process, it has been used as chiral pool to prepare sphingosine and other phytosphingosines. Several syntheses of Derythro-sphingosine from D-ribo-phytosphingosine have been achieved in a concise manner (Scheme 20). 45 The common point of these syntheses was selective protection of C1-hydroxy and C2-amine groups followed by elimination of a cyclic sulfate intermediate (95, 97 and 99) to exclusively construct trans olefin. Aside from elimination of a cyclic sulfate intermediate, epoxide (101) 46 and alcohol (104) 47 were also eliminated to give trans olefin. Different from the above protocols, van Boom 48 has employed stereoselective transformation of 102 into the corresponding (Z)-enol triflate (103) followed by a regiospecific reduction to install trans double bond. Among these syntheses, Overkleeft 45c has provided a short synthetic route with highest overall yield (67%) to date.

Chirality from D-tartric acid and its diester

Synthesis Review

2017-01-23 page

In 2004, Basu 50 employed two contiguous hydroxyl groups of Dtartrate diester as chiral template to accomplish the synthesis of protected D-erythro-azidosphingosine (113) (Scheme 22). The highlight of this work was selective benzoylation of azidotriol 111 and exclusive formation of trans olefin via cross metathesis. However, compound 113 was obtained in a low yield (36%). Another formal synthesis of D-erythro-azidosphingosine from Dtartrate diester was reported by Panza 51 in 2002 taking advantage of Julia olefination of sulfone 115 to construct trans olefin in a E/Z ratio of 5:1 in 53% yield. Bittman 52 has achieved the synthesis of Lg-lyxo-phytosphingosine (5) employing diastereoselective addition of the Grignard reagent to aldehyde 117 derived from D-tartric acid to give a mixture of 118a and 118b in a ratio of 9:1 (Schem 23). Another crucial step was convertion of diol to azido 119 by Mitsunobu's procedure. According to the same synthetic procedure for 5, Dribo-phytosphingosine (2) was also prepared from compound 118b which was obtained from 118a via Mitsubobu reaction. Shiozaki 53 has provided an alternative access to D-erythrosphingosine (1) and Lg-lyxo-phytosphingosine (5) using chiral βlactam 120 derived from D-tartrate diester as chiral template (Scheme 24). Ring-opening reaction of β-lactam 120 by 4tolyltetrade-cylsulfone followed by elimination afforded key ketone 122 in a satisfactory yield (93%). Isomerization of ketone 122 to enol triflate 123, then reductive elimination and deprotection yielded N-Boc-D-erythro-sphingosine (88a) in a moderate yield. On the other hand, diastereoselective reduction of 122 by LiEt3BH gave protected aminotriol 124 in good yield and stereoselectivity, which further converted into in an excellent yield (96%).

Scheme 24 Syntheses of Lg-lyxo-phytosphingosine (5) and N-Boc-D-erythrosphingosine (88a) from D-tartrate diester.

Chirality from other chiral precursors

Concise, efficient and enantiodivergent syntheses of D-and Lerythro-sphingosine (1 and ent-1) were achieved by Merino et al in 2006. 54 As shown in scheme 25, the key steps involved a stereocontrolled Mannich-type reaction between D-glyceraldehyde nitrone 125 and 2-silyloxy silylketene acetal, and a trans-selective Wittig olefination. Interestingly, Mannich-type reaction conducted in the presence of SnCl2, Yb(OTf)3 , or Zn(OTf)2 gave compound 126a as major product , on the other hand, when using SnCl4 as promoter gave compound 126b as major product. L-erythrosphingosine (ent-1) was prepared according to the same procedure as described for 1 from 126a. Aside from the above chiral precursors, Sarabia and his coauthors employed L-methionine as chiral pool to synthesize cyclic sulfonium salt 133, which was successfully applied to synthesize Derythro-sphingosine (1) and D-ribo-phytosphingosine (2) over 7 steps, 13 steps, respectively (Scheme 27). 56 Highlight of this work was stereoselective formation of epoxide amide (134, 135 or 138) and regioselective epoxide-opening reaction by amino or azide nucleophile at C2 position.

Scheme 27 Syntheses of D-erythro-sphingosine (1) and D-ribo-phytosphingosine (2) from L-methionine.

Chiral auxiliary

Chiral sulfur auxiliary

A high-yielding and facile synthesis of D-erythro-sphingosine (1) was achieved by Castillón et al in 2008 in 42% overall isolated yield with 6 steps (Scheme 27). 56 The success of this work was employing asymmetric sulfur ylide reaction between the sulfonium salt 139 and the aldehyde 140 to construct the epoxide 141 with the desired configuration. In addition, E-selective CM for chain extension and a regio-, stereoselective intramolecular epoxideopening reaction to form oxazolidinone 142 were also crucial for the synthesis. Aside from the above sulfur auxiliary, Wei et al 58a has introduced a novel chiral N-tert-butanesulfinamide 145 for cross-coupling with bulky long-chain aliphatic aldehydes 144 derived from D-glutamic acid to stereoselectively install amino functionality at C2 position and hydroxyl functionality at C3 position, thus, an efficient synthesis of D-ribo-phytosphingosine (2) was accomplished (Scheme 29) over 5 steps in 27% overall yield from known compound 143. 58b Scheme 29 Synthesis of D-ribo-phytosphingosine (2) from D-glutamic acid.

Chiral N-containing auxiliary

Similar to Wei's protocol, 58a chiral iminoglycinate 147 was used by Bundle et al 59 to condense with alehyde to build amino group at C2 position and hydroxyl group at C3 postion, therefore, iminoglycinate condensed with acrolein to give truncated sphingosine 149 in a concise manner. On the other hand, iminoglycinate condensed with aldehyde containing a R or Sconfiguration hydroxyl group to give D-ribo-phytosphingosine (2) and L-lyxo-phytosphingosine (5) in more than 45% overall yield in both cases (Scheme 30).

Scheme 30 Syntheses of truncated sphingosine (149), D-ribo-phytosphingosine (2) and L-lyxo-phytosphingosine (5) from chiral iminoglycinate (147).

