Ambiphilic Ferrocenes

Dr Léa Radal

Dr Petr Vosáhlo

Dr Julien Roger

Dr Hélène Cattey

Prof. Dr Régine Amardeil

Prof. Dr Ivana Císařová

Petr Štěpnička
email: petr.stepnicka@natur.cuni.cz

Prof. Dr Nadine Pirio
email: nadine.pirio@u-bourgogne.fr

Jean-Cyrille Hierso
email: jean-cyrille.hierso@u-bourgogne.fr

Jean-Cyrille Hierso

J.-C Hierso

Dr J.-C Hierso

Highly Functionalized Brønsted Acidic/Lewis

Lorsqu'un écrit scientifique issu d'une activité de recherche financée au moins pour moitié par des dotations de l'État, des collectivités territoriales ou des établissements publics, par des subventions d'agences de financement nationales ou par des fonds de l'Union européenne est publié dans un périodique paraissant au moins une fois par an, son auteur dispose, même après avoir accordé des droits exclusifs à un éditeur, du droit de mettre à disposition gratuitement dans un format ouvert, par voie numérique, sous réserve de l'accord des éventuels coauteurs, la version finale de son manuscrit acceptée pour publication, dès lors que l'éditeur met lui-même celle-ci gratuitement à disposition par voie numérique ou, à défaut, à l'expiration d'un délai courant à compter de la date de la première publication. Ce délai est au maximum de six mois pour une publication dans le domaine des sciences, de la technique et de la médecine et de douze mois dans celui des sciences humaines et sociales.

The synthesis of highly functionalized ferrocenes bearing both acidic/basic functions (PR2, R = Ph, Cy, i-Pr, and carboxylic acids) and additional structuring substituents (bulky t-Bu groups) is reported along with their coordination behavior and solid-state structures.

Introduction

The chemistry of functionalized ferrocenes evolved from the simple but widely applied chelating diphosphane, 1,1'bis(diphenylphosphanyl)ferrocene (dppf), [1,2] to the synthesis of tailored ditopic hemilabile ferrocenyl phosphane ligands. [1a, 3 , 4 ] Early on, the synthesis of phosphanyl ferrocenes bearing an additional polar group R on the other ring of the ferrocene backbone (Scheme 1a) has been pursued by the Štěpnička group, with R being carboxyl group. [5] This work was further extended to numerous carboxamides, [ 6 ] amidosulfonates, [ 7 ] and other compounds that can serve as hybrid ligands and/or synthetic building blocks. [8] A general limiting aspect of the synthesis of hemilabile ferrocenes still remains an access to highly functionalized polysubstituted ferrocene derivatives as starting materials. These can bear various functional and structuring spectator substituents, and their preparation clearly involves synthetic difficulties arising mainly from the need of multiple functionalization and functional group compatibility. In this context, the Hierso group designed highly functionalized hybrid ligands and polyphosphanes (1,1',3,3-tetrafunctionalized) using dialkylated 1,1'-tertbutylferrocene as a scaffold, [ 9 ] and cousin compounds with geminally dimethylated substituents (Scheme 1b). [ 10 ] These compounds were used as ligands promoting metal-catalysed C-C and C-X bond formation (X = O, S, etc.). [11] New functionalized ferrocenes, which include both P,B-and N,B-ambiphiles were also developed as molecular systems that combine Lewis acidic and Lewis basic groups on a common ferrocene platform. [12,13] A further functionalization at ferrocene core was demonstrated to introduce novel properties such as planar chirality and steric control over the conformation of the metallocene backbone. [11, 13, 14] By combining these approaches, we examined herein synthetic pathways to new hybrid ferrocene ligands, tert-butylated phosphanylferrocene carboxylic acids and their corresponding aldehydes, that combine Brønsted acidic and Lewis basic functional moieties in their molecules, searching for an enhanced steric control within these compounds. Different synthetic pathways were investigated and we identified the most convenient road. The ligands were converted to gold(I) complexes and to phosphane selenides to further establish the properties and mutual influence of the functional groups in the prepared compounds. All these species were characterized by multinuclear NMR and by single-crystal X-ray diffraction analysis.

Lorsqu'un écrit scientifique issu d'une activité de recherche financée au moins pour moitié par des dotations de l'État, des collectivités territoriales ou des établissements publics, par des subventions d'agences de financement nationales ou par des fonds de l'Union européenne est publié dans un périodique paraissant au moins une fois par an, son auteur dispose, même après avoir accordé des droits exclusifs à un éditeur, du droit de mettre à disposition gratuitement dans un format ouvert, par voie numérique, sous réserve de l'accord des éventuels coauteurs, la version finale de son manuscrit acceptée pour publication, dès lors que l'éditeur met lui-même celle-ci gratuitement à disposition par voie numérique ou, à défaut, à l'expiration d'un délai courant à compter de la date de la première publication. Ce délai est au maximum de six mois pour une publication dans le domaine des sciences, de la technique et de la médecine et de douze mois dans celui des sciences humaines et sociales. Scheme 1. Functionalized ferrocenylphosphane hybrid derivatives (conformations encountered in the solid state structures are depicted).

Results and Discussion

The synthesis of the target compound 10 (Scheme 2) has been achieved by ring opening of 1-phenyl-1-phospha- [START_REF]Štěpnička in Ferrocenes: Ligands, Materials and Biomolecules[END_REF]ferrocenophane with phenyllithium, [ 15 ] followed by in situ carboxylation with excess solid carbon dioxide. This procedure, previously used to synthesize Hdpf (1), [5a] was adopted for the synthesis of 10 starting from the convenient precursor 1,1'-di-tertbutylferrocene 7 (Scheme 2). [12, 14] The tert-butylated ferrocenophane 9 was characterized in CDCl3 solution by a 31 P NMR peak at δP -15.1 ppm, similar to its non-tert-butylated counterpart. [16] Ring opening of 9 was successfully achieved with PhLi, but it was found to be troublesome by using other nucleophiles like t-BuLi. Acid 10 was next obtained by in situ carboxylation with solid or gaseous carbon dioxide and acidic workup. Characterization of 10 in CDCl3 solution by multinuclear NMR data showed diagnostic peaks (δP = -19.5, δC(COOH) = 177.6), which are consistent with those of the non-tert-butylated analogue (δP = -17.6, δC(COOH) = 177.2 in CDCl3). [5a] The synthesis of 10 via ferrocenophane opening lacks modularity, especially with respect to substituents at the phosphorus atom and, in addition, showed in our hands severe purification troubles. Because we also aimed at modifying Lewis basicity of phosphane PR2 function via changing the R groups, we explored the use of dilithium intermediate 8 [12] to diastereoselectively generate 1,1'-dibromo-3,3'-di-tert-butylferrocene ( 11) [12] (Scheme 2). Compound 11 can be further used in sequential lithiation/electrophilic trapping with a variety of chlorophosphanes [5b, 17 ] to prepare a range of electronically distinct functional phosphanes. The phosphanylferrocene bromide intermediates were isolated in their free (12)(13)(14) or in Pprotected form as borane adducts (15)(16)(17), which are both suitable for further synthesis. This is illustrated by the preparation of free acid 10 and its borane adduct 18 and, similarly, by the preparation of the corresponding aldehydes 21 and 23. [18] Compound 21 can be both OH-reduced and phosphane-protected by one-pot use of BH3.SMe2 in excess, providing alcohol 22. In the case of acid 18, we further demonstrated that the borane protecting group can be removed by using conventional methods. [19] Herein, removal of the BH3 moiety with 1,4-diazabicyclo[2.2.2]octane (DABCO, 2.2 equiv) in toluene (80 °C, 18 h) provided acid 10 in 62% yield after acidic workup and chromatographic purification. Operationally simpler heating of adduct 18 in ethanol (10 h) afforded the deprotected acid in a considerably better 90% yield. [20] Lorsqu'un écrit scientifique issu d'une activité de recherche financée au moins pour moitié par des dotations de l'État, des collectivités territoriales ou des établissements publics, par des subventions d'agences de financement nationales ou par des fonds de l'Union européenne est publié dans un périodique paraissant au moins une fois par an, son auteur dispose, même après avoir accordé des droits exclusifs à un éditeur, du droit de mettre à disposition gratuitement dans un format ouvert, par voie numérique, sous réserve de l'accord des éventuels coauteurs, la version finale de son manuscrit acceptée pour publication, dès lors que l'éditeur met lui-même celle-ci gratuitement à disposition par voie numérique ou, à défaut, à l'expiration d'un délai courant à compter de la date de la première publication. Ce délai est au maximum de six mois pour une publication dans le domaine des sciences, de la technique et de la médecine et de douze mois dans celui des sciences humaines et sociales. The molecular structures of 10 and 18 (Figure 1) were determined by single-crystal X-ray diffraction analysis, ascertaining that both compounds are racemic, retaining the configuration of their precursor 11. Compound 10 crystallized with two structurally independent but very similar molecules, whereas in the structure of the corresponding borane adduct 18 there is only one structurally independent molecule. Although the orientation of the functional substituents in the molecules of 10 and 18 seems to be locked by the bulky auxiliary substituents, the ferrocene scaffolds preserves Lorsqu'un écrit scientifique issu d'une activité de recherche financée au moins pour moitié par des dotations de l'État, des collectivités territoriales ou des établissements publics, par des subventions d'agences de financement nationales ou par des fonds de l'Union européenne est publié dans un périodique paraissant au moins une fois par an, son auteur dispose, même après avoir accordé des droits exclusifs à un éditeur, du droit de mettre à disposition gratuitement dans un format ouvert, par voie numérique, sous réserve de l'accord des éventuels coauteurs, la version finale de son manuscrit acceptée pour publication, dès lors que l'éditeur met lui-même celle-ci gratuitement à disposition par voie numérique ou, à défaut, à l'expiration d'un délai courant à compter de la date de la première publication. Ce délai est au maximum de six mois pour une publication dans le domaine des sciences, de la technique et de la médecine et de douze mois dans celui des sciences humaines et sociales. some of its conformational flexibility. This is shown by the torsion angles P•••Ct1•••Ct2•••CO2H, which are found at 52.32(3) ° and 53.16(3) ° for 10 (two molecules) vs. 133.37(5) ° for 18, corresponding roughly to an opened 1,2-conformation and a 1,3conformation, respectively (see Tables 1 and2). Despite the presence of bulky auxiliary substituents, the carboxyl groups remain roughly coplanar with the planes of their parent cyclopentadienyl rings (twisting <5°). However, the ferrocene cyclopentadienyl rings are considerably more tilted in 18 (8.73(8)° ) than in 10 (2.12(7) ° and 3.80(7) °). In both structures, the individual molecules assemble into the characteristic dimers via pairs of the O-HO hydrogen bonds between the carboxyl groups from either the independent molecules (10; OO = 2.6363( 14), 2.6279( 14) Å) or a molecule and its inversion-related counterpart (18; OO = 2.599(2) Å).

We also determined the solid-state structures of the borane protected bromide 17 with R = i-Pr (Figure 2), the boraneprotected aldehyde obtained thereof (25; Figure 3) and its analogue with cyclohexyl substituents at phosphorus atom (24; Figure 4). Selected characteristic structural parameters for these species are reported in Table 1. The general geometric features of ferrocenes 17, 24 and 25 are comparable. In particular, the introduced functional groups Fg (bromide, carboxylic acid and aldehyde, respectively) are similarly oriented with respect to the phosphane moiety with P•••Fg spatial distances fairly close, ranging between approximately 4.5 and 4.9 Å in the series. The roughly opened 1,2-conformation in these compounds corresponds well to the conformation found for acid 10 and, conversely, differs from the 1,3-conformation determined for its borane adduct 18.

P•••Ct1•••Ct2•••Fg (°)
63.50(2) -56.42( 6) 56.05 (6) tilt angle (°) [b] 2.68(6) 3.91 (13) 3.47(12) [a] Ct1 and Ct2 are the centroids of the phosphane-and Functional groups Fg-substituted cyclopentadienyl rings, respectively. [b] Dihedral angle of the least-squares cyclopentadienyl planes.

The electron-donating character of phosphanes is often estimated by measuring the 1 JP,Se coupling constants of their Lorsqu'un écrit scientifique issu d'une activité de recherche financée au moins pour moitié par des dotations de l'État, des collectivités territoriales ou des établissements publics, par des subventions d'agences de financement nationales ou par des fonds de l'Union européenne est publié dans un périodique paraissant au moins une fois par an, son auteur dispose, même après avoir accordé des droits exclusifs à un éditeur, du droit de mettre à disposition gratuitement dans un format ouvert, par voie numérique, sous réserve de l'accord des éventuels coauteurs, la version finale de son manuscrit acceptée pour publication, dès lors que l'éditeur met lui-même celle-ci gratuitement à disposition par voie numérique ou, à défaut, à l'expiration d'un délai courant à compter de la date de la première publication. Ce délai est au maximum de six mois pour une publication dans le domaine des sciences, de la technique et de la médecine et de douze mois dans celui des sciences humaines et sociales. selenide derivatives. [14a] An increase in these coupling constants indicates an increase in the s character of the phosphorus lonepair orbital, that is, an electron-withdrawing effect of the phosphane on the selenium. We have converted acid 10 into the corresponding phosphane selenide 10Se (Scheme 3), which was structurally characterized (Figure 5). The 1 JPSe value of 736 Hz determined for 10Se (d(P-Se) = 2.1045(9) Å) was identical to that of the cousin compound, 1,1'-bis(diphenylphosphanylselenoyl)-3,3'-di(tert-butyl)ferrocene, [14a] indicating that the carboxyl function exerts little influence over phosphorus in this case. In the solid state, the selenation led to an opening of the ferrocene backbone as indicated by the torsion angle P 9) ° (vs. 52.32(3) ° for 10), which makes the carboxyl group and the phosphane substituent more distant. Consequently, a stronger intermolecular hydrogen bonding is operating in the structure of 10Se (OO distances are shorter at 2.595(4) Å), which is arguably due to a lower steric congestion around the carboxyl group (see Figures 1 and5). Conversely, ferrocene cyclopentadienyl rings in 10Se are more tilted ( 9.02( 13) °) than in the parent phosphane 10 (2.12(7) °and 3.80( 7) °). The coordination of 10 to gold(I) was achieved by reacting the hybrid with [AuCl(SMe2)], leading in quantitative yield to complex 26 (Scheme 3) for which an X-ray structure analysis has also been solved (Figure 6). In the solid state, the molecules of the Au(I) complex 26 associate analogously to the free ligand 10 (cf. Fig. 1) into hydrogen bonded dimers having comparable O1O2i distances of 2.671( 5) Å and O2O1i distances of 2.651( 5) Å. In comparison with selenation of the phosphane moiety, the coordination to gold seems to have a smaller impact on the conformation of the ambiphilic ligand, despite the higher covalent radius of gold. The gold complex presents a linear P-Au-Cl geometry (angles found to be 178.04( 5 4) Å, while no bonding is detected. We anticipate that this proximity might be used in assisting catalytic processes in the sphere of gold and studies into this direction are currently ongoing. Conversely, in the solid-state distances between gold centers are above 7 Å, precluding any aurophilic interaction. The crystal packing showed the high hindrance provided by the ferrocene ligand, which keeps the Au centers away from each. Lorsqu'un écrit scientifique issu d'une activité de recherche financée au moins pour moitié par des dotations de l'État, des collectivités territoriales ou des établissements publics, par des subventions d'agences de financement nationales ou par des fonds de l'Union européenne est publié dans un périodique paraissant au moins une fois par an, son auteur dispose, même après avoir accordé des droits exclusifs à un éditeur, du droit de mettre à disposition gratuitement dans un format ouvert, par voie numérique, sous réserve de l'accord des éventuels coauteurs, la version finale de son manuscrit acceptée pour publication, dès lors que l'éditeur met lui-même celle-ci gratuitement à disposition par voie numérique ou, à défaut, à l'expiration d'un délai courant à compter de la date de la première publication. Ce délai est au maximum de six mois pour une publication dans le domaine des sciences, de la technique et de la médecine et de douze mois dans celui des sciences humaines et sociales.

•••Ct1•••Ct2•••Fg = 132.50(
Table 2. Selected geometric parameters for phosphanylferrocene carboxylic acid 10 and its derivatives.

Parameter [a] 10 10Se 18 26

Fe-Ct1 (Å) 1.6603(6) 1.6597(6) 1.6513(15) 1.6711(11) 1.653(2) 1.664(2) Fe-Ct2 (Å) 1.6613(7) 1.6612(7) 1.6669(17) 1.6548(10) 1.6533(19) 1.6563( 18) 4.1974(13) P2: 4.1070 (13) 5.989(4) 6.0188(23) P1: 4.328( 5) P2: 4.317( 5)

P•••CO2H (Å) P1:
OO (Å) [b] 2.6363( 14

P•••Ct1•••Ct2••• Fg (°)
52.32(3) 53. 16(3) 132.50( 9) 133.37( 5) 52.99( 13) -52.90 (13) tilt angle (°) [c] 2.12(7) 3.80( 7) 9.02( 13) 8.73( 8) 1.2( 7) 2.4( 5)

[a] Ct1 and Ct2 are the centroids of the phosphane-and Fg-substituted cyclopentadienyl rings, respectively. [b] H-bonding distance. [c] Dihedral angle of the least-squares cyclopentadienyl planes.

Conclusions

We report here a convenient synthetic pathway to the family of Brønsted acidic/Lewis basic ambiphilic ferrocene ligands, in particular to highly functionalized tert-butylated phosphanylferrocenecarboxylic acids bearing various phosphane substituents (PR2 = PPh2, Pi-Pr2, PCy2), and their corresponding aldehydes. The role we originally attributed to the tert-butyl groups was a steric control over the conformation of the metallocene backbone, possibly enforcing a close proximity between the phosphane and carboxyl groups that could be then transferred to coordination compounds synthesized from these ligands. However, the resulting situation was more complex than we anticipated. Indeed, although the orientation of the functional substituents seems to be locked by the bulky auxiliary substituents (e.g., in the case of acid 10), the ferrocene scaffolds retains partly its conformational flexibility as it was demonstrated by its backbone conformational changes in the solid-state structures, noted upon converting phosphane 10 into borane adduct 18 or selenide 10Se. Conversely, upon coordination to gold, the expected conformational control is clearly visible in the solid state (complex 26). Another noticeable point is that besides intramolecular steric interactions, the polarity of tert-butylated ferrocene carboxylic acids induce significant intermolecular hydrogen bonding, which controls the supramolecular assembly of the ferrocene derivatives in the solid state and perhaps also in solution. On-going work is focused at taking profit of these structural features exerted by the reported original ditopic ligands in cooperative transition metal catalysis.

Experimental Section

Materials and Methods. All reactions were performed under argon or nitrogen by using standard Schlenk techniques. Anhydrous THF was obtained from a Puresolv MD5 solvent drying system. Solvents used for chromatography and crystallizations were of reagent grade and were used without additional purification. NMR spectra were recorded at 25°C on a Varian UNITY Inova 400 (400 MHz in Prague), or 1 H NMR (500 and 600 MHz in Dijon) and 13 C NMR (101, 126 or 151 MHz) spectra were recorded with a Bruker AVANCE III instrument spectrometers. Chemical shifts (/ppm) are given relative to internal tetramethylsilane ( 1 H and 13 C NMR) or to external, 85% aqueous H3PO4 ( 31 P NMR). FTIR spectra were recorded on a Nicolet 6700 spectrometer in the range 400-4000 cm -1 . ESI mass spectra were obtained with a Bruker Compact Q-TOF spectrometer. Elemental analyses were performed using a Perkin-Elmer PE 2400 CHN analyzer (in Prague), or a Thermo Electron Flash EA 1112 Series (in Dijon).

Synthesis of 10 from bromo derivative 12. Bromide 12 [12] (0.40 g, 0.71 mmol) was dissolved in dry THF (10 mL) and the solution was cooled in a dry ice/ethanol bath. n-Butyllithium (0.49 mL of 1.6 M solution, 0.78 mmol) was added and the mixture was stirred with continuous cooling for 30 min. Next, crushed dry ice (ca. 40 g) was added and the mixture was allowed to warm to room temperature (ca. 90 min). Following acidification with methanolic HCl (1.1 mL of 1.25 M, 1.4 mmol), the volatiles were removed under reduced pressure. The obtained crude product was purified column chromatography (silica gel column, dichloromethane-methanol 50:1). The first minor band was discarded and the following major one collected and evaporated to give acid 10 as an amorphous, red-brown solid (yield 82%, 0.31 g). Additional purification could be achieved through crystallization from hot hexane. 1 H NMR (400 MHz, CDCl3): δ 7.60 -7.55 (2 H,m,Ph),m,Ph),m,Ph),4.73 (1 H, dd, J = 2.6, 1.3 Hz, Cp), 4.57 (1 H, t, J = 1.5 Hz, Cp), 4.29 (1 H, dd, J = 2.6, 1.7 Hz, Cp), 4.17 (1 H, dt, J = 2.8, 1.4 Hz, Cp), 4.10 (1 H, dd, J = 2.5, 1.5 Hz, Cp), 3.90 (1 H, dt, J = 2.5, 1.3 Hz, Cp), 1.26 (9 H, s, t-Bu), 1.04 (9 H, s, t-Bu). 13 Synthesis of 10 via ferrocenophane 9. An oven-dried, two-neck reaction flask was charged with 1,1'-di-tert-butylferrocene [12] (7; 1.0 g, 3.35 mmol) and anhydrous pentane (5 mL). Distilled N,N,N',N'-tetramethyl-1,2-Lorsqu'un écrit scientifique issu d'une activité de recherche financée au moins pour moitié par des dotations de l'État, des collectivités territoriales ou des établissements publics, par des subventions d'agences de financement nationales ou par des fonds de l'Union européenne est publié dans un périodique paraissant au moins une fois par an, son auteur dispose, même après avoir accordé des droits exclusifs à un éditeur, du droit de mettre à disposition gratuitement dans un format ouvert, par voie numérique, sous réserve de l'accord des éventuels coauteurs, la version finale de son manuscrit acceptée pour publication, dès lors que l'éditeur met lui-même celle-ci gratuitement à disposition par voie numérique ou, à défaut, à l'expiration d'un délai courant à compter de la date de la première publication. Ce délai est au maximum de six mois pour une publication dans le domaine des sciences, de la technique et de la médecine et de douze mois dans celui des sciences humaines et sociales.

diaminoethane (1 mL, 6.7 mmol) was added and the resulting solution was cooled at -80 °C. tert-Butyllithium (4.0 mL of 1.7 M solution in pentane, 6.8 mmol) was introduced and the reaction mixture was warmed up to room temperature while stirring overnight. Then, the mixture was filtered and the solid was washed twice with pentane to obtain adduct 1,1'-dilithio-3,3'-ditert-butylferrocene-N,N,N',N '-tetramethyl-1,2-diaminoethane (8) as an orange solid (57% yield, 813 mg). Next, adduct 8 (812.6 mg, 1.91 mmol) and anhydrous pentane (14.5 ml) were cooled at -80 °C. Dichlorophenylphosphane (0.27 mL, 2.0 mmol) in pentane (18.3 mL) was introduced drop by drop and the reaction mixture was warmed up to room temperature overnight. Following quenching with distilled water (15 mL), the organic layer was separated, washed with distilled water, dried over anhydrous magnesium sulfate and, finally, evaporated. Crude product was obtained as a mixture of 89% of 3,3'-di-tert-butyl,1-phenyl-1'phosphaferrocenophane ( 9), 7% of 1,1'-di-tert-butylferrocene (7) and 4% of N,N,N',N'-tetramethyl-1,2-diaminoethane (NMR yields) and was not further purified. For 9: 1 H NMR (500 MHz, CDCl3): δ 7.64 (2 H, ddd, J = 8.0, 6.5, 1.3 Hz), 7.42 (2 H, td, J = 7.7, 2.0 Hz), 7.35 -7.27 (1 H, m), 4.50 (2 H, q, J = 1.4 Hz), 4.32 (1 H, dd, J = 2.4, 1.3 Hz), 4.30 (1 H, dt, J = 2.3, 1.0 Hz), 4.28 (1 H, dd, J = 2.4, 1.4 Hz), 4.25 (1 H, t, J = 1.4 Hz), 1.19 (9 H, s), (9 H, s). 31 P{ 1 H} NMR (202 MHz, CDCl3): δ -15.14 (s). Impurities did not allow unambiguous assignment of the 13 C NMR signals.

Compound 9 (683.3 mg, 1.69 mmol,  90% pure) and diethyl ether (10 mL) was cooled at -80 °C. Phenyllithium (7.5 mL of 0.39 M solution in diethyl ether, 2.93 mmol) was introduced dropwise and the mixture was stirred for 30 min with continuous cooling and then 40 min at room temperature. After carbon dioxide gas was bubbled through the solution of the lithiated intermediate with stirring at 0 °C for 6 h, the mixture was quenched with saturated 5% aqueous NaOH. The crude product obtained after acidification was found difficult to purify. Hence, pure 10 was obtained by using a procedure similar to the synthesis of 19 described below. Yield: 44.0 mg (5%); for analytical data see above.

Synthesis of 10 by deborylation of adduct 18. Under argon, compound 18 (0.15 g, 0.27 mmol) was dissolved in degassed absolute ethanol (5.5 mL) and the solution was heated at reflux for 10 h under argon. The reaction mixture was cooled to room temperature and concentrated under vacuum. The crude product was purified by column chromatography (silica gel, hexane-ethyl acetate 5:1). First minor band was discarded and the following orange band was collected and evaporated to afford deprotected acid 10 as an orange solid. Yield: 128 mg (90%). The analytical data were identical to those given above.

Synthesis of 10Se from 10. An oven-dried, two-neck reaction flask was charged with 1'-(diphenylphosphanyl)-3,3'-di-tert-butylferrocene-1carboxylic acid (10; 26.3 mg, 0.05 mmol) and an excess of selenium black (60 mg, 0.76 mmol) in anhydrous dichloromethane (0.5 mL). The mixture was stirring for 24 h at room temperature then the solution was filtered to obtain 10Se in quantitative yield as testified by multinuclear NMR analysis. Crystal used for structure determination was obtained from CH2Cl2heptane. 1 H NMR (500 MHz, CDCl3): δ 7.84 -7.76 (2 H,m,Ph),m,Ph),m,Ph),m,Ph), (1 H, m, Cp), 4.64 (1 H, t, J = 1.5 Hz, Cp), 4.51 (1 H, dt, J = 3.0, 1.5 Hz, Cp), 4.42 (1 H, q, J = 2.1 Hz, Cp), 4.33 (1 H, dd, J = 2.7, 1.6 Hz, Cp), 4.31 (1 H, dt, J = 2.8, 1.6 Hz, Cp), 1.33 (9 H, s, t-Bu), 0.99 (9 H, s, t-Bu). 13 C{ 1 H} NMR (126 MHz, CDCl3): δ 177. [START_REF] Sheldrick | ShelXT-Integrated space-group and crystal-structure determination[END_REF]134.49 (d,J = 78.5 Hz),133.47 (d,J = 77.7 Hz), 132.58 (d, J = 10.9 Hz), 132.04 (d, J = 10.9 Hz), 131.48 (d, J = 2.9 Hz), 131.20 (d, J = 2.9 Hz), 128.43 (d, J = 3.0 Hz), 128.33 (d, J = 3.0 Hz), 108.05 (d, J = 7.9 Hz), 74.90, 74.21, 73.98 (d, J = 26.9 Hz), 72.60 (d,J = 10.9 Hz),72.04,71.27,70.70,70.43,69.28,31.79,31.73,31.13,30.49

Synthesis of 15.

An oven-dried, two-neck reaction flask was charged with 1,1'-dibromo-3,3'-di-tert-butylferrocene (11; 0.46 g, 1.0 mmol) and anhydrous THF (8 mL) and the resulting solution was cooled with a dry ice/ethanol bath to ca. -75°C. n-Butyllithium (0.63 mL of 1.6 M solution in hexane, 1.0 mmol) was introduced and the mixture was stirred for 30 min with continuous cooling. Chlorodiphenylphosphane (0.20 mL, 1.1 mmol) to the lithiated intermediate and the stirring was continued at -75°C for 30 min and at room temperature for additional 90 min. Next, neat BH3SMe2 (0.11 mL, 1.2 mmol) was added and the mixture was stirred for 30 min before quenching with saturated aqueous NaHCO3 (Caution! Gas evolution.). The mixture was partitioned between diethyl ether (20 mL) and brine (10 mL). The organic layer was separated, washed with brine, dried over anhydrous magnesium sulfate and evaporated with chromatographic silica gel to afford crude product in pre-adsorbed form. The crude product was transferred to the top of a silica gel column. Elution with hexanediethyl ether (5:1) removed a minor yellow band containing the starting dibromide and non-polar by-products. Changing the eluent to a hexanediethyl ether 1:1 mixture led to the development of a major band due to the product, which was collected and evaporated to afford analytically pure adduct 15 as an orange yellow solid (0.45 g, 78%). 1 H NMR (400 MHz, CDCl3): δ 7.65-7.57 (2 H, m, Ph), 7.50-7.33 (8 H, m, Ph), 4.53 (1 H, td, J = 2.5, 1.4 Hz, Cp), 4.36 (1 H, t, J = 1.5 Hz, 1 H, Cp), 4.28-4.26 (2 H, m, Cp), 4.04 (1 H, dt, J = 2.3, 1.4 Hz, Cp), 3.97 (1 H, dd, J 2.5, 1.6, Cp), 1.32 (9 H, s, t-Bu), 1.02 (9 H, s, t-Bu), 1.70-0.80 (3 H, br m, BH3). 13

Synthesis of 16.

An oven-dried, two-neck reaction flask was charged with 1,1'-dibromo-3,3'-di-tert-butylferrocene (11; 0.5 g, 1.1 mmol) and anhydrous THF (3.2 mL) and the resulting solution was cooled at -80 °C. n-Butyllithium (0.68 mL of 1.6 M solution in hexane, 1.08 mmol) was introduced and the mixture was stirred for 1 h with continuous cooling. Chlorodicyclohexylphosphane (0.24 mL, 1.08 mmol) in anhydrous THF (1.3 mL) was added to the lithiated intermediate and the stirring was continued at room temperature overnight. Then, BH3SMe2 (0.7 mL of 2.0 M solution in THF, 1.4 mmol) was added and the mixture was stirred for additional 1 h before quenching with saturated aqueous NaHCO3. The reaction mixture was partitioned between diethyl ether (20 mL) and brine (10 mL). The organic layer was separated, washed with brine, dried over anhydrous magnesium sulfate and evaporated. The crude product was purified by chromatography over a silica gel column using hexanedichloromethane (4:1) as the eluent. The first minor band was discarded containing the starting 1,1'-di-tert-butylferrocene and the following major Lorsqu'un écrit scientifique issu d'une activité de recherche financée au moins pour moitié par des dotations de l'État, des collectivités territoriales ou des établissements publics, par des subventions d'agences de financement nationales ou par des fonds de l'Union européenne est publié dans un périodique paraissant au moins une fois par an, son auteur dispose, même après avoir accordé des droits exclusifs à un éditeur, du droit de mettre à disposition gratuitement dans un format ouvert, par voie numérique, sous réserve de l'accord des éventuels coauteurs, la version finale de son manuscrit acceptée pour publication, dès lors que l'éditeur met lui-même celle-ci gratuitement à disposition par voie numérique ou, à défaut, à l'expiration d'un délai courant à compter de la date de la première publication. Ce délai est au maximum de six mois pour une publication dans le domaine des sciences, de la technique et de la médecine et de douze mois dans celui des sciences humaines et sociales.

one collected and evaporated to give compound 16 as an orange solid. Yield: 0.34 g (54%). 1 H NMR (600 MHz, CDCl3): δ 4.47 (2 H,s,Cp),4.38 (1 H,s,Cp),4.19 (1 H,s,Cp),4.05 (1 H,s,Cp),3.87 (1 H,s,Cp),m,Cy),1.99 (1 H,d,J = 13 Hz,Cy),m,Cy),m,Cy), 1.64 (1 H,d,J = 12.5 Hz,Cy),m,Cy),1.29 (9 H,s,m,Cy),1.22 (9 H,s,m,Cy), 0.78 -0.28 (3 H,br m,BH3). 13 C{ 1 H} NMR (151 MHz, CDCl3): δ 106.48 (d, J = 5.0 Hz), 102.50, 78.44, 74.38 (d, J = 10.7 Hz), 72.44 (d, J = 3.6 Hz), 71.22 (d, J = 55.0 Hz), 71.18, 69.15, 68.98 (br m), 64.81, 33.83 (d, J = 33.7 Hz), 32.76 (d, J = 34.6 Hz), 32.03, 31.96, 31.64, 31.06, 30.67, 28.20, 27.72 (d, J = 2.5 Hz), 27.57, 27.34, 27.33, 27.31, 27.26, 27.07, 27.03, 27.00, 27.00, 26.95, 26.23, 26.18, 22.84. 31

Synthesis of 17.

An oven-dried, two-neck reaction flask was charged with 1,1 '-dibromo-3,3'-di-tert-butylferrocene (11; 3.23 g, 7.09 mmol) and anhydrous THF (20.8 mL) and the resulting solution was cooled at -80 °C. n-Butyllithium (4.34 mL of 1.6 M solution in hexane, 6.95 mmol) was introduced and the mixture was stirred for 1 h with continuous cooling. Chloro-diisopropylphosphane (1.1 mL, 6.95 mmol) in anhydrous THF (7.0 mL) was added to the lithiated intermediate and the stirring was continued at room temperature overnight. The mixture was partitioned between dichloromethane and brine. The organic layer was separated washed with brine, dried over anhydrous magnesium sulfate and finally evaporated. The crude product was purified by chromatography over silica gel column using hexane-dichloromethane (11:9) as the eluent. First minor band was discarded and the following orange band was collected and evaporated to give compound 14 as an orange solid. Yield: 2.402 g (71%). In the following step, an oven-dried, two-neck reaction flask was charged with 1-(diisopropylphosphanyl)-1'-bromo-3,3'-di-tert-butylferrocene (14; 1.09 g, 2.21 mmol) and anhydrous dichloromethane (20 mL). BH3SMe2 (1.66 mL of 2.0 M solution in THF, 3.32 mmol) was added and the mixture was stirred for 1.5 h before quenching with distilled water. The mixture was partitioned between dichloromethane and brine. The organic layer was separated, washed with brine, dried over anhydrous magnesium sulfate and, finally, evaporated. The obtained crude product was purified by recrystallization from hot heptane to give compound 17 as orange crystals. Yield: 0.67 g (60%). 1 H NMR (600 MHz, CDCl3): δ 4.52 (1 H,s,Cp),4.47 (1 H,s,Cp),4.39 (1 H,s,Cp),4.19 (1 H,s,Cp),4.06 (1H,s,Cp),3.4 (1 H,s,Cp), (1 H, d of sept, J = 7.0, 11.4 Hz, i-Pr), 2.11 -2.03 (1 H, d of sept, J = 6.7, 10.7 Hz, i-Pr), m,1.29 (9 H,s,1.22 (9,H,s,dd, J = 7.0, 14.9 Hz, i-Pr), 0.95 -0.92 (3 H, dd, J = 7.0, 14.0 Hz, i-Pr), 0.84 -0.29 (3 H, br m, BH3). 13 C{ 1 H} NMR (151 MHz, CDCl3): δ 106.71 (d,J = 4.4 Hz),102.6,78.3,74.28 (d,J = 11.3 Hz),72.23 (d,JPC = 3.9 Hz),71.12 (d,J = 55.1 Hz),71.11,69.48 (br s),69.04,64.99,31.95,31.65,31.03,30.66,23.97 (d,J = 33.7 Hz),23.75 (d,J = 35.0 Hz),18.95,18.16 (d,J = 2.2 Hz)

Synthesis of 18.

Adduct 15 (0.58 g, 1.0 mmol) was dissolved in anhydrous THF (15 mL) in an oven-dried reaction flask. The stirring solution was cooled to approximately -75°C with a dry ice/ethanol bath and treated with n-butyllithium (0.69 mL of 1.6 M in hexane, 1.1 mmol). After stirring for 30 min, crushed dry ice (ca. 40 g) was added and the mixture was allowed to warm to room temperature during 90 min. Next, it was acidified with methanolic HCl (1.76 mL of 1.25 M solution) and concentrated under reduced pressure. The oily residue was taken up with dichloromethane (20 mL) and the solution washed with brine (2× 15 mL), dried over MgSO4 and evaporated. The crude product was purified by chromatography over a silica gel column using dichloromethane-methanol (50:1) as the eluent. The first minor band was discarded and the following major one collected and evaporated to give compound 18 as an orange powder. Yield: 0.42 g (78%). Crystals used for structure determination were obtained from chloroform-hexane. (d, J = 65.3 Hz), 69.86, 68.85, 31.66, 31.56, 30.97, 30.35. 31

Synthesis of 19.

An oven-dried, two-neck reaction flask was charged with 1-(dicyclohexylphosphanyl)-1'-bromo-3,3'-di-tert-butylferrocene-borane (16; 51.9 mg, 0.09 mmol) in anhydrous diethyl ether (1.5 mL) and the resulting solution was cooled at -80 °C. n-Butyllithium (0.09 mL of 1.6 M solution in hexane, 0.14 mmol) was introduced and the mixture was stirred for 1 h with continuous cooling. Next, the carbon dioxide gas was passed through the reaction mixture while stirring and cooling to 0 °C for 6 h, before quenching with 5% aqueous NaOH. The mixture was partitioned between diethyl ether and distilled water. The organic layer was separated, washed with brine, dried over anhydrous magnesium sulfate and, finally, evaporated. The aqueous layer was collected and few drops of 6 M HCl were added to obtain a red precipitate. The red precipitate was dissolved in dichloromethane, and the solution was dried over MgSO4 and evaporated. The residue was purified by chromatography over silica gel column using dichloromethane-methanol (99:1) as the eluent. First minor band was the 1-(dicyclohexylphosphanyl)-3,3'-di-tert-butylferrocene and the following band was collected and evaporated to give compound 19 as an orange solid. Yield: 19 mg (38%). 1 H NMR (500 MHz, CDCl3): δ 4.88 (1 H, dd, J = 2.6, 1.3 Hz, Cp), 4.78 (1 H, t, J = 1.5 Hz, Cp), 4.44 (1 H, d, J = 2.6, 1.6 Hz, Cp), 4.40 (1 H, dt, J = 2.9, 1.5 Hz, Cp), 4.23 (1 H, q, J = 1.5 Hz, Cp), 4.17 (1 H,dt,J = 2.5,1.3 Hz,Cp),m,Cy),m,Cy),m,Cy),m,Cy),1.30 (9 H,s,1.28 (9 H,s,m,Cy), 0.79 -0.2 (3 H, br m, BH3). 13 C{ 1 H} NMR (126 MHz, CDCl3): δ 177.52 (s, COOH), 107.65, 106.67 (d,J = 5.6 Hz),73.36 (br m),72.16,71.24 (d,J = 53.6 Hz),70.93 (d,J = 6.8 Hz),69.99,69.84,68.88,68.65 (br m),33.67 (d,J = 34.1 Hz),33.04 (d,J = 34.1 Hz),32.00,31.62,31.03,30.92,27.82 (d,J = 6.3 Hz), 27.59 (d, J = 10.0 Hz), 27.14 (d,J = 5.8 Hz),27.08,27.06,27.03,26.94 (d,J = 4.8 Hz),26.84,26.20. 31 P{ 1 H} NMR (202 MHz, CDCl3): δ 24.92 (s). 11 B{ 1 H} NMR (160 MHz, CDCl3): δ -Lorsqu'un écrit scientifique issu d'une activité de recherche financée au moins pour moitié par des dotations de l'État, des collectivités territoriales ou des établissements publics, par des subventions d'agences de financement nationales ou par des fonds de l'Union européenne est publié dans un périodique paraissant au moins une fois par an, son auteur dispose, même après avoir accordé des droits exclusifs à un éditeur, du droit de mettre à disposition gratuitement dans un format ouvert, par voie numérique, sous réserve de l'accord des éventuels coauteurs, la version finale de son manuscrit acceptée pour publication, dès lors que l'éditeur met lui-même celle-ci gratuitement à disposition par voie numérique ou, à défaut, à l'expiration d'un délai courant à compter de la date de la première publication. Ce délai est au maximum de six mois pour une publication dans le domaine des sciences, de la technique et de la médecine et de douze mois dans celui des sciences humaines et sociales.

Synthesis of 20.

Compound 20 was prepared similarly to 19, starting with 1-(diisopropylphosphanyl)-1 '-bromo-3,3'-di-tert-butylferrocene-borane (17; 200 mg, 0.39 mmol), anhydrous diethyl ether (8 mL) and n-butyllithium (0.37 mL of 1.6 M solution in hexane, 0.59 mmol). Yield: 88 mg (45%), orange solid. Crystals used for structure determination were obtained from chloroform-hexane. 1 H NMR (500 MHz, CDCl3): δ 4.90 (1 H,dd,J = 2.6,1.3 Hz,Cp),4.82 (1 H, t, J = 1.5 Hz, Cp), 4.50 (1 H, q, J = 1.9 Hz, Cp), 4.46 (1 H,dd,J = 2.7,1.6 Hz,Cp),4.20 (1 H,d,J = 1.5 Hz,Cp),4.19 (1 H,dd,J = 2.8,1.5 Hz,Cp),d of sept,J = 7.2,10.6 Hz,d of sept,J = 7.1,11.7 Hz,1.30 (9 H,s,, 1.28 (9 H,s,, m,, 1.03 (3 H,dd,J = 14.6,7.1 Hz,, 0.75 -0.36 (3 H,br m,BH3). 13 C{ 1 H} NMR (126 MHz, CDCl3): δ 177.53 (s, COOH), 107.82, 107.14 (d,J = 5.4 Hz,74.19 (br m),72.42,71.46 (d,J = 55 Hz),70.54 (d,J = 5.1 Hz),70.11,69.61,68.79,68.34 (br m),31.98,31.6,31.04,30.96,24.38 (d,J = 34.9 Hz),23.71 (d,J = 34.0 Hz),18.56,18.12,18.08 (d,J = 2.4 Hz) [12] (50 mg, 0.1 mmol, 31 P δ -17.77) dissolved in dichloromethane (2 mL) was cooled at 0°C. Then, BH3SMe2 (0.08 mL of 2.0 M solution in THF, 0.15 mmol) was added and the mixture was stirred for 2 h at room temperature before quenching with distilled water. The mixture was partitioned between dichloromethane and brine. The organic layer was separated, washed with brine, dried over anhydrous magnesium sulfate and, finally, evaporated to give compound 22 as a yellow oil. Yield: 35 mg (67%). 1 H NMR (500 MHz, CDCl3): δ 7.65 (2 H,ddd,J = 11.0,8.3,1.5 Hz,Ph),m,Ph),4.40 (1 H,dt,J = 2.4,1.2 Hz,Cp),4.30 (1 H,dd,J = 2.4,1.3 Hz,Cp),m,Cp and CH2OH),4.13 (1 H, t, J = 1.5 Hz, Cp), 4.01 (1 H,dd,J = 2.5,1.6 Hz,Cp),1.87 (1 H,s,OH),1.29 (9 H,s,, 0.86 (10 H,s,. 13 C{ 1 H} NMR (126 MHz, CDCl3): δ 133.5 (d, J = 9.6 Hz), 132.51 (d, J = 9.5 Hz), 132.554(d, J = 59.2 Hz), 131.25 (d, J = 2.5 Hz), 130.82 (d, J = 2.4 Hz), 130.67 (d, J = 59.3 Hz), 128.76 (d, J = 10.0 Hz), 128.57 (d, J = 10.2 Hz), 106.12 (d, J = 7.3 Hz), 103.13, 88.59, 72.5 (d, J = 6.0 Hz), 70.23 (d, J = 12.3 Hz), 69.47, 69.06 (d, J = 6.6 Hz), 68.59 (d, J = 68.5 Hz), 67.12, 66.20, 60.33, 31.56, 31.31, 31.11, 30.33. 31

Synthesis of 23.

Adduct 15 (67 mg, 0.12 mmol) was dissolved in anhydrous THF (2 mL) in an oven-dried reaction flask. The stirring solution was cooled to approximately -80°C and treated with n-butyllithium (0.1 mL of 1.6 M in hexane, 0.16 mmol). After stirring for 20 min, neat DMF (0.05 mL, 0.65 mmol) was added and the mixture was stirred for 30 min at -80°C. The mixture was allowed to warm to room temperature during 90 min before quenching with brine (2 mL) and 3M HCl (2 mL). Subsequently, the mixture was extracted with ethyl acetate and washed with brine, dried over MgSO4 and evaporated. The crude product was purified by chromatography over a silica gel column using pentane-ethyl acetate (19:1) as the eluent. The two first minor bands were discarded and the following major one collected and evaporated to give compound 23 as a red solid. Yield: 24.3 mg (39%). 1 H NMR (500 MHz, CDCl3): δ 9.33 (1 H,s,CHO),m,Ph),4.79 (1 H,dd,J = 2.8,1.3 Hz,Cp),4.70 (1 H, t, J = 1.5 Hz, Cp), 4.45 (2 H, p, J = 2.0 Hz, Cp), 4.33 (1 H,dt,J = 2.8,1.4 Hz,Cp),4.21 (1 H, q, J = 1.6 Hz, Cp), 1.28 (9 H, s, t-Bu), 1.04 (9 H,s,. 13 C{ 1 H} NMR (126 MHz, CDCl3): δ 195.38 (s, CHO), 133.00 (d,J = 4.3 Hz),132.92 (d,J = 4.5 Hz),131.46 (d,J = 15.7 Hz),131.40 (d,J = 3.9 Hz),131.14 (d,J = 2.3 Hz)

Synthesis of 24.

Compound 24 was prepared similarly to 23, starting with 1-(dicyclohexylphosphanyl)-1'-bromo-3,3'-di-tert-butylferrocene-borane (16; 100 mg, 0.17 mmol), anhydrous THF (5 mL), n-Butyllithium (0.25 mL of 1.6 M solution in hexane, 0.4 mmol) and anhydrous DMF (0.11 mL, 1.43 mmol). The crude product was purified by chromatography over a silica gel column using pentane-ethyl acetate (9:1). Yield: 61.8 mg (67%), red solid. Crystals used for structure determination were obtained from hot heptane. (d,J = 53.2 Hz),71.02,70.20,70.17 (d,J = 6.3 Hz),69.11,67.58 (d,J = 6.3 Hz),33.29 (d,J = 34.0 Hz),33.27 (d,J = 33.8 Hz),31.93,31.53,31.06,30.91,28.02,27.93,27.85,27.72,27.13 (d,J = 4.4 Hz), 27.05 (d, J = 2.2 Hz), 26.97 (d,J = 3.3 Hz),26.89,26.18,26.13. 31 P{

Synthesis of 25.

Compound 25 was prepared similarly to 23, starting with 1-(dicyclohexylphosphanyl)-1'-bromo-3,3'-di-tert-butylferrocene-borane (17; 200 mg, 0.39 mmol), anhydrous THF (4 mL), n-Butyllithium (0.29 mL of 1.6 M solution in hexane, 0.47 mmol) and anhydrous DMF (0.155 mL, 1.95 mmol). Yield: 107.1 mg (60%), red solid. Crystals used for structure determination were obtained from hot heptane. (d, J = 5.4 Hz), 78.52, 73.86 (d, J = 8.2 Hz), 71.24 (d, J = 52.6 Hz), 71.09, 70.51, 69.89 (d, J = 5.4 Hz), 68.78, 67.67 (d, J = 6.2 Hz), 31.88, 31.52, 31.07, 30.89, 23.89 (d, J = 34.3 Hz), 23.56 (d, J = 34.3 Hz), 18.49, 18.12, 18.01, 17.83. 31 Lorsqu'un écrit scientifique issu d'une activité de recherche financée au moins pour moitié par des dotations de l'État, des collectivités territoriales ou des établissements publics, par des subventions d'agences de financement nationales ou par des fonds de l'Union européenne est publié dans un périodique paraissant au moins une fois par an, son auteur dispose, même après avoir accordé des droits exclusifs à un éditeur, du droit de mettre à disposition gratuitement dans un format ouvert, par voie numérique, sous réserve de l'accord des éventuels coauteurs, la version finale de son manuscrit acceptée pour publication, dès lors que l'éditeur met lui-même celle-ci gratuitement à disposition par voie numérique ou, à défaut, à l'expiration d'un délai courant à compter de la date de la première publication. Ce délai est au maximum de six mois pour une publication dans le domaine des sciences, de la technique et de la médecine et de douze mois dans celui des sciences humaines et sociales.

Synthesis of 26.

A thin glass tube was charged under air with 1'-(diphenylphosphanyl)-3,3'-di-tert-butylferrocene-1-carboxylic acid (10; 26.3 mg, 0.05 mmol) and chloro(dimethylsulfide)gold(I) (14.7 mg, 0.05 mmol) in 0.5 mL of dichloromethane-D2. The mixture was stirring for a few minutes at room temperature to form gold complex 26 in quantitative yield as testified multinuclear NMR monitoring. Crystal used for structure determination was obtained from CH2Cl2heptane. 1 H NMR (500 MHz, CD2Cl2): δ 7.81 -7.74 (2 H,m,Ph),m,Ph),m,Ph),4.88 (1 H,dd,J = 2.7,1.3 Hz,Cp),4.55 (1 H, t, J = 1.5 Hz, Cp), m,Cp),4.37 (1 H,dd,J = 2.7,1.6 Hz,Cp),4.27 (1 H,td,J = 2.5,1.4 Hz,Cp),1.34 (9 H,s,, 0.93 (9 H,s,. 13 (d, J = 11.6 Hz), 108.64 (d, J = 9.1 Hz), 107.65, 74.80 (d, J = 18.8 Hz), 73.18, 72.56 (d, J = 7.7 Hz), 71.03, 70.48 (d, J = 71.5 Hz), 70.27, 69.46, 31.58, 31.38, 31.35, 30.65. 31

Synthesis of 27.

Compound 27 was prepared similarly to 26, starting with 20 (25.5 mg, 0.05 mmol), chloro(dimethylsulfide)gold(I) (14.7 mg, 0.05 mmol) and dichloromethane-D2 (0.5 mL) to form gold complex 26 in quantitative yield as evidenced by 1 H and 31 P NMR analysis. H NMR (500 MHz, CD2Cl2): δ 9.54 (1 H,s,CHO),m,Ph),m,Ph),4.80 (1 H,dd,J = 2.8,1.3 Hz,Cp),4.64 (1 H, t, J = 1.4 Hz, Cp), 4.51 (1 H, t, J = 2.2 Hz, Cp), 4.46 (1 H,dt,J = 2.6,1.4.6 Hz,Cp),4.43 (1 H,td,J = 2.8,1.4 Hz,Cp),4.28 (1 H,dt,J = 3.1,1.5 Hz,Cp), 1.28 (9 H, s, t-Bu), 1.07 (9 H,s,. 13 (d, J = 7.7 Hz), 108.92, 78.95, 75.22 (d, J = 12.5 Hz), 72.16 (d, J = 13.2 Hz), 71.54, 70.57 (d, J = 70.9 Hz), 70.24, 69.88, 69.83 (d, J = 8.1 Hz), 31.63, 31.38, 31.23, 30.86. 31 X-Ray crystallography. Full-sphere diffraction data for 10 and 18 were collected with a Bruker D8 VENTURE Kappa Duo PHOTON100 instrument equipped with a IµS micro-focus X-ray tube. The data for other compounds 10Se, 17, 24, 26 and 27 were recorded with a Bruker D8 Venture diffractometer and the data for 20 and 25, on a Bruker CCD diffractometer, All the diffractometers were equipped with Cryostream Cooler (Oxford Cryosystems) and Mo K radiation was used in all cases.

The structures were solved by direct methods (SHELXT) [ 21 ] and then refined using a full-matrix least squares routine based on F 2 with SHELXL-2014/2018. [ 22 ] All non-hydrogen atoms were refined with anisotropic displacement parameters. OH and BH hydrogens were identified on the difference electron density maps and were refined as riding atoms wit Uiso(H) set to a multiple of Ueq of the bonding atom. Hydrogens residing on carbon atoms were included in their theoretical positions and refined similarly. Relevant crystallographic data, data collection and structure refinement parameters are presented in Supporting Information. CCDC 1877094-1877102 (10,10Se,17,18,24,25,26 and 27 respectively) contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Scheme 2 .

 2 Scheme 2. Synthesis of tert-butylated phosphanyl ferrocene carboxylic acids and aldehydes (TMEDA = N,N,N',N'-tetramethyl-1,2-diaminoethane). Compounds 7, 8, 11, 12, 14 and 21 have been synthesized from reported procedures. Intermediary 13 was not isolated.

Figure 1 .

 1 Figure 1. View of the molecular structure of 10 (top) and 18 (bottom). Hydrogen atoms except those in OH groups, are omitted for clarity. Ortep at 30% probability level.

Figure 2 .

 2 Figure 2. View of the molecular structure of 17. 30% probability level.

Figure 3 .

 3 Figure 3. View of the molecular structure of 25. 30% Probability level.

Figure 4 .

 4 Figure 4. View of the molecular structure of 24. 30% Probability level.

Scheme 3 .

 3 Scheme 3. Coordination chemistry of ferrocenyl hybrids to selenium or gold.

Figure 5 .

 5 Figure 5. View of the molecular structure of 10Se (symmetry transformation: (i) 2-x, 2-y, 2-z). Hydrogen atoms (except those in the OH groups) are omitted except those in OH groups. 30% Probability level.

  ) ° and 176.45(5) °). Interestingly, the changed conformation of the ferrocene ligand in complex 26 results in close proximity between the Brønsted acidic carboxylic function and the gold center with distances Au•••O=COH = 3.509(4), 3.442(3) Å and Au•••C(O)OH = 3.777(5), 3.720(

Figure 6 .

 6 Figure 6. Views of the molecular structure of 26 (only one of the two molecules present in the asymmetric unit is shown here). A dichloromethane molecule and H atoms were omitted for clarity (except for OH). Each complex formed a centrosymmetric dimer. Bonding distance P-Au: 2.2249(12) and 2.2235(11) Å. 30% Probability level.

  C{ 1 H} NMR (101 MHz, CDCl3): δ 133.35 (d, J = 9.8 Hz), 132.59 (d, J = 9.5 Hz), 132.36 (d, J = 59.2 Hz), 131.03 (d, J = 59.2 Hz), 130.92 (d, J = 2.5 Hz), 130.64 (d, J = 2.4 Hz), 128.53 (d, J = 10.1 Hz), 128.38 (d, J = 10.1 Hz), 107.57(d, J = 6.4 Hz), 102.61, 77.42, 73.79 (d, J = 7.3 Hz), 73.35 (d, J = 10.7 Hz), 71.21, 70.14 (d, J = 66.

1 H

 1 NMR (400 MHz, CDCl3): δ 7.60 -7.54(2 H, m, Ph), m, Ph), 4.74 (1 H, dd, J = 2.6, 1.4 Hz, Cp), 4.62 (1 H, t, J = 1.5 Hz, Cp), 4.47 (1 H, td, J = 2.4, 1.4 Hz, Cp), 4.34 (1 H, dd, J = 2.7, 1.6 Hz, Cp), 4.32 -4.28 (2 H, m, Cp), 1.34 (9 H, s, t-Bu), 1.02 (9 H, s, t-Bu), 1.60 -0.80 (3 H, br m, BH3). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 176.94, 132.84 (d, J = 9.5 Hz), 132.77 (d, J = 9.8 Hz), 131.67(d, J = 58.6 Hz), 131.14 (d, J = 59.2 Hz), 130.93 (d, J = 2.5 Hz), 130.80 (d, J = 2.4 Hz), 128.51 (d, J = 4.9 Hz), 128.41 (d, J = 4.9 Hz), 107.98 (d, J = 6.4 Hz), 107.87, 72.75 (d, J = 8.2 Hz), 71.82, 71.77 (d, J = 9.2 Hz), 70.94 (br s), 70.20, 70.08

  P{ 1 H} NMR (162 MHz, CDCl3): δ 17.21 (br s). IR (Nujol): max 2637 m, 2601 m, 2554 m, 2397 m, 2369 m, 2341 m, 1675 vs (COOH), 1490 m, 1479 s, 1435 m, 1396 w, 1342 w, 1311 m, 1261 s, 1218 w, 1199 w, 1180 m, 1132 w, 1109 m, 1077 m, 1058 m, 1047 w, 1036 w, 1028 w, 999 w, 967 m, 940 w, 922 m, 903 w, 884 w, 862 m, 853 m, 837 w, 830 w, 816 w, 787 w, 752 m, 744 m, 736 m, 701 m, 694 m, 673 w, 639 m, 622 w, 613 w, 593 w, 564 m, 530 w, 511 m, 492 m, 472 m, 444 w, 432 w cm -1 . Anal. Calc. for C31H38FeBO2P (540.26): C 68.92, H 7.09%. Found: C 68.79, H 7.23%. ESI+ MS calc. for C31H38FeBO2NaP ([M + Na] + ): 563.1944, found: 563.1947.

  C{ 1 H} NMR (126 MHz, CD2Cl2): δ 174.32 (s, COOH), 135.08 (d, J = 14.3 Hz), 133.26 (d, J = 13.5 Hz), 132.47 (d, J = 2.6 Hz), 132.02 (d, J = 63.0 Hz), 131.60 (d, J = 2.6 Hz), 130.82 (d, J = 65.0 Hz), 129.55 (d, J = 12.0 Hz), 129.22

1

 1

  C{ 1 H} NMR (126 MHz, CD2Cl2): δ 194.22 (s, COOH), 134.31 (d, J = 13.9 Hz), 133.99 (d, J = 14.0 Hz), 132.23 (d, J = 2.7 Hz), 132.17 (d, J = 2.7 Hz), 131.66 (d, J = 55.2 Hz), 131.15 (d, J = 55.5 Hz), 129.55 (d, J = 2.0 Hz), 129.45 (d, J = 1.9 Hz), 109.16

Table 1 . Selected geometric parameters for 17, 24 and 25. Parameter (Fg) a 17 (Fg = Br) 24 (Fg = C27) 25 (Fg = C21)

 1

	Fe-Ct1 (Å)	1.6558(7)	1.6540(9)	1.6615(10)
	Fe-Ct2 (Å)	1.6579(7)	1.6577(10)	1.6619(10)
	P-B (Å)	1.9153(18)	1.914(2)	1.917(3)
	P•••Fg (Å)	4.9064(5)	4.621(2)	4.562(2)
	Ct1-Fe-Ct2 (°)	176.88(4)	175.45(5)	175.68(5)
	P-C-Ct1 (°)	170.22(12)	166.00(16)	170.03(16)
	R-C-Ct2 (°)	176.58(13)	175.8(2)	178.7(2)

  C{ 1 H} NMR (101 MHz, CDCl3): δ 177.62, 140.21 (d, J = 12.2 Hz), 137.92 (d, J = 11.3 Hz), 135.04 (d, J = 21.1 Hz), 132.35 (d, J = 18.6 Hz), 129.08, 128.28 (d, J = 7.6 Hz), 127.98 (d, J = 5.8 Hz), 127.69, 106.99 (d, J = 5.5 Hz), 106.45, 76.56 (d, J = 10.4 Hz), 73.75(d, J = 26.9 Hz), 71.31, 71.09, 70.24, 69.26, 69.02, 68.77, 31.52, 31.37 (d, J = 0.9 Hz), 30.86, 30.51. 31 P{ 1 H} NMR (162 MHz,

	CDCl3): δ -19.49. IR (Nujol): max 3087 w, 3071 w, 3055 w, 3041 w, 2637
	w, 2598 w, 2548 w, 1668 vs, 1586 m, 1571 w, 1477 s, 1446 m, 1437 m,
	1338 w, 1302 m, 1252 s, 1219 w, 1182 m, 1172 m, 1099 w, 1080 m, 1070
	m, 1049 m, 1039 w, 1028 m, 998 w, 978 w, 940 w, 921 m, 902 w, 866 m,
	850 m, 821 m, 783 w, 750 m, 740 s, 697 s, 680 w, 671 w, 626 w, 611 w,
	558 m, 532 w, 509 m, 493 m, 466 m, 440 w cm -1 . Anal. Calc. for
	C31H35FeO2P (526.43): C 70.73, H 6.70%. Found C 70.63%, H 6.63%.
	ESI+ MS calc. for C31H36FeO2P ([M + H] + ): 527.1797, found: 527.1788.

  .

	31 P{ 1 H} NMR (202 MHz, CDCl3): δ 31.35 ( 1 JPSe = 736 Hz). Anal. Calc. for
	C31H35FeO2PSe•1.7 dichloromethane •0.8 heptane (844.45): C 55.43%,
	H 6.22%. Found C 55.43%, H 6.22%. ESI+ MS calc. for C31H35FeO2PSe
	([M + H] + ): 629.0787, found: 629.0762.

  P{ 1 H} NMR (243 MHz, CDCl3): δ 24.70 (d, 1 JBP = 71.8 Hz). 11 B{ 1 H} NMR (193 MHz, CDCl3): δ -42.26 (t, 1 JPB = 71.25 Hz). Anal. Calc. for C30H49BBrFeP•0.5 H2O •0.3 hexane (634.68):

	C 60.18%, H 8.83%. Found C 59.94%, H 9.00%. ESI+ MS calc. for
	C30H49BBrFeNaP ([M + Na] + ): 609.2095, found: 609.2106.

  42.41 (s). Anal. Calc. for C31H50BFeO2P•0.15 dichloromethane•0.15 hexane (578.03): C 66.60%, H 9.14%. Found C 66.30%, H 9.67%. ESI+ MS calc. for C31H50BFeNaO2P ([M + Na] + ): 575.2889, found: 575.2892.

of 22 from 21. Compound 21

  , 17.82. 31 P{ 1 H} NMR (202 MHz, CDCl3): δ 30.96 (s). 11 B{ 1 H} NMR (160 MHz, CDCl3): δ -42.92 (s). IR (ATR): 2960 m, 2870 m, 2382 m, 1662 vs (COOH), 1477 s, 1461 s, 1385 w, 1359 m, 1302 w, 1254 s, 1177 m, 1141 w, 1071 m, 1044 m, 921 m, 877 w, 854 w, 832 w, 746 m, 688 m, 659 w, 607 m cm -1 . Anal. Calc. for C25H42BFeO2P•0.8 H2O•0.5 hexane (524.35): C 63.49%, H 9.63%. Found C 63.59%, H 9.67%. ESI+ MS calc. for C25H42BFeNaO2P ([M + Na] + ): 495.2263, found: 495.2239.

	Synthesis

  P{ 1 H} NMR (202 MHz, CDCl3): δ 15.96 (d, 1 JBP = 51.6 Hz). 11 B{ 1 H} NMR (160 MHz, CDCl3): δ -38.46 (s). This oily product did not furnish satisfactory EA. ESI+ MS calc. for C31H40BFeNaOP ([M + Na] + ): 549.2157, found: 549.2148.

  , 130.99 (d, J = 17.4 Hz), 128.86 (d, J = 10.2 Hz), 128.75 (d, J = 10.0 Hz), 108.37, 108.31 (d, J = 6.2 Hz), 78.46, 74.53 (d, J = 9.7 Hz), 71.33 (d, J = 8.1 Hz), 71.05, 70.42 (d, J = 65.5 Hz), 69.75, 69.57, 68.57 (d, J = 7.2 Hz), 31.48, 31.02, 30.70. 31 P{ 1 H} NMR (202.38 MHz, CDCl3): δ 15.84 (d, 1 JBP = 47.6 Hz). 11 B{ 1 H} NMR (160.39 MHz, CDCl3): δ -38.25 (s). Anal.

	Calc. for
	C31H38BFeOP•0.7 H2O•0.9 pentane (601.84): C 70.85%, H 8.41%. Found
	C 70.73%, H 8.49%. ESI+ MS calc. for C31H38BFeNaOP ([M + Na] + ):
	547.2000, found: 547.1988.

  P{ 1 H} NMR (202 MHz, CD2Cl2): δ 26.48 (s). Anal. Calc. for C31H35AuClFeO2P (758.85): C 49.07%, H 4.65%. Found C 49.25%, H 4.65%. ESI+ MS calc. for C31H35AuClFeNaO2P ([M + Na] + ): 781.0976, found: 781.0979.

  P{ 1 H} NMR (202 MHz, CD2Cl2): δ 28.30 (s). Anal. Calc. for C31H35AuClFeOP•0.8 H2O (757.27): C 49.38%, H 4.89%. Found C 49.28%, H 5.02%. ESI+ MS calc. for C31H35AuClFeNaOP ([M + Na] + ): 765.1027, found: 765.1023.

Acknowledgments

This work was supported by the ANR-PRC 2016 program (ALCATRAS, ANR-16-CE07-0001-01), the CNRS, Universite ́ de Bourgogne, Conseil Re ́gional de Bourgogne through the plan d'actions re ́gional pour l'innovation (PARI) and the fonds europe ́en de de ́veloppement regional (FEDER), and by the Charles University Research Centre program No. UNCE/SCI/014. Keywords: Ferrocenyl phosphanes • Brønsted acids • Hybrid ligands • Coordination compounds • Structures


Keywords: fish, neurotrophins, brain, encephalon, zebrafish neurons

Nerve Growth Factor (NGF), a member of the neurotrophin family, was initially described as neuronal survival and growth factor, but successively has emerged as an active mediator in many essential functions in the central nervous system of mammals. NGF is synthesized as a precursor pro-NGF and is cleaved intracellularly into mature NGF. However, recent evidence demonstrates proNGF is not a simple inactive precursor, but is also secreted outside the cells and can exert multiple roles.

.Despite the vast literature present in mammals, studies devoted to NGF in the brain of other vertebrate models are scarce. Zebrafish is a teleost fish widely known for developmental genetic studies and is well established as model for translational neuroscience research. Genomic organization of zebrafish and mouse NGF are highly similar, and zebrafish NGF protein has been reported in a mature and two precursors forms. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the NGF mRNA and protein distribution in the adult zebrafish brain and to characterize the phenotype of NGF positive cells.

NGF mRNA was visualized by in situ hybridization on whole mount brains. NGF protein distribution was assessed on microtomic sections by using an antiserum against NGF, able to recognize proNGF in adult zebrafish brain as demonstrated also in previous studies. To characterize NGF positive cells, anti-NGF was employed on microtomic slides of aromatase B transgenic zebrafish (where radial glial cells appeared fluorescent) and by means of double immunolabelling against NGF/PCNA (proliferation marker) and NGF/MAP2 (neuronal marker).

NGF mRNA and protein were widely distributed in the brain of adult zebrafish and their pattern of distribution of positive perikaryal was overlapping, both in males and females, with few slight differences. Specifically, the immunoreactivity to the protein was observed in fibers over the entire encephalon. MAP2 immunoreactivity was present in the majority of NGF positive cells, throughout the zebrafish brain. PCNA and aromatase B cells were not positive to NGF, but they were closely A c c e p t e d m a n u s c r i p t intermingled with NGF cells. In conclusion, our study demonstrated that mature neurons in the zebrafish brain express NGF mRNA and store proNGF.

INTRODUCTION

Nerve Growth Factor (NGF) is the first identified factor belonging, together with Brain Derived Neurotrophic Factor (BDNF), Neurotrophin (NT) 3 and NT 4/5, the neurotrophin family. As all members of the family, NGF is synthesized as a precursor form (pro-NGF) and either secreted outside the cells, as pro-and mature NGF, or cleaved intracellularly to mature NGF. Active forms of proand mature NGF are homodimers. To date, three types of NGF receptors are known: TrkA, p75 and sortilin. TrkA, the high affinity receptor, is a member of receptor tyrosine kinases family which includes receptor TrkB (for BDNF and NT 4/5) and TrkC (for NT 3). p75NTR is a member of the tumor necrosis factor receptor (TNFR) superfamily and can transduce signals of all neurotrophins.

Sortilin is a member of the family of Vps10p-domain transmembrane receptors, and was earlier characterized as a receptor for neurotensin. While TrkA mediates trophic signalling of the mature form of NGF, p75NTR bifunctionally mediates: (a) a signal to neuronal survival, specially when binding to mature NGF and acting together with TrkA; or (b) the induction of neuronal death when forming a receptor complex with sortilin and mediating proNGF signal (for a review see [START_REF] Niewiadomska | The cholinergic system, nerve growth factor and the cytoskeleton[END_REF]. NGF, initially described as neuronal survival and growth factor, encompasses roles regarding the density of innervation, synthesis of neurotransmitters and neuropeptides and cell body size, axonal sprouting and dendritic arborization (for a review see [START_REF] Minnone | NGF and Its Receptors in the Regulation of Inflammatory Response[END_REF].

The brain of fish possesses, beyond structural organization similar to all vertebrates, extremely high adult neurogenesis and astonishing regenerative properties. These peculiarities make the fish brain suitable for discovering regenerative mechanisms probably suppressed in mammals during evolutive adaptation [START_REF] Panula | The comparative neuroanatomy and neurochemistry of zebrafish CNS systems of relevance to human neuropsychiatric diseases[END_REF]Cacialli et al 2018b). Among teleost fish, zebrafish (Danio rerio) is widely used as model species for developmental genetic studies and functional mechanisms of numerous genes responsible of human diseases (D 'Angelo et al., 2016). In addition, zebrafish brain has been largely utilized in numerous studies devoted to adult neurogenesis [START_REF] Pellegrini | Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish[END_REF] A c c e p t e d m a n u s c r i p t regenerative ability after injury [START_REF] Cosacak | Regeneration, Plasticity, and Induced Molecular Programs in Adult Zebrafish Brain[END_REF][START_REF] Alunni | A comparative view of regenerative neurogenesis in vertebrates[END_REF]Cacialli et al 2018 a,b).

In fish species, homologs of mammalian neurotrophins have been identified, and an additional member, named NT6/7, probably originated by a duplication of the ray-finned fish NGF [START_REF] Dethleffsen | Insert-containing neurotrophins in teleost fish and their relationship to nerve growth factor[END_REF]. NT6/7 has been identified also in zebrafish [START_REF] Nilsson | Neurotrophin-7: a novel member of the neurotrophin family from the zebrafish[END_REF]. Genomic organization of zebrafish and mouse NGF are highly similar, and zebrafish NGF protein has been reported in a mature form of 194 aminoacid. [START_REF] Dethleffsen | Insert-containing neurotrophins in teleost fish and their relationship to nerve growth factor[END_REF]. In addition, two proNGF isoforms have been described in zebrafish: isoform 1 (NP_001338647.1) and isoform 2 (NP_954680.2), respectively of 224 and 261 a.a.. [START_REF] Dethleffsen | Insert-containing neurotrophins in teleost fish and their relationship to nerve growth factor[END_REF].

The distribution pattern of neurotrophins and their Trk receptors was described in the brain of adult fish (Dalton et al 2009a,b, D'Angelo et al 2012, D'Angelo et al 2014a, D'Angelo et al 2014b[START_REF] Cacialli | BDNF Expression in Larval and Adult Zebrafish Brain: Distribution and Cell Identification[END_REF], D'Angelo et al 2016[START_REF] Gatta | Neurotrophins and Their Trk-Receptors in the Cerebellum of Zebrafish[END_REF]. Furthermore, BDNF appeared involved in repairing mechanisms of adult zebrafish after traumatic brain injury (Cacialli et al 2018 a,b;[START_REF] Lucini | BDNF, Brain, and Regeneration: Insights from Zebrafish[END_REF].

In the present study, we evaluated NGF mRNA and protein distributionin the brain of zebrafish and identified neurons as the NGF source cells. Thanks to the conserved adult neurogenesis in fish brain and the potential involvement of neurotrophin in the regenerative ability, the identification of NGF neurons in the adult brain of zebrafish could represent a useful tool to evaluate its involvement in the regenerative process after injury or chemical/genetic induced degeneration.

In situ hybridization (ISH)

Oligonucleotide primers used to amplify and clone cDNA for the production of NGF ISH probes are: forward 5'-ACATGTACCATGAGGAGCAC-3'; reverse 5'-GTCGCTGGTGTGTGGAAAAT-3' (708 bp; NM_001351718.1). For preparation of NGF digoxigenin (DIG) labeled antisense riboprobe, the vector pCRII-TOPO containing NGF was linearized by BamHI digestion and digoxigenin-labeled riboprobe was prepared using in vitro transcription with T7 RNA polymerase. For sense riboprobe the vector containing NGF was linearized by Not1 and digoxigenin-labeled riboprobe was prepared using in vitro transcription with SP6 RNA polymerase.

ISH was performed on whole mount brains as previously described by [START_REF] Adolf | Conserved and acquired features of adult neurogenesis in the zebrafish telencephalon[END_REF]; [START_REF] Diotel | Comprehensive Expression Map of Transcription Regulators in the Adult Zebrafish Telencephalon Reveals Distinct Neurogenic Niches[END_REF]. Briefly, six zebrafish brains (males and females) were excised and fixed in paraformaldehyde (PFA 4%) dissolved in phosphate-buffered saline (PBS), for 24 hours (h) at 4°C. Then, the brains were dehydrated in a methanol/PBS concentration (25%; 50%; 75%; 100%) and stored at -20°C. After rehydrating through methanol/PBS gradient series and washed in PBS, the brains were incubated for 40 minutes (min) in PBS containing proteinase K (10 µg/ml) at room temperature (RT). After post-fixation in 4% PFA for 20 min and washes in PBS, brains were then A c c e p t e d m a n u s c r i p t prehybridized for 1 h and incubate overnight at 65°C in the hybridization buffer (pH 6) containing the DIG-labeled probe. Then, brains were washed in SCC 2x/formamide 50% and SSC 0.2x, and preincubated with blocking buffer for 3 h and then overnight with antidigoxigenin-AP, Fab fragments (1:5,000; Roche, NJ; Cat# 11093274910, RRID: AB_514497) at 4°C. The next day the brain sections were washed with PBS before staining with NBT/BCIP buffer (pH 9.5).

Whole mount stained brains were embedded in agar 2% and photographed with a Digital camera equipped on Zeiss Stemi. Then, the embedded whole mount stained brains were transversally sectioned with a razor blade or vibratome and the sections were mounted on the slide.

The specificity of the ISH labelling was demonstrated by using sense riboprobe, that showed absence of any staining.

Immunohistochemistry (IHC)

Imunohistochemical procedures were performed following detailed suggestions reported by de [START_REF] De Girolamo | Neuropeptide localization in nonmammalian vertebrates[END_REF]. Six adult zebrafish brains (male and female) were fixed in Bouin's solution for 24 h and processed for paraffin embedding. Transverse microtome sections were mounted on poly-lysine slides. Sections were deparaffinised in xylene, rehydrated through graded ethanol, treated with 3% H2O2 for 30 min and rinsed in PBS (pH 7.4) followed by antigen retrieval in sodium citrate buffer (pH 6; 80 °C) for 30 min.

Single immunohistochemistry

After rinsing 2 times in 0.2% Triton PBS (PBT), non-specific binding was blocked by treating sections with 1/5 normal goat serum (Vector, Burlingame, CA, USA, cod S-1000-20) for 30 min at RT. Then, sections were incubated over night at RT in a humidified chamber with rabbit antibody against NGF (Santa Cruz Biotechnology, CA, USA, cod sc-549. It recognizes N-terminus of the mature chain of NGF of human origin) diluted 1/100 or 1/300, respectively depending on the secondary antibody specified at following point a) or b). The next day, the sections were washed several times in PBT and alternatively incubated with a) Alexa Fluor® goat anti-rabbit 488 (1:200; Invitrogen Molecular probes, Eugene, OR, REF: A-11037; RRID: AB_10561549) for 2 h at RT in a dark and humidified chamber or b) EnVision-horseradish anti-peroxidase (HRP)-system (Dako, Santa Barbara, CA, cod. K4002). This system is based on a HRP labeled polymer conjugated with goat anti-rabbit IgG.

Sections treated with Alexa Fluor® goat anti-rabbit 488, after three washes in PBT, were mounted with the medium Vectashield (Vector) containing 4,6-diamino-2-phenylindole (DAPI), to visualize cell nuclei. Sections treated with EnVision-HRP-system were immersed in a fresh solution of 10 μg of 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich Corporation, St. Louis, MO, USA, cod. D5905) in 15 ml of a 0.5 mol, Tris buffer, pH 7.6, containing 1.5 ml of 0.03% H2O2. Then, sections were dehydrated and mounted.

For immunohistochemistry on cyp19a1bGFP transgenic zebrafish (glial cell marker, [START_REF] Pellegrini | Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish[END_REF]Tong et al., 2008) Moreover, the incubation of NGF antiserum preincubated with its homologous antigen showed no immunoreactivity, and NGF antiserum preincubated with its heterologous antigens did not modify the normal pattern of immunostaining.

Double immunolabelling

Double immunocytochemical staining NGF/PCNA and NGF/MAP2 was performed as follows: dewaxed and rehydrated consecutive sections were rinsed in PBS, and incubated for 48 h at RT. PCNA (proliferative cell nuclear antigen), antibody at diluted 1:100, was used to detect proliferative cells (Clone PC10; Dako, Glostrup, Denmark; REF: M0879; RRID: AB_2160651). This antibody is a marker of proliferating cells in vertebrate species, including zebrafish [START_REF] Pellegrini | Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish[END_REF][START_REF] Marz | Regenerative Response Following Stab Injury in the Adult Zebrafish Telencephalon[END_REF][START_REF] Cacialli | BDNF Expression in Larval and Adult Zebrafish Brain: Distribution and Cell Identification[END_REF]2018). MAP2 (Microtube-Associated Protein2) antibody, diluted RRID: AB_10561549). Tissue sections were washed in PBS-Triton 0,2%, and slides were mounted with the Vectashield medium containing DAPI for nuclei counterstaining (Vector Laboratories, Burlingame, CA). Controls for double immunolabelling were performed by incubating the sections with one of the two primary antisera and with the mismatched secondary antibodies.

Microscopy

The stained sections were photographed using a Nikon Eclipse 90i microscope, an epifluorescence microscope Olympus equipped with a DP71 digital camera and Leica DM6B, SN: 449492.

The digital raw images were optimized for image resolution, contrast, evenness of illumination, and background by using Adobe Photoshop CS5 (Adobe Systems, San Jose, CA, USA).

A c c e p t e d m a n u s c r i p t RESULTS NGF mRNA (Fig 1A ) and protein were widely distributed in the brain of adult zebrafish(Tab. 1). The pattern of distribution of positive perikarya was overlapping, both in males and females, with few slight differences (Table 1). Specifically, the immunoreactivity to the protein was observed in fibers over the entire encephalon. Thus, regions characterized only by presence of fibers, such as the glomerular layer of olphactory bulbs and deep and white zone of the optic tect, showed positivity to the protein. Based on these general considerations, for sake of the simplicity, the term NGF in place of NGF mRNA and NGF protein was used in the following description of results. The anatomical terminology follows "Neuroanatomy of the zebrafish brain" by [START_REF] Wulliman | Neuroanatomy of the Zebrafish Brain. A topological atlas[END_REF].

Telencephalon

The olfactory bulbs showed moderate quantity of NGF (Fig 1C ,D). The cells of both external and internal cellular layer and fibers of the glomerular layer resulted positive.

In the whole telencephalon, more intense NGF positivity was seen in the ventral telencephalon (Fig In the tegmentum, NGF was observed in cells of central nucleus of semi-circular torus and superior reticular formation.

Rhombencephalon

The cerebellar body was intensely reactive (Fig 1F). NGF positivity was observed in numerous large cells of the Purkinje layer and in few cells of the molecular layer of valvula and body (Fig. 7A -C 1 ).

Few weak stained cells were observed in the granular eminence.

In the medulla oblongata, few large cells containing NGF and belonging to the inferior reticular formation were seen.

Characterization of NGF containing cells

In order to identify the nature of NGF positive-cells in the brain of adult zebrafish, we carried out immunohistochemical staining against NGF on slides of aromatase B transgenic fish (cyp19a1b GFP), where radial glial cells appeared green fluorescent. Also, we performed double labelling using antibodies against NGF/PCNA (proliferation marker) and NGF/MAP2 (neuronal marker). Aromatase The serum raised against NGF was previously characterized [START_REF] Gatta | Neurotrophins and Their Trk-Receptors in the Cerebellum of Zebrafish[END_REF], where brain homogenates of adult zebrafish showed only a band of 25 kDa, corresponding to the molecular weight of proNGF isoform 1 (NP_001338647.1) [START_REF] Gatta | Neurotrophins and Their Trk-Receptors in the Cerebellum of Zebrafish[END_REF]. Accordingly, the presence of proNGF was also reported in the brain of the teleost N. furzeri, by employing the same antiserum(D 'Angelo et al 2014). On the other hand, this antiserum detected the mature form of NGF in different organs of fish, such as gut [START_REF] Lucini | Neurotrophin-like immunoreactivity in the gut of teleost species[END_REF] and kidney [START_REF] Arcamone | Distribution of NGF and NT-3-like protein immunoreactivity in the teleost kidney[END_REF]. Taking into consideration that the employed NGF antiserum is able to detect both pro-and mature NGF in fish tissues, our results suggest that that only the proNGF form is present in the brain of zebrafish. Remarkably, in mammalian brain, precursor and intermediate forms of NGF are expressed [START_REF] Fahnestock | The precursor pro-nerve growth factor is the predominant form of nerve growth factor in brain and is increased in Alzheimer's disease[END_REF][START_REF] Fahnestock | The nerve growth factor precursor proNGF exhibits neurotrophic activity but is less active than mature nerve growth factor[END_REF], and has been demonstrated to be actually the predominant form of NGF in central nervous system (CNS) [START_REF] Fahnestock | The precursor pro-nerve growth factor is the predominant form of nerve growth factor in brain and is increased in Alzheimer's disease[END_REF], whereas mature NGF appears to be lacking.

The co-presence of pro-NGF and the neuronal marker MAP2 immunoreactivity throughout the brain of adult zebrafish demonstrates that pro-NGF containing cells are neurons. This result was further confirmed by the absence of the glial marker aromatase B and proliferative marker PCNA.

Consistently with our observations, in the teleost fish N. furzeri, NGF was morphologically detected in neurons widespread throughout all brain regions (D 'Angelo et al., 2014). Only some glial cells lining the mesencephalic and rhomboencephalic ventricles of N. furzeri seemed to express NGF. At opposite, in goldfish NGF was almost totally localized in radial glia cells lining the ventricles (Benowitz and Shashoua, 1979). Species specific characteristics could explain the different results achieved in zebrafish, N. furzeri and goldfish. In agreement with our results, in the rat brain, NGF production was reported in neurons, predominantly localized in GABAergic neurons of the cortex, hippocampus, striatum and basal forebrain [START_REF] Lauterborn | NERVE GROWTH-FACTOR MESSENGER-RNA IS EXPRESSED BY GABAERGIC NEURONS IN RAT HIPPOCAMPUS[END_REF][START_REF] Lauterborn | NGF messenger-RNA is expressed by gabaergic but not cholinergic neurons in rat basal forebrain[END_REF][START_REF] Pascual | Expression of nerve growth factor and neurotrophin-3 mRNAs in hippocampal interneurons: Morphological characterization, levels of expression, and colocalization of nerve growth factor and neurotrophin-3[END_REF][START_REF] Bizon | Subpopulations of striatal interneurons can be distinguished on the basis of neurotrophic factor expression[END_REF][START_REF] Sofroniew | Nerve growth factor signaling, neuroprotection, and neural repair[END_REF][START_REF] Biane | Nerve growth factor is primarily produced by GABAergic neurons of the adult rat cortex[END_REF]. NGF was also reported in neuronal populations of adult monkey brain [START_REF] Hayashi | Expression of the gene for nerve growth-factor (NGF) in the monkey central nervous system[END_REF][START_REF] Zhang | Immunohistochemical distribution of NGF, BDNF, NT-3, and NT-4 in adult rhesus monkey brains[END_REF]. However, oligodendroglial progenitors A c c e p t e d m a n u s c r i p t derived from human embryonic stem cells are a source of NGF [START_REF] Zhang | Intracellular sphingosine 1-phosphate mediates the increased excitability produced by nerve growth factor in rat sensory neurons[END_REF][START_REF] Althaus | Glial cells as targets and producers of neurotrophins[END_REF].

Remarkably in adult zebrafish, another member of neurotrophin family, BDNF, was also expressed in neuronal populations of the whole brain [START_REF] Cacialli | BDNF Expression in Larval and Adult Zebrafish Brain: Distribution and Cell Identification[END_REF] and of telencephalon after injury (Cacialli et al 2018).

NGF expression was comparable between zebrafish and N. furzeri (D 'Angelo et al 2014), despite some slight differences in the neuroanatomy, whereas substantial differences in NGF cell localization and distribution were seen between goldfish and zebrafish as previously described. In rat brain, NGF levels resulted consistent in all regions with the highest presence in cortical areas [START_REF] Hoener | Nerve growth factor (NGF) content in adult rat brain tissues is several-fold higher than generally reported and is largely associated with sedimentable fractions[END_REF][START_REF] Sakamoto | Developmental changes in the NGF content in the A c c e p t e d m a n u s c r i p t brain of young, growing, low-birth-weight rats[END_REF]. Specifically, the pattern of distribution of both NGF mRNA [START_REF] Shelton | Studies on the expression of the beta-nerve growth-factor (NGF) gene in the central nervous system level and regional distribution of NGF messenger RNA suggest that NGF functions as a trophic factor for several distinct populations of neurons[END_REF] and protein [START_REF] Nishio | Detailed distribution of nerve growth-factor in rat-brain determined by a highly sensitive enzyme-immunoassay[END_REF] was described throughout the rat brain, with the highest intensity in the neocortex, the hippocampal pyramidal layer and striatum (Gall andIsackson, 1989, Rylett and[START_REF] Rylett | ROLE OF NEUROTROPHINS IN CHOLINERGIC-NEURON FUNCTION IN THE ADULT AND AGED CNS[END_REF]. Notably, observations in adult zebrafish brain do not support the fact that the forebrain is the NGF prelavent containing region.

In our study, both NGF mRNA and protein were detected in the perikaryon, and only proNGF protein was distributed along neuronal prolongations. Although the cellular co-presence of NGF mRNA and protein was not investigated, the overlapping pattern of distribution of NGF mRNA and protein throughout the zebrafish brain suggests that NGF expression and translation take place in mature neurons. The presence of NGF protein along neuronal prolongments could be retrogradely transported, according to the classical view on neurotrophins considered as target-derived retrogradely transported substances, but also anterogradely transported, accordingly to a vast literature. The idea that NGF may be produced locally was suggested since two decades ago (Lauterborn et al 1991, Conner and[START_REF] Conner | Distribution of nerve Growth factor-like immunoreactive neurons in the adult-rat brain following colchicine treatment[END_REF] and activity-dependent release of NGF and its effect on synaptic plasticity was postulated (Blöchl andThoenen, 1995, 1996;[START_REF] Wu | Nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 are sorted to dense-core vesicles and released via the regulated pathway in primary rat cortical neurons[END_REF]. Finally, Guo and collaborators (2012) ProNGF in the CNS is released in the extracellular space [START_REF] Bruno | Activity-dependent release of precursor nerve growth factor, conversion to mature nerve growth factor, and its degradation by a protease cascade[END_REF] and induces activation of the apoptotic machinery with subsequent death of different neuronal populations, mostly after injury and neurodegenerative disorders (for a review see [START_REF] Costa | To the end of the line: Axonal mRNA transport and local translation in health and neurodegenerative disease[END_REF].

The present results demonstrate that also in zebrafish brain NGF is synthesized in perykaria, however future studies are necessary to test whether proNGF is anterogradely transported and released in the extracellular space, at terminal ending of NGF positive fibers.

In conclusion, our study demonstrated that mature neurons of the zebrafish brain express NGF mRNA and store proNGF. Experimental studies reported proNGF as inhibition factor of the proliferation of neural stem cells isolated from postnatal mouse hippocampus, causing cell cycle arrest in the G0/G1 phase [START_REF] Guo | proNGF inhibits proliferation and oligodendrogenesis of postnatal hippocampal neural stem/progenitor cells through p75NTR in vitro[END_REF]. Thus, it is tempting to speculate that proNGF in the zebrafish brain, where cell proliferation is considerably high and persist along the entire lifespan, could represent a key negative regulator factor of this process.

  Animals and brain dissection Animals used in this study were housed in zebrafish facilities (INRA LPGP, BIOSIT, Rennes, France, agreement number: B 35-238-6) under standard conditions of photoperiod (14/10) and temperature (28°C). This project was approved by the local animal care and Westerfield ethics committee (Comité Rennais d'Ethique en matière d'Expérimentation Animale, Rennes, France), under the number EEA B-35-040. Zebrafish did not receive medical treatment prior or during the experience. No deaths occurred in the facilities before the euthanasia of animals used for in situ hybridization and immunohistochemistry experiments. Fish were suppressed with an overdose of tricaine methanesulfonate (MS-222).

  A c c e p t e d m a n u s c r i p t

  , NGF antibody was detected with Alexa Fluor® goat anti-rabbit 594 (1:200; Invitrogen Molecular probes, Eugene, OR, USA, REF: A-11037; RRID: AB_10561549). The specificity of IHC, assessed by substitution of NGF antiserum, secondary antibody fluorescent dye -conjugated or the EnVision with PBS or normal serum, achieved no specific immunostaining.

  , was used to detect neurons, (sc-74422 MAP-2 (A-8) Santa Cruz Biotechnology, Santa Cruz, CA, USA). After rinsing in PBS, the sections were incubated for 2 h at RT with mixture of the secondary antibodies directed against rabbit and mouse IgG. a) Alexa fluor® goat anti-mouse 594 (1:200; Invitrogen Molecular probes, Eugene, OR, REF: A-11005; RRID: AB_10561507; b) Alexa Fluor® goat anti-rabbit 594 (1:200; Invitrogen Molecular probes, Eugene, OR, USA, REF: A-11037;

  1C, D) and in the posterior zone dorsal telencephalic area (Fig 1B, C). Positive cells were distributed in the medial (Fig 3B, B 1 ), dorsal (Fig 8B, D), lateral (Fig 2A 1 , 3C, C 1 , 10 B, D), central (Fig 11B, D) and posterior part of dorsal part of telencephalic area. In the ventral telencephalic area, small round cells were seen in the dorsal and ventral part. Diencephalon Numerous intensely stained cells were seen in the anterior parvocellular preoptic nucleus (Fig 1C, 2B 1 ), and in the posterior parvocellular preoptic nucleus (Fig 9 B, D). Weak signal was detected in few cells of magnocellular preoptic nucleus. In dorsal and ventral habenular nucleus few positive cells were detected. High density of NGF positive cells were observed in the ventro-medial and ventro-lateral thalamic nuclei (Fig 9B, D). Few and weak positive cells were seen in the central posterior thalamic nucleus. Numerous positive cells were detected in the posterior tuberal nucleus. In the lateral and medial preglomerular nuclei, positive cells and some fibers were detected.

  In the hypothalamus, the ventral zone of periventricular hypothalamus showed intense positivity in the whole mount brain (Fig 1E) and numerous NGF positive cells were seen in histological sections (Fig 2C 1 , 5B-C 1 ). In the dorsal (Fig 1E) and caudal zone of periventricular hypothalamus moderate positivity was seen. Large NGF positive cells belonging to the diffuse nucleus of the inferior lobe were seen (Fig 6 A-B 1 ). Few NGF positive cells were also present in the mammillary body and in the nucleus of the medial longitudinal fascicle Mesencephalon NGF positive fibers were present in the longitudinal tori and the optic tect, particularly in the deep white zone and superficial white zone. Numerous small NGF positive cells appeared scattered in the periventricular grey zone (Fig 4, B 1 , B 2 ).

  positive cells, distributed along ventricles, were closely intermingled with NGF positive cells along telencephalic (Fig 8 B -D) and diencephalic (Fig 9B-D) ventricles. PCNA-positive cells were A c c e p t e d m a n u s c r i p t positioned along the ventricular lining of the brain, and NGF was detected in the cytoplasm of cells very close to PCNA labelled cells (Fig 10B-D). MAP2 immunoreactivity was present in the majority of NGF positive cells, throughout the zebrafish brain (Fig 11B-D).

  the neuroanatomical distribution of NGF mRNA and protein in zebrafish.

  demonstrated, by immunohistochemical, ELISA and electrophysiological analyses, anterograde delivery of NGF in hippocamposeptal system of mice.

  Author's contribution PC conceived and planned the experimentation, acquired and analyzed data; CG, AL, AP acquired and analyzed data; LDA, PdG, EP critically revised the manuscript; CL analyzed data and wrote the paper.

LEGENDS

(A -C) Representative section taken from the zebrafish atlas (Wullimann et al., 1996). NGF positive cells are represented by blue dots. (A 1 -C 1 ) NGF positive cells in the dorsal part of telencephalon (A 1 ), in the anterior parvocellular preoptic nucleus (B 1 ) and along the ventral zone of periventricular hypothalamus (C 1 ). Hv: ventral zone of periventricular hypothalamus; PPa: anterior parvocellular preoptic nucleus; Tel: telencephalon. Scale bar: 50 μm. (A, B) Representative sections taken from the zebrafish atlas (Wullimann et al., 1996). NGF positive cells are represented by green dots. Cells positive to NGF in the posterior zone of dorsal telencephalic area (A 1 ) and in periventricular gray zone of optic tect (B 1 ). (A 2 , B 2 ) NGF positive cells co-marked with DAPI. TelV: telencephalic ventricle; Scale bar: 50 μm.