Dr William Erb
email: william.erb@univ-rennes1.fr

Prof Min Wen

Jean Pierre Hurvois

Florence Mongin

Dr Yury S Halauko

Oleg A Ivashkevich

Dr Vadim E Matulis

Thierry Roisnel

Pierre Jean

Prof F Hurvois

Dr T Mongin

Roisnel

Prof. O A Ivashkevich

O-Isopropylferrocenesulfonate: synthesis of polysubstituted derivatives and electrochemical study

Few studies have been reported on the synthesis of ferrocenesulfonates up to now. Here, we describe the use of ortho-deprotometalation and "halogen dance" reactions to easily reach original derivatives. Further functionalizations using crosscoupling and metal/halogen exchange were also performed from iodinated derivatives. Finally, while the electrochemical properties of selected ferrocenesulfonates were evaluated and compared with the corresponding sulfonamide derivatives, halogen bonds were observed at the solid state for iodinated derivatives of ferrocenesulfonates.

10.

Introduction

Since its discovery in 1952, 1 ferrocene has been established as one of the most important organometallics. Synthetic developments around this sandwich compound are inescapable. Indeed, due to the relative stability of ferrocene and an easy reversible one-electron oxidation process, derivatives have found applications in numerous fields including catalysis and material science. [START_REF] Togni | Ferrocenes: Homogeneous Catalysis, Organic Synthesis[END_REF] While many ferrocene derivatives functionalized with usual functional groups have heavily been studied, syntheses of ferrocenesulfonic acids remain scarce in the literature. The parent compound can be obtained by sulfonation of ferrocene, and be isolated either as the free acid, 3 or more conveniently as its ammonium 4 or 4-toluidinium sulfonate 5 salt.

Monosubstituted ferrocene sulfonic acids/sulfonates have been used as anion dopants in the formation of cationic polymers due to their good charge-discharge properties. 6 Their high electrocatalytic activity also made ferrocenesulfonates useful as mediators for detection (e.g. of sulfides), 7 and as electron carriers in redox enzyme catalysis. [7][8] Few applications in catalysis have also been reported for phosphino derivatives of ferrocenesulfonic acid. Indeed, 2phosphinoferrocenesulfonic acids appeared as an attractive class of ligands for chemists working in the field of catalysis, and in particular those in search of efficient ligands for palladiumcatalyzed alkene (co)polymerization. 9 These original compounds can be easily prepared from 4-toluidinium ferrocenesulfonate by a deprotolithiation-trapping sequence involving its initial conversion into the corresponding lithium salt. 9 Interestingly, O-alkyl 2-phosphinoferrocenesulfonates have also been identified as ligands for catalysis in water. They were for example employed as soluble catalysts, in the presence of a ruthenium source, in the ortho-arylation of (2-pyridyl)benzene. 10 Besides, their gold(I) complexes proved hydrophilic enough to catalyze the addition of carboxylic acids to non-activated internal alkynes in aqueous media. 11 .

However, despite the potential applications of O-alkyl ferrocenesulfonates, very few studies have been dedicated to their functionalization. In 2002, Metallinos and Snieckus reported the enantioselective deprotometalation of O-isopropyl-and O-(2,4-dimethyl-3-pentyl)ferrocenesulfonates in the presence of n-, iso-or sec-butyllithium and (-)-sparteine. While moderate enantiomeric excesses (ee) were observed, the recourse to a double asymmetric induction from enantiopure Omenthylferrocenesulfonates led to diastereoisomeric ratios up to 91:9. 12 To our knowledge, this represents the only reported study dedicated to the functionalization of O-alkyl ferrocenesulfonates.

In continuation with our work dedicated to the synthesis of original polysubstituted ferrocenes, 13 we recently used tertiary sulfonamides to promote functionalization of the remaining positions of the substituted ferrocene cyclopentadienyl ring. 14 Here, we report how isopropylsulfonate can be used efficiently to direct not only deprotolithiation but also "halogen dance" reaction toward original ferrocene derivatives. While we mainly worked in racemic series, attempts to enantioselectively functionalize Oisopropylferrocenesulfonate are also presented.

Results and Discussion

Synthesis of the functionalized ferrocenes

We first attempted the synthesis of O-isopropylferrocenesulfonate (1) from ferrocene by using a deprotometalation-trapping sequence. Indeed, Willis and co-workers showed the possible use of 1,4-diazabicyclo[2.2.2]octane-bis(sulfur dioxide) (DABSO) as a sulfur dioxide surrogate to intercept different organometallic compounds. 15 However, after treatment of ferrocenyllithium (prepared by reacting ferrocene with tert-butyllithium) 16 with DABSO, conversion of the putative lithium sulfinate to ferrocenesulfonyl chloride (2) by using sulfuryl chloride, and addition of sodium isopropoxide, the expected sulfonate 1 could not be detected (Scheme 1, top right).

Therefore, we rather took advantage of our recently optimized one-pot synthesis of ferrocenesulfonyl chloride (2) following an efficient sulfonation-chlorination sequence (75% yield). 17 Similarly to a previously reported protocol, 12 we treated 2 with in situ prepared sodium isopropoxide in tetrahydrofuran (THF) and isolated the corresponding sulfonate 1 in 88% yield (Scheme 1, left and bottom).

Scheme 1. Synthesis of racemic 2-iodo-O-isopropylferrocenesulfonate (3) and "halogen dance" precursor 5.

It is known that alkyl sulfonates behave as very efficient deprotometalation directing groups. 18 In addition, in the ferrocene series, the pKa values calculated within DFT framework (see Experimental Section for computational details) for Oisopropylferrocenesulfonate (1; see Figure 1) show that the isopropylsulfonate function is more able to stabilize 2lithioferrocenes when present at the position next to the metal than diisopropylcarboxamide (as already observed in the benzene series 19 ), and even than dimethylsulfonamide. 14 and more commonly used N,N-diisopropylferrocenecarboxamide. 20 As a consequence, as shown by Metallinos and Snieckus from O-menthyl-2-(trimethylsilyl)ferrocenesulfonate, 12 and by Sierra, Cadierno and co-workers from O-isopropylferrocenesulfonate, 10- 11 alkyllithiums (n-or sec-butyllithium) are basic enough to achieve the deprotonation of 1 in THF at -78 °C. Accordingly, reacting 1 with n-butyllithium in THF at -80 °C for 1 h followed by addition of molecular iodine led to the formation of the iodide 3 in 84% yield (Scheme 2, top). In order to progress toward original 1,3-disubstituted and 1,2,4-trisubstituted ferrocenes, the lithio intermediate was also intercepted by chlorotrimethylsilane to provide 4 in high yield. When this 2-silylated derivative 4 was subjected to a second metalation-iodination sequence, as above, the 1,2,3-trisubstituted ferrocene 5 was isolated in 79% yield (Scheme 2, bottom). Scheme 2. Synthesis of racemic 2-iodo-O-isopropylferrocenesulfonate (3) and "halogen dance" precursor 5.

"Halogen dance" is an elegant way to transfer a heavy halogen (in general iodine or bromine, the former being more appropriate) from a position of a (hetero)aromatic compound to another one. 21 The reaction, based on the conversion of a less stable arylmetal to a more stable one, is generally performed by means of hindered lithium amides such as lithium 2,2,6,6tetramethylpiperidide (LiTMP) and lithium diisopropylamide.

Since our preliminary results in the ferrocene series using dialkylcarboxamides as directing groups (DG), 20,22 we have disclosed more appropriate substituents (fluorine, 23 chlorine 23a and dialkylsulfonamides 14 ) to direct "halogen dance" by exerting acidifying and/or coordinating effect(s) that stabilize the lithio product. These substituents are efficient when combined with a protecting group (PG) that avoids competitive deprotometalation at the otherwise free position next to the DG. Furthermore, as trimethylsilyl is not a DG for deprotometalation, both the free position next to the DG and the next one are protected from deprotonation (Scheme 3). Scheme 3. General reaction scheme for a "Halogen dance" reaction.

That 2-iodo-O-isopropyl-5-(trimethylsilyl)ferrocenesulfonate ( 5) is a relevant substrate to attempt "halogen dance" was confirmed by a pKa value between I and SO2OiPr in 6 (30.7) lower than the one next to I in 5 (34.2) (see Figure 2), and by a lower Gibbs free energy of the corresponding anion for 6 compared to 5 (by 31 kJ mol -1 ). In addition, when the pKa values of 4-iodo-Oisopropyl-2-(trimethylsilyl)ferrocenesulfonate (6) were compared 10.1002/ejic.202100448

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European Journal of Inorganic Chemistry with those of 4-iodo-N,N-dimethyl-2-(trimethylsilyl)ferrocenesulfonamide and 1-fluoro-4-iodo-2-(trimethylsilyl)ferrocene, which are substrates suitable to undertake "halogen dance", a lower pKa value at the free position next to the alkyl sulfonate was recorded for 6, making 5 even more promising than the corresponding dimethylsulfonamide and fluoride. 14 The "halogen dance" reaction was thus performed, as described previously, 14,23 by treating the 1,2,3-trisubstituted ferrocene 5 with LiTMP (1.1 equiv) in THF at -50 °C for 2 h. After subsequent methanolysis, the expected 1,2,4-trisubstituted derivative 6 was isolated in 83% yield (Scheme 4, top). The same approach also allows for the synthesis of 1,2,3,4-tetrasubstituted ferrocenes containing an alkyl sulfonate, when an electrophile is introduced after the "halogen dance" step. This was exemplified, by using chlorotrimethylsilane to intercept the lithiated intermediate, during the synthesis of 3-iodo-O-isopropyl-2,5bis(trimethylsilyl)ferrocenesulfonate (7). The product was isolated in a moderate 46% yield, partly due to the formation of Oisopropyl-2,5-bis(trimethylsilyl)ferrocenesulfonate (7') (Scheme 4, left). The latter seems to result from a competitive iodine/lithium exchange between LiTMP and 5, followed by electrophilic trapping, as already noticed previously. 24 Scheme 4. Ferrocene "halogen dance" reaction from 1,2,3-trisubstituted 5 to 1,2,4-trisubstituted 6 and 1,2,3,4-tetrasubstituted 7, and subsequent deprotection of 6 toward 1,3-disubstituted 8.

Removal

of the silyl protecting group, using tetrabutylammonium fluoride in THF at room temperature, finally furnished 3-iodo-O-isopropylferrocenesulfonate (8) in 84% yield (Scheme 4, right). This compound constitutes a new original 1,3disubstituted ferrocene scaffold, prone for further functionalization.

From the iodinated derivatives of Oisopropylferrocenesulfonates, post-functionalizations can be considered.

For example, 4-iodo-O-isopropyl-2-(trimethylsilyl)ferrocenesulfonate (6) was involved in a Suzuki-Miyaura cross-coupling with 4-fluorophenylboronic acid. Thus, in the presence of caesium fluoride, Pd(dba)2 (dba = dibenzylideneacetone) and SPhos (2-(dicyclohexylphosphino)-2',6'-dimethoxybiphenyl), the 4-arylated product 9 was isolated in 77% yield (Scheme 5, top).

Iodine is also a suitable group to introduce molecular diversity through halogen/metal exchange. This was demonstrated from 3iodo-O-isopropylferrocenesulfonate (8) upon treatment by tertbutyllithium and subsequent quenching with tri(isopropyl)borate. As pinacol boronic esters are more stable towards hydrolysis and easier to purify, trans-esterification with pinacol was finally done to isolate the product 10 in 55% yield (Scheme 5, bottom). Scheme 5. Examples of post-functionalization (top: Suzuki-Miyaura crosscoupling; bottom: halogen/metal exchange followed by electrophilic trapping) to increase molecular diversity in the ferrocenesulfonate family.

Monosubstituted ferrocenes being prochiral substrates, several studies have been dedicated to their enantioselective deprotometalation with recourse to either alkyllithium-ligand chelates or chiral lithium amides. 25 The most successful achievements were reported from N,Ndiisopropylferrocenecarboxamide by using nBuLi•(-)-sparteine (up to 99% ee) 26 or nBuLi•(+)-sparteine surrogate (92%), 27 from (dialkylaminomethyl)ferrocenes by using either nBuLi•(R,R)-N,N,N',N'-tetramethylcyclohexane-1,2-diamine ((R,R)-TMCDA; up to 80% ee), 28 or isopropyllithium•(R,R)-TMCDA (up to 98%), 29 and from boron trifluoride-activated aminoferrocenes by using nBuLi•(R,R)-N,N'-dimethyl-N,N'-di(3-methylbutyl)cyclohexane-1,2-diamine (up to 80% ee). 30 As regarding O-isopropylferrocenesulfonate (1), Metallinos and Snieckus attempted its enantioselective deprotolithiation by using n-, iso-or sec-butyllithium as their chelates with (-)sparteine in ethers (Et2O or tBuOMe), but always obtained 10.1002/ejic.202100448

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European Journal of Inorganic Chemistry moderate results (ee < 58%). 12 We further tested both chelates nBuLi•(R,R)-TMCDA and sBuLi•(R,R)-TMCDA in THF at -80 °C for 1 h toward 1, but low enantioselectivities were recorded (ee < 17%) after subsequent trapping of the lithio products by chlorotrimethylsilane (Scheme 6). Scheme 6. Enantio-enriched deprotonation of 1 by using alkyllithium-(R,R)-TMCDA chelates. [a] Traces of 1 and 7' were also detected in the crude. [b] 1 and 7' were also obtained in 2 and 4% yield, respectively.

Besides alkyllithium-ligand chelates, chiral lithium amides can also be used to perform the enantioselective deprotometalation of prochiral ferrocenes. Such a possibility was investigated by Price and Simpkins in 1995 with moderate success. 31 The authors employed lithium bis[(S)-1-phenylethyl]amide [(S)-PEALi] in THF by using chlorotrimethylsilane in excess as in situ trap. While ferrocene diphenylphosphine oxide could be converted into its 2silylated derivative in 54% ee, most of the tested ferrocenes either did not react (those substituted by CH2OH, CH2OMe, CH2OCH2OMe, PPh2 and SPh) or only gave racemic mixtures (SO2Ph, CONiPr2).

Because alkyl sulfonates are strong directing groups, we reasoned that the functionalization of 1 could work by this way. We therefore applied the procedure reported by Price and Simpkins, and added 1 to a mixture of lithium bis[(R)-1phenylethyl]amide [(R)-PEALi] (1 equiv) and chlorotrimethylsilane (3 equiv) in THF at -80 °C. After 30 min at this temperature and subsequent hydrolysis, the 2-silylated derivative 4 was isolated in a low 7% yield and a moderate 20% ee, the rest being 1 (91% recovery) (result not shown).

In order to avoid unwanted trapping of the base by chlorotrimethylsilane, and to favor deprotonation, 32 we next treated a mixture of 1 and chlorotrimethylsilane (1 equiv) by (R)-PEALi in THF at -10 °C for 30 min before hydrolysis. This time, 4 was obtained in 70% yield, but in a low 4% ee. The 2,5bis(trimethylsilyl) derivative 7', formed competitively under these conditions (and isolated in 11% yield), became the major product by increasing the amount of chlorotrimethylsilane to 2 or 3 equivalents (Scheme 7). Scheme 7. Attempts to enantioselectively deprotonate 1 by using (R)-PEALi in the presence of chlorotrimethylsilane as in situ trap. [a] Substrate 1 was also recovered (16% yield).

By following studies on the use of chiral lithium-zinc mixed amides and combinations to reach the same goal, 33 we showed in 2019 that zinc-based in situ traps generally give better results than chlorotrimethylsilane. 34 In particular, we evidenced [Zn{(S)-PEA}2], in situ prepared from ZnCl2•TMEDA and (S)-PEALi in a 1:2 ratio, as the best one to this purpose. Indeed, upon addition of (S)-PEALi (1 equiv) to a mixture of [Zn{(S)-PEA}2] (1 equiv) and N,N-diisopropylferrocenecarboxamide in THF at -70 °C before slow warming to -10 °C (over 3 h) and iodolysis, the corresponding 2-iodo derivative was obtained in 96% yield and 69% ee. By applying comparable reaction conditions to Oisopropylferrocenesulfonate (1), the 2-iododerivative 3 was obtained in 65% yield and 64% ee (Scheme 8). Admittedly, we did not reach the 82% ee already reported from (-)-(1R,2S,5R)-Omenthylferrocenesulfonate by using sec-butyllithium•(-)sparteine, 12 but we could get this result without recourse to double asymmetric induction.

Scheme 8. Attempts to enantioselectively deprotonate 1 by using (R)-PEALi in the presence of [(R)-PEA]2Zn as in situ trap. [a] Substrate 1 was also recovered (26% yield).

Electrochemical characterization

The electrochemical behavior of sulfur-containing ferrocene derivatives has been the subject of very few studies. In the course of a study on the use of alkylated potassium ferrocenesulfonates as electrocatalysts for sulfide detection, Lawrence and coworkers recorded in water the oxidation peak potentials of potassium 2,1'-dimethylferrocenesulfonate, and potassium 2ethyl-, 2-butyl-and 2-tert-butylferrocenesulfonates. They evidenced a little effect of the chain length on the oxidation potential while the presence of a second alkyl group onto ferrocene logically led to a lower oxidation potential. 7 In 1997, Kagan and co-workers studied by cyclic voltammetry the electrochemical properties of different ferrocene sulfides, sulfoxides and sulfones. In all cases, they recorded a one-electron reversible system, in accordance with the formation of stable ferricinium species. The E1/2 values (vs. SCE) recorded in acetonitrile were found to be dependent on the number of oxygens at sulfur, with increasing ferrocene redox potentials from sulfides to sulfones (e.g., 0.450 V for Fc-SPh, 0.605 for Fc-S(O)Ph, 0.695 for Fc-S(O)2Ph; Fc = ferrocenyl). Furthermore, the E1/2 values were slightly dependent on the electron-withdrawing or -donating character of the substituent on sulfur (e.g., 0.450 V for Fc-SPh, 0.440 for Fc-S-4-tolyl, 0.471 V for Fc-4chlorophenyl). 35 Due to the lack of data on both O-alkylferrocenesulfonates and N,N-dialkylferrocenesulfonamides, we studied by cyclic voltammetry the electrochemical behavior of some of the Oisopropylferrocenesulfonates here synthesized, as well as N,Ndimethylferrocenesulfonamides 14,17 and (N-10.1002/ejic.202100448

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European Journal of Inorganic Chemistry pyrrolidino)