Ishikawa 60 has reported a synthesis of D-erythro-sphingosine (1) using chirality transfer from chiral guanidinium ylide 151 to 3alkenyl aziridine-2-carboxylate (153a and 153b) to build chiral amino alcohol unit in 1 (Scheme 31). In this work, chiral guanidinium ylide 151 reacted with α,β-unsaturated aldehyde 152 to give a mixture of cis/trans (ca. 1:1) aziridine-2-carboxylate without diastereoselectivity. However, both diastereoisomers can be further converted into compound 1 via oxazolidinone intermediates (155a or 155b). To note, cis-aziridine-2-carboxylate (153b) was subjected to ring-opening reaction followed by SN2 type substitution to invert the hydroxyl configuration at the C3 position.

Asymmetric reactions

A number of syntheses of sphingosines and phytosphingosines have been accomplished from achiral starting materials using asymmetric reactions to set chiral hydroxyl groups, or chiral amino groups, or both. The extensive literatures searching revealed they could be classified into five categories: Sharpless dihydroxylation, Sharpless epoxidation and Shi's epoxidation, catalytic asymmetric aldol reaction, Sharpless kinetic resolution, asymmetric aminohydroxylation and amination.

Sharpless dihydroxylation reaction

Very recently, Sudalai et al 61 has reported an enantioselective synthesis of tetraacetyl-D-ribo-phytosphingosine ( 16) starting from 1-hexadecanol taking advantage of L-proline-catalyzed α-amino-oxylation, Horner-Wardsworth-Emmons olefination for E-selective olefin and Sharpless asymmetric dihydroxylation as the pivotal reactions (Scheme 32). Besides, regioselective sulfonylation of diol 157 at the α-position of the ester was also necessary for the synthesis. Prior to Sudalai's synthesis, Bittman 62 employed a similar synthesis of 16 via the common intermediate 156 from 1hexadecene. Sharpless dihydroxylation reaction was utilized twice by Bittman to install the three continous stereogenic centers in 16.

Minor difference from Sudalai's strategy was that the synthesis of α-aizdoester 158 was carried out through regioselective α-azidation of the cyclic sulfate of dihydroxyl ester 157. This strategy was also sucessfully employed by Bittman to synthesize D-erythrosphingosine (1) commencing with 1-pentadecyne. Matheu has reported efficient and high-yielding syntheses of Derythro-sphingosine (1) and D-ribo-phytosphingosine (2) via common cyclic sulfate intermediate 164 over 7 steps in 44%, 53% overall yield, respectively (Scheme 33). 64 The desired chirality was obtained by dynamic kinetic resolution of butene epoxide and sequential Sharpless hydroxylation. Elimination of compound 164 gave compound 1, on the other hand, ring-opening by BzOH, CsCO3 further provided compound 2.

Scheme 33 Syntheses of D-erythro-sphingosine (1) and D-ribo-phytosphingosine (2) from butadiene monoepoxide.

Sharpless epoxidation and Shi's epoxidation reaction

Somfai et al 65 has reported the synthesis of Bn-protected Derythro-sphingosine (168) utilizing Shi's epoxidation, regioselective opening reactions of vinyl epoxide 166 and vinylaziridine 167 in the allylic position as crucial steps (Scheme 34). Notably, no regioseletivity was observed during Shi's epoxidation of diene 165 so as to give a mixture of 2,3-epoxy olefin 166 and 4,5-epoxy olefin. Later, Somfai et al described an improved strategy to synthesize Derythro-sphingosine (1) from divinylcarbinol over 5 steps in 51% overall yield. Highlight of the strategy was Sharpless epoxidation, subsequent Payne rearrangement, a regioselective ring-opening reaction and E-selective cross-metathesis for chain extension. Righi et al 66 has described an efficient approach to synthsize Derythro-sphingosine (1) and D-lyxo-phytosphingosine (5) based on Wittig olefination, stereoselective addition of the common aldehyde 174, respectively (Scheme 35). The aldehyde 174 was derived from (Z)-4-benzyloxy-2-buten-1-ol employing Sharpless epoxidation and following regioselective ring-opening reactions as key steps. Notably, Wittig olefination of 174 didn't gave trans olefin in a satisfactory selectivity (E/Z=7/3).

Scheme 35 Syntheses of D-erythro-sphingosine (1) and D-lyxo-phytosphingosine ( 5) from (Z)-4-benzyloxy-2-buten-1-ol.

Asymmetric aldol reaction

In 2006, Enders's group 67a reported concise and straightforward syntheses of D-arabino-phytosphingosine (3) and protected L-ribophytosphingosine (181) via the common ketone 178 with 6 steps in 49%, 38% overall yield, respectively (Scheme 36). The stereogenic centers at C3, C4 positions were introduced by (S)-prolinecatalyzed aldol reaction of 2,2-dimethyl-1,3-dioxan-5-one and pentadecanal with excellent diastereo-and enantiomeric excess (>99%, 95% respectively). While the configuration at C2 position was installed by stereoselective reduction of ketone. To improve the diastereoselectivity during the reduction, hydroxyl group at C4 position was protected as its silyl ether. On the other hand, the adol reaction was catalyzed by (R)-proline giving compound ent-178 with 59% yield and good diastereo-and enantioselectivity (>99%, 95% respectively), and compound ent-178 further converted into D-ribo-azidophytosphingosine (182). 67b Kobayashi 68 has reported an efficient synthesis of L-erythrosphingosine (ent-1) from (E)-4-((tert-butyldiphenylsilyl)oxy)but-2enal over 7 steps with 16% overall yield. The key steps involve a chiral zirconium complex catalyzed aldol reaction of aldehyde by silicon enolate 183 and cross coupling of acetate with Grignard reagent (Scheme 37). Notably, the aldol reaction successfully introduced the desired chiral hydroxyl and amino groups of compound 184 in a high yield (95%) and moderate stereoselectivity (anti/syn=8/2).

Scheme 37 Synthesis of L-erythro-sphingosine (ent-1) from (E)-4-((tert-butyldiphenylsilyl)oxy)but-2-enal.

Sharpless kinetic resolution

Barua 69 has reported a facile and flexible synthesis of D-ribophytosphingosine (2) from achiral trans-cinnamaldehyde over 13 steps in 15.6% overall yield (Scheme 38). The stereocenters at C3, C4 positions were obtained by Sharpless kinetic resolution of homoallylic alcohol 186, while the stereocenter at C2 postion was obtained by a regioselective epoxide-opening reaction. Another highlight of the synthesis was oxidative cleavage of phenyl ring by NaIO4/ RuCl3•H2O .

Scheme 38 Synthesis of D-ribo-phytosphingosine (2) from trans cinnamaldehyde.

Like Barua's protocol, Kumar 70 has also employed Sharpless kinetic resolution to introduce the stereocenter at C3 postion in the syntheses of N-Boc-L-threo-sphingosine (88b) and tetraacetyl-Darabino-phytosphingosine (66) (Scheme 39). And the syn stereochemistry of amino group at C2 position was installed by a tethered aminohydroxylation in a moderate yield (65-66%). Both compound 88b and 66 were synthesized over 8 steps in 8%, 11% overall yield, respectively.

Scheme 39 Syntheses of N-Boc-L-threo-sphingosine (88b) and tetraacetyl-Darabino-phytosphingosine (66) from 1-pentadecyne and 1-pentadecanol, respectively.

Asymmetric aminohydroxylation and amination

In 2008, Davies et al reported divergent and efficient syntheses of N,O,O-triacetyl-D-erythro-sphingosine (18), tetraacetyl-D-lyxophytosphingosine (ent-48) and tetraacetyl-D-ribo-phytosphingosine ( 16) from the common intermediate oxazolidine aldehyde 197 (Scheme 40). 71 Wittig olefination of oxazolidine aldehyde 197 leaded to give compound 18, and the E-selectivity (E/Z=94/6) was carried out by quenching the reaction with methanol. On the other hand, addition of compound 197 by Grignard reagent gave a 90:10 mixture of alcohol 199a and 199b, which further converted into compound ent-48 and 16, respectively. Highlight of the protocol was highly diastereoselective conjugate addition of unsaturated ester 195 followed by in situ enolate oxidation with (camphorsulfonyl)oxaziridine (CSO). Same as Davies's protocol, Han 72 also employed an asymmetric aminohydroxylation reaction of α,β-unsaturated ester 200 to introduce the stereocenters at C2, C3 postions with high regioselectivity (>20:1) and enantioselectivity (>99%). While the stereochemistry at C4 position was set by a high diastereoselective (>10:1) addition of aldehyde 201 by Grignard reagent. Thus, Nacetyl L-xylo-phytosphingosine (202) was obtained with 5 steps in 22% overall yield (Scheme 41). An alternative synthesis of 202 could also be achieved through a two-step manipulation on compound 203. On the other hand, the stereochemical interconversion of the hydroxyl group at C4 postion was carried out by treatment of compound 203 with MsCl/Et3N via oxazine intermediate 204, which further converted into N-acetyl L-arabinophytosphingosine (206).

In 2013, Helmchen 73 reported a novel synthesis of D-erythrosphingosine (1) in 9 linear steps and 5% overall yield (scheme 42).

Highlight of the scheme was a chiral iridium-catalyzed allylic amination to set the chiral carbamate 207 in a high yield (87%) and enantioselectivity (98%). The other pivotal reactions involved ringclosing metathesis (RCM) and stereoselective epoxidationregioselective elimination reaction.

Conclusions

Sphingosines and phytosphingosines have drawn increased attention from the synthetic chemists' community in recent years because of their various physiological activities. Two key points of theses syntheses were introduction of stereochemistry and extension of a long aliphatic chain. Chiral pool approaches, chiral auxiliary and asymmetric reactions are the three main strategies to set stereogenic centers. Among them, chiral pool strategy which was concise and efficient with short synthetic route, high yield, and high optical purity usually seemed to be more acceptable. Meanwhile, chiral auxiliary and asymmetric reactions provided candidate strategies to synthesize sphingosines and phytosphingosines from various starting materials, which could be more flexible and straightforward in some cases. And the protocol for chain elongation often employed Wittig olefination, olefin cross metathesis and nucleophilic addition by Grignard reagent. The protocols with higher yield, shorter synthetic route, higher enantioselectivity, higher diastereoselective, and better versatility for the syntheses of the library of sphingosines and phytosphingosines are still expected in future.

Scheme 7

 7 Scheme 7 Syntheses of compound 1, 16 and 48 from D-glucose and its derivatives.

Scheme 8

 8 Scheme 8 Syntheses of tetraacetyl-D-xylo-and D-ribo-phytosphingosine (10 and 16) starting from the methyl β-glycosides of D-GlcNAc (53a) and D-AllNAc (53b), respectively.

Scheme 9

 9 Scheme 9 Syntheses of D-erythro-sphingosine (1) and D-ribo-azidophytosphingosine (59) from 1,3-O-benzylidene-D-threose.

Scheme 10

 10 Scheme 10 Synthesis of tetraacetyl-D-ribo-phytosphingosine (16) from 3,4,6tri-O-benzyl-D-galactal.

Scheme 14

 14 Scheme 14 Syntheses of compound 1, 18, 79 and 80 from L-serine methyl ester HCl.

Scheme 16

 16 Scheme 16 Syntheses of D-xylo-(4) and D-arabino-phytosphingsine (3) from L-serine methyl ester.

Scheme 20

 20 Scheme 20 Synthesis of D-erythro-sphingosine (1) from D-ribo-phytosphingosine (2) through different routes.D-ribo-phytosphingosine works as important chiral pool, it was also used to prepare other phytosphingosines. Kim reported highyielding and concise syntheses of D-arabino-(3), D-lyxo-(5), and Dxylo-phytosphingosine (4) from D-ribo-phytosphingosine (2) (Scheme 21).49 The configurational inversion of C-4 of compound 2 which leaded to afford D-lyxo-phytosphingosine (5) was carried out via cyclic sulfate intermediate 105 or oxonium ion intermediate 108 attacked by nucleophiles. On the other hand, inversion of C-3 and C4 leaded to D-arabino-phytosphingosine (3), and inversion of C-3 leaded to D-xylo-phytosphingosine (4). Both were carried out via mesylate intermediate attacked by nucleophiles (H2O).

Scheme 22

 22 Scheme 22 Syntheses of compound 113 and 116 from D-tartrate diester.

Scheme 23

 23 Scheme 23 Syntheses of Lg-lyxo-phytosphingosine (5) and D-ribo-phytosphingosine (2) from D-tartric acid.

Scheme 25

 25 Scheme 25 Syntheses of D-and L-erythro-sphingosine (1 and ent-1) from Dglyceraldehyde nitrone. Rao's group 55a has reported a stereoselective synthesis of N,O,O,O-tetraacetyl D-lyxo-phytosphingosine (ent-48) over 10 steps from a known intermediate 55b derived from L-ascorbic acid. The crucial reactions involved Grignard addition on epoxide 129 and stereoselective addition of chiral imine 131 by vinylmagnesium bromide (Scheme 26).

Scheme 26

 26 Scheme 26 Synthesis of N,O,O,O-tetraacetyl D-lyxo-phytosphingosine (ent-48) from L-ascorbic acid.

Scheme 28

 28 Scheme 28 Synthesis of D-erythro-sphingosine (1) from chiral sulfur auxiliary.

Scheme 31

 31 Scheme 31 Synthesis of D-erythro-sphingosine (1) from chiral guanidinium ylide 151.

Scheme 32

 32 Scheme 32 Syntheses of tetraacetyl-D-ribo-phytosphingosine (16) and Derythro-sphingosine (1).

Scheme 34

 34 Scheme 34 Syntheses of Bn-protected D-erythro-sphingosine (168) and Derythro-sphingosine (1).

Scheme 36

 36 Scheme 36 Syntheses of D-arabino-phytosphingosine (3), protected L-ribophytosphingosine (181) and D-ribo-azidophytosphingosine (182) from 2,2dimethyl-1,3-dioxan-5-one.

Scheme 40

 40 Scheme 40 Syntheses of N,O,O-triacetyl-D-erythro-sphingosine (18), tetraacetyl-D-lyxo-phytosphingosine (ent-48) and tetraacetyl-D-ribo-phytosphingosine (16) from cis-but-2-ene-1,4-diol.

Scheme 41

 41 Scheme 41 Syntheses of N-acetyl L-xylo-phytosphingosine (202) and N-acetyl L-arabino-phytospchingosine (206) from α,β-unsaturated ester 200.

Acknowledgment

This work was supported by Natural Science Foundation of Hubei Province of China (No. 2015CFB333) for Youth Fund and Hubei Chenguang Talented Youth Development Foundation, China.

Biographical sketches

Yangguang Gao was born in 1983 and grew up in Chaohu, China. He obtained his Ph.D degree in Organic Chemistry at University of Chinese Academy of Sciences in 2014. At the same year, he took up his current position as assistant research fellow at Jianghan University. Now, his research interests include carbohydrate chemistry and total synthesis of natural products with exellent biological activities.

Xianran He was born in


Leticia P Granja

Eduardo D Martínez

Horacio Troiani

Clément Sanchez

Galo J A A Soler Illia

Magnetic Gold Confined in Ordered Mesoporous Titania Thin Films: a Noble Approach for Magnetic devices

In the last decade, the surprising magnetic behavior of gold nanoparticles has been reported. This unexpected property is mainly attributed both to size and surface effects.

Mesoporous thin films are ideal matrices for metallic nanoparticles inclusion due to their highly accessible and tailorable pore systems that lead to completely tunable chemical environments. Exploiting these features, we synthesized Au nanoparticles within mesoporous titania thin films (film thickness ∼150 nm and pore diameter ∼5 nm), and we studied their magnetic properties under confinement. We present here the results of the magnetization as a function of temperature and magnetic field for this system, Page 1 of 19 ACS Paragon Plus Environment ACS Applied Materials & Interfaces which are consistent with the previously reported for free (unconfined) thiol-capped gold nanoparticles. The successful inclusion of stable magnetic Au nanoparticles within transparent mesoporous thin films opens the gates for the application of these nanocomposites in 2D microdevices technology and magneto-optical devices.

INTRODUCTION

Gold nanoparticles (GNPs) have been intensively studied due to their wide possibilities of applications in optics, catalysis and sensing, among other fields. In the last decade, it was surprisingly found that GNPs could also present magnetic properties, which has opened a thriving research area. [START_REF] Nealon | Magnetism in Gold Nanoparticles[END_REF] These unusual magnetic properties have been ascribed to the nanoparticles size or surface functionalization, and many different GNPs systems have been studied by magnetometry techniques. [START_REF] Donnio | Very Slow High-Temperature Relaxation of the Remnant Magnetic Moment in 2 nm Mesomorphic Gold Nanoparticles[END_REF][START_REF] Lloveras | Magnetic Interactions in Spin-Labeled Au Nanoparticles[END_REF][START_REF] Hori | Diameter Dependence of Ferromagnetic Spin Moment in Au Nanocrystals[END_REF] Although the details are still to be clarified, the chemically induced magnetism is considered the main responsible for the magnetic moment in GNPs or clusters. The emergence of the magnetic properties has been studied by varying the stabilizing ligand. 5,6 It is nowadays well established that in general noble metal nanoclusters and nanoparticles can form high spin states, and a size dependence of the magnetic properties has been observed in surface-capped nanoparticles or clusters based on non-magnetic metals (i.e. Au, Ag, Cu, Pd or Pt). 7,[START_REF] Roduner | Magnetic Properties and the Superatom Character of 13-Atom Platinum Nanoclusters[END_REF][START_REF] Maitra | Ferromagnetism Exhibited by Nanoparticles of Noble Metals[END_REF] Although some works have proposed alternative mechanisms, such as self-sustained persistent currents of conduction electrons, at this moment there is general consensus about the orbital nature of this magnetic moment, [START_REF] Nealon | Magnetism in Gold Nanoparticles[END_REF][START_REF] Gréget | Magnetic properties of gold nanoparticles: a room-temperature quantum effect[END_REF] The appearance of magnetization in capped GNPs was proposed to be associated with a spin symmetry breaking related with the 5d and 6s electrons of the Au atoms involved in the chemical bond with the ligands. The presence of the capping agents at the surface changes the number of uncompensated spins, which in turn modifies the relative spin densities at the Fermi energy, thus creating a non-zero magnetic moment. 6 Maitra et al. [START_REF] Maitra | Ferromagnetism Exhibited by Nanoparticles of Noble Metals[END_REF] evidenced experimentally that the saturation magnetization increases with capping and depends on the strength of the metal-sulfur bond. Although the origin of GNPs magnetism has not been fully elucidated yet, and there is a lack of reproducibility of the magnetic behavior between reports, nowadays the magnetism of gold at the nanoscale is accepted. [START_REF] Nealon | Magnetism in Gold Nanoparticles[END_REF][START_REF] Suzuki | Measurement of a Pauli and Orbital Paramagnetic State in Bulk Gold Using X-Ray Magnetic Circular Dichroism Spectroscopy[END_REF] At present, the role of the surface and the interface of noble metal thin layers are being particularly considered in magnetic devices research. As an example, magnetic proximity effects have been reported at the Pt/Y 3 Fe 5 O 12 interfaces in magnetoresistance devices. [START_REF] Liang | Influence of Interface Structure on Magnetic Proximity Effect in Pt/Y 3 Fe 5 O 12 Heterostructures[END_REF][START_REF] Vélez | Magnetoresistance in Thin Metal Films with Strong Spin-Orbit Coupling[END_REF] Moreover, it was recently found that magnetization is induced in the Au thin layer of superconducting spin valves. [START_REF] Flokstra | Remotely Induced Magnetism in a Normal Metal Using a Superconducting Spin-Valve[END_REF] In the light of these findings, magnetic structures based in noble metal nanoparticles is becoming a growing field, and might inspire the design of novel magnetic, opto-magnetic and spintronic devices.

Materials with highly controlled pore size are very interesting matrices for the immobilization of magnetic metallic nanospecies. Platinum clusters have been immobilized in zeolites, which affect the hydrogen adsorption properties, leading to controlling the catalytic and magnetic behaviors. [START_REF] Jensen | Support Effects on Hydrogen Desorption, Isotope Exchange, Chemical Reactivity and Magnetism of Platinum Nanoclusters in KL Zeolite[END_REF] Very recently, the magnetism of GNPs within hollow mesoporous silica particles has been exploited in multifunctional nanoparticles for theranostics. [START_REF] Hembury | Gold-Silica Quantum Rattles for Multimodal Imaging and Therapy[END_REF] In order to fully exploit their potential in 2D magnetic devices such as magnetic sensors and memory devices, GNPs need to be efficiently incorporated into thin films, permitting an easy positioning and handling of the magnetic species, which is still a challenge. 17 Ordered mesoporous thin films 18 arise as a promising matrix to solve this limitation, by using them as robust frameworks to be filled with the nanoparticles desired, which results in enhanced nanoparticle stability and high accessibility for analytes. [START_REF] Angelomé | Synthesis and Applications of Mesoporous Nanocomposites Containing Metal Nanoparticles[END_REF][START_REF] Wolosiuk | Silver Nanoparticle-Mesoporous Oxide Nanocomposite Thin Films: a Platform for Spatially Homogeneous SERS-Active Substrates with Enhanced Stability[END_REF] In addition, simple and affordable patterning methods that rely on chemical or lithographic routes can be applied to these composites in order to specifically locate the nanoparticles in the micron or submicron scale. [START_REF] Fuertes | Controlled Deposition of Silver Nanoparticles in Mesoporous Single-or Multilayer Thin Films: From Tuned Pore Filling to Selective Spatial Location of Nanometric Objects[END_REF][START_REF] Martínez | Electrical Conductivity in Patterned Silver-Mesoporous Titania Nanocomposite Thin Films: Towards Robust 3D Nano-Electrodes[END_REF] In this work, we present an efficient and robust method to incorporate magnetic Au nanoparticles within the pore system of highly ordered mesoporous titania thin films [START_REF] Crepaldi | Controlled Formation of Highly Organized Mesoporous Titania Thin Films: From Mesostructured Hybrids to Mesoporous Nanoanatase TiO 2[END_REF] combining a soft reduction method and thiol capping. Controlled reduction and capping process were essential towards obtaining stable magnetic GNPs confined inside the mesopores. The method is highly reproducible and leads to protected GNPs, whose magnetic properties are constant throughout the samples and conserved along time.

These nanocomposites permit to create magnetically functionalized mesoporous thin films, and open the gate for exploiting GNPs properties in thin film related applications such as opto-magnetic [START_REF] Suda | Reversible Phototuning of the Large Anisotropic Magnetization at the Interface between a Self-Assembled Photochromic Monolayer and Gold[END_REF] and spintronic devices.

EXPERIMENTAL

Synthesis of mesoporous titania thin films

Mesoporous TiO 2 thin films (MTTF) were synthesized by dip-coating, using a titania sol in the presence of a supramolecular polymer template. [START_REF] Crepaldi | Controlled Formation of Highly Organized Mesoporous Titania Thin Films: From Mesostructured Hybrids to Mesoporous Nanoanatase TiO 2[END_REF] A stock solution of TiCl 4 in ethanol was prepared by adding 19 g of TiCl 4 in 181 g of absolute ethanol in a cold bath. To 20 g of this solution, it was added 0.56 g of Brij58 as supramolecular template and 1.8 g of water to promote the hydrolysis of the Ti precursor. The molar ratio between components TiCl 4 :Brij58:H 2 O:EtOH was 1:0.05:10:40. The prepared sol was heated up to 32 °C and dip coated at a withdrawal speed of 3.0 mm•s -1 in a controlled environment of 35% relative humidity (RH). Double side polished silicon wafers (University Wafers, USA) were used as substrates, therefore mesoporous films were deposited simultaneously over the two sides of the substrates.

After deposition, the films were kept for 24 hours in a chamber at 50% RH to promote stabilization of the mesostructure, and then submitted to 60 °C for 30 minutes, 130 °C for additional 30 minutes and finally calcined at 350 °C for 2 h. [START_REF] Roncaroli | Mesoporous Thin Films of TiO 2 on Attenuated Total Reflection Crystals. An In Situ Fourier-Transform Infrared Study of the Kinetics and Equilibrium of Adsorption and Photocatalysis of Carboxylic Acids[END_REF] Films were then washed with acetone and immersed for two hours in an ethanol:water 1:1 v:v mixture under strong agitation in order to remove any residue from the pyrolysis of the surfactant. Films were finally dried at 130 ºC. Mesoporous TiO 2 films templated with Brij 58 will be referred hereafter as TB.

Loading mesoporous films with Au nanoparticles

Mesoporous TiO 2 films were loaded with Au by controlling the nucleation and growth of GNPs in cycles of adsorption and reduction of AuCl 4 -. The procedure, described and analyzed in detail in previous work, [START_REF] Sánchez | Optical properties of Au nanoparticles included in mesoporous TiO2 thin films: a dual experimental and modeling study[END_REF] was performed by immersing the films in a 1 mM aqueous solution of HAuCl 4 •3H 2 O at pH 4, controlled by addition of NaOH. After 1 minute, films were extracted, washed with deionized water, and immersed in NaBH 4 10 mM aqueous solution for another minute. This reduction step cycle (RS) was repeated 16 times in total. An intermediate step was added after each two reduction steps to favor the adsorption of a thiol molecule by immersing the films in an ethanol solution of octanethiol (OT, Sigma-Aldrich, 98.5%) 10 µM for 30 minutes. The objective of this procedure was to limit the growth of the GNPs by thiol capping, and favor the development of new nucleation sites instead. Particular care was set in avoiding contact of the precursor solutions and samples with iron or any other magnetically active substances during the synthesis and measurement procedures, in order to discard the presence of magnetic impurities.

Thin Film Characterization

Mesoporous thin films thickness, optical constants and porosity were characterized by spectroscopic ellipsometry in a commercial SOPRA GES5 multispectral ellipsometer in microspot configuration; the data was analyzed by the use of Winelli II software. Ellipso-porosimetry was performed in order to determine film pore volume and pore diameter, by means of controlled condensation of water vapor inside porosity and

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

measure of the subsequent change in refractive index of the film, following Boissière et al. [START_REF] Boissiere | Porosity and Mechanical Properties of Mesoporous Thin Films Assessed by Environmental Ellipsometric Porosimetry[END_REF] Field emission-scanning electron microscopy (FE-SEM) images were obtained with a ZEISS LEO 982 GEMINI field emission electron microscope using an in-lens detector to improve resolution. Energy Dispersive Spectroscopy (EDS) was performed at the FE-SEM, to quantify the amount of Au in the samples by resolving the atomic ratio Au:Ti(r). High resolution transmission electron microscopy (HR-TEM) was performed in a Philips CM 200 electron microscope operating at 200 kV equipped with a LaB 6 emission filament and an ultra-thin objective lens for high resolution images. Samples for HR-TEM measurements were prepared by depositing a small amount of pieces scratched off the films onto an ethanol drop placed on carbon coated copper grids.

Magnetization of the GNP@MTTF sample was measured in a MPMS Quantum Design SQUID magnetometer. It was studied as a function of temperature (T) and applied magnetic field (H) in the ranges of 5 -300 K and up to 5 T respectively.

RESULTS

Spectroscopic ellipsometry was used to determine the thickness and refractive index at 633 nm and 0% relative humidity (RH) of the TB films, finding values of 147 ± 6 nm and 1.701 ± 0.004 respectively. By controlling the RH in a closed chamber around the sample, ellipsometry could be used to obtain a water adsorption isotherm from which the porosity and the pore size distribution (PSD) could be extracted. The results presented in Fig. 1 show a type IV isotherm with an H2 hysteresis loop, characteristic of a mesoporous structure formed by pores with restrictions. The porous volume fraction obtained of 32% is formed by pores of 4.9 ± 0.9 nm in diameter, connected by open necks of 3.2 ± 0.4 nm wide. Spectroscopic ellipsometry [START_REF] Thompkins | Spectroscopic Ellipsometry and Reflectometry[END_REF] was also exploited to study the optical properties of GNP@MTTF at 0% RH. It was found that the plasmonic features of GNPs are retained together with the magnetic properties. This subject is discussed in-depth at the SI.

Electronic microscopy was used to analyze the framework of MTTF and the GNP@MTTF samples (see Fig. 2). The characteristic long range highly ordered mesoporous structure obtained for TB films [START_REF] Crepaldi | Controlled Formation of Highly Organized Mesoporous Titania Thin Films: From Mesostructured Hybrids to Mesoporous Nanoanatase TiO 2[END_REF] is displayed by TEM (Fig. 2-A) and small angle X-Ray scattering (SAXS) at 3° grazing incidence (inset of Fig. 2-A). The GNP@MTTF nanocomposite displays a homogeneous dispersion of 4-6 nm spherical GNPs located evenly across and along the film thickness (Fig. 2-B to 2-F, calculated from TEM images, average of 200 nanoparticles).

Considering the film porosity, the density of the TiO 2 matrix and the atomic ratio Au:Ti(r) obtained by EDS, an estimated value of the gold filling fraction (F) can be calculated using Eq. 1; [START_REF] Sánchez | Optical properties of Au nanoparticles included in mesoporous TiO2 thin films: a dual experimental and modeling study[END_REF] where Vp is the accessible porous fraction (Vp = 0.32 from Fig. 1), δ Au and M Au are respectively the bulk density and the molecular weight of gold (19.3 g cm -3 , 196.96 g mol -1 ), δ TiO2 is the bulk density of anatase TiO 2 (3.8985 g cm -3 ), which is the main crystallographic phase present in the films,Error! Bookmark not defined. and M TiO2 is the molecular weight (79.89 g mol -1 ). The filling fraction F represents the volume fraction of the porosity (Vp) of the film occupied by GNPs, from which the total film volume occupied by GNPs (volume filling fraction, VFF) is calculated. The number of Au atoms in each square millimeter of film, 147 nm thick, was also calculated. The resulting values of the EDS analysis, F and VFF are summarized in The magnetization (m) as a function of the applied magnetic field (H) was measured at different temperatures for the GNP@MTTF sample. The magnetization measurements of film samples are always made up by the sum of the film and the substrate contributions. In our case, we aimed to elucidate the relevant features of the magnetic results inherent to the GNPs, which are confined within the TB film deposited on both sides of a double side polished (DSP) silicon substrate. The crude data information

100 ) 1 ( (%) 2 2 × - = p TiO Au p Au TiO V M V M r F δ δ
obtained from the magnetometer is shown, for 300 K and 5 K, at the inset of Fig. 3-B.

The magnetic response is governed by a linear negative-slope which is independent of temperature, due to the diamagnetic contribution of the TiO 2 film, the Si substrate and the sample mounting. Fig. 3-A and 3-B display the magnetization results once the diamagnetic slope was subtracted. Note that the resolution range used to measure in the magnetometer is conditioned by the whole magnetic signal (Inset of Fig. 3-B), which is about ten times higher than the non-linear features extracted from them (Fig. 3-A and3-B). Therefore these measurements of the magnetization of GNPs included inside the thin film oxide matrix are more indirect, and consequently more noisy and weaker, than the measurements of the GNPs alone, which are already widely reported.

Within this framework, it was confirmed that the magnetization results displayed here are reproducible from sample to sample and the magnetic properties of the samples were preserved along the several months that the experiments lasted. In Fig 3-A and3-B are compared two session measurements for 300 K and 5 K respectively, where it can be appreciated clear differences with temperature. These are important results considering sample-to-sample reproducibility and the stabilization of the GNPs embedded in the porous matrix, which is a well documented feature in metal nanoparticle-mesoporous oxide nanocomposite systems. [START_REF] Wolosiuk | Silver Nanoparticle-Mesoporous Oxide Nanocomposite Thin Films: a Platform for Spatially Homogeneous SERS-Active Substrates with Enhanced Stability[END_REF] The magnetic behavior of the TB films without GNPs was also studied. It was found that the magnetization is mostly independent of temperature and that there are no clear differences between the zero field cooling (ZFC) and field cooling (FC) curves (Figure 4). Slight deviations from the diamagnetic behavior at low temperatures are consistent with the electronic characteristics previously reported for TiO 2 [START_REF] Senftle | Magnetic Suceptibility of tetragonal Titanium Dioxide[END_REF][START_REF] Chauvet | Magnetic Properties of the Anatase Phase of TiO 2[END_REF] and doped silicon. [START_REF] Sarachik | Magnetic Properties of Boron-Page 18 of 19 ACS Paragon Plus Environment ACS Applied Materials & Interfaces Doped Silicon[END_REF][START_REF] Sonder | Magnetic Properties of N-Type Silicon[END_REF] Following the same analysis performed with the GNP@MTTF results, the negative linear contribution was deducted from the m(H) curves (inset A of Fig. 4) of the TB film. Comparing with the obtained for GNP@MTTF, which saturates at 8×10 -4 emu/g at 300 K (normalized by the sample mass), the non-linear component of the m(H) of the TB film (see Inset B of Fig. 4) is independent of T and does not exceed 4.88×10 With the aim of comparing our findings with those already reported for other GNPs systems, we estimated the magnetic moment (M) per Au atom from the Au content obtained by EDS (see Table 1). Figure 5-A displays M(H) results measured at different temperatures between 300 K and 5 K. It can be observed that M increases and the hysteresis appears with the decrease of temperature. The coercive field (H c ) reaches a value of H c = 0.045 T at 5 K (see inset of Fig. 5-A), in good agreement with the values previously reported in literature. [START_REF] Nealon | Magnetism in Gold Nanoparticles[END_REF][START_REF] Hori | Diameter Dependence of Ferromagnetic Spin Moment in Au Nanocrystals[END_REF]5 The saturation magnetic moment (m s ) is ∼ 0.07 µ B / Au atom at 5 K. Regarding the small mass of GNPs present in the MTTF structure and the ∼50% uncertainty involved in the estimation, it is remarkable that the m s obtained is well included within the wide range, 0.002 to 0.3 µ B / Au atom, reported for similar thiol-capped GNPs. [START_REF] Nealon | Magnetism in Gold Nanoparticles[END_REF][START_REF] Hori | Diameter Dependence of Ferromagnetic Spin Moment in Au Nanocrystals[END_REF][START_REF] Hembury | Gold-Silica Quantum Rattles for Multimodal Imaging and Therapy[END_REF] In order to understand the magnetic behavior involved in these systems, the M(H) curves of Fig. 5-A were plotted together as a function of H/T in Fig. 5-B. It can be noted that the M(H/T) reversible loops merge into the same curve above 100 K indicating the presence of a superparamagnetic state. At low temperatures, it can be appreciated in Fig.

5-B that M(H/T) curves deviates from this behavior and hysteresis in M(H) appears

below 50 K, suggesting that the system would become blocked.

As a complement of the former observations, Figure 6 displays the results of the magnetization as a function of temperature, performed with H = 0.1 T, for the Zero Field Cooling (ZFC) and Field Cooling (FC) measurement modes. It can be seen that ZFC and FC curves join into the same M(T) above 150 K, reinforcing the idea of a low temperature blocked state. Dutta et al. [START_REF] Dutta | Magnetism in Dodecanethiol-Capped Gold Nanoparticles: Role of Size and Capping Agent[END_REF] had previously reported a blocking temperature of ∼50 K in ∼5 nm dodecanethiol-caped GNPs. Looking more in detail, a peak can be appreciated at ∼80 K and ∼45 K respectively for FC and ZFC curves. This behavior has been previously observed in thiol-capped GNPs and seems to be directly associated with the magnetic mechanisms present in these systems. [START_REF] Maitra | Ferromagnetism Exhibited by Nanoparticles of Noble Metals[END_REF][START_REF] Wu | Quantum Spins in Mackay Icosahedral Gold Nanoparticles[END_REF] Furthermore, low T magnetic interactions are evidenced in the non-zero magnetic moment for H = 0. Regarding the GNPs size, the presence of remnant magnetization and coercive field could not be related with an inner magnetic domain structure. Thus, the interactions between nanoparticles emerge as the main candidates to explain the magnetic behavior observed at low T. [START_REF] Nealon | Magnetism in Gold Nanoparticles[END_REF] Regarding the consensus about a chemically induced magnetism, favored by the interactions with OT at the Au-S bonding, [START_REF] Hori | Diameter Dependence of Ferromagnetic Spin Moment in Au Nanocrystals[END_REF]6 the main contribution to the magnetization for each particle could be originated at the superficial Au atoms. [START_REF] Maitra | Ferromagnetism Exhibited by Nanoparticles of Noble Metals[END_REF] In this direction, we could consider only the external monatomic Au shell of the particles, calculated for the particle size distribution (inset of Fig. 2-B). The value we obtained for m s in this way is ∼0.3 µB/superficial Au atom, which is significantly higher than the one reported by Crespo et al., who estimated 0.036 µB/Au-S bonding for dispersed thiol-capped GNPs of 1.4 nm size. 5

It is usually assumed in the literature that the m s dispersion reported could be related with a non-homogenous magnetic behavior of the GNPs within a same sample. In our case, the MTTF mesostructure would confine the GNPs and limit their size. This confinement along with the procedure design for the OT capped GNPs synthesis would contribute together to obtain a more homogenous magnetic behavior for our GNP@MTTF samples. Moreover, Donnio et al. [START_REF] Donnio | Dendronized Ferromagnetic Gold Nanoparticles Self-Organized in a Thermotropic Cubic Phase[END_REF] reported ferromagnetic-like behavior for dendrimer-coated GNPs self-assembled in a cubic array presumably related with the presence of the mesoscale order. This would suggest that also the order of the mesoporous matrix could be relevant in our case. Further experiments are in due course to shed light on this subject.

In summary, we present here for the first time the magnetic response of GNPs embedded and in-situ generated in semiconductor TiO 2 mesoporous thin films. We demonstrate that the magnetic results obtained from the GNP@MTTF are consistent with previous reports for dispersed thiol-capped GNPs systems, manifesting low temperature remnant magnetization. These highly stable and reproducible magnetic nanostructures formed in ordered mesoporous thin films, are compatible with current microelectronics fabrication processes. These results also show the importance of the materials processing (in this case, the in-situ synthesis-reduction-capping cycle) in the magnetic properties of the nanocomposite system. Inclusion of stable GNPs in accessible mesoporous matrices opens the door to extending the applications of GNPs beyond the present nanoparticle domains, inspiring the design of novel magnetic and opto-magnetic thin film devices.

Page 10 of 19 ACSFigure 1 .Figure 2 .Figure 3 .

 19123 Figure 1. Water adsorption-desorption isotherm (A) and PSD (B) of mesoporous thin film TB/Si dip-coated at 3 mm•s -1 obtained by spectroscopic ellipsometry.

Figure 4 .Figure 5 .

 45 Figure 4. Magnetization (m) as a function of temperature (T) for the TB film, measured in field cooling (FC) and zero field cooling (ZFC) modes with H = 0.1 T. Inset A: m(H) as obtained from the SQUID for 300 K and 5 K. Inset B: The same m(H) results after the subtraction of the negative slope of the diamagnetic contribution.

Figure 6 .

 6 Figure 6. Zero Field Cooling (ZFC) and Field Cooling (FC) magnetic moment (M) as a function of temperature (T), measured with an applied field (H) of 0.1 T, for the GNP@MTTF sample. Hollow and filled symbols correspond to ZFC and FC curves respectively. The diamagnetic contribution was already subtracted.

Table 1 .

 1

	Eq. 1

F (%) VFF (%) Atomic ratio Au:Ti Au atoms/mm 2

	22.6	7.3	0.27	6.3×10 14

Table 1

 1 Composition of MTTF after gold infiltration and thiol capping analyzed by ellipsometry and EDS.

of 19 ACS Paragon Plus Environment ACS Applied Materials & Interfaces

	-4
	emu/g. Therefore, it should not be interpreted as a hysteresis loop or any other reliable
	magnetic behavior. It is known that for small magnetic signals, the measurements are

very sensitive to some measurements settings. One of the most critical is the SQUID center procedure, what introduces in many cases very confusing artifacts.

[START_REF] Sawicki | Sensitive SQUID Magnetometry for Studying Manomagnetism A[END_REF]

Page 8

ACS Paragon Plus Environment

Crespo, P.; Litrán, R.; Rojas, T.C.; Multigner, M.; de la Fuente, J.M.; Sánchez-López, J. C.;

Garitaonandia, J. S.; Insausti, M.; Goikolea, E.; Suzuki, M.; Cashion, J. D.; Kawamura, N.;

ACKNOWLEDGEMENTS

This work was funded by PIP 112-200801-00038 (CONICET), PICT 2087 and 2015-3526 (ANPCyT) and ECOS CONICET-CNRS (A08E05). L.P.G., G.J.A.A.S.I. and H. T are CONICET staff members. We thank Solange Di Napoli and Andrea Barral for fruitful discussions. Magnetometry measurements were performed in a SQUID belonging to the National Magnetism System (MINCYT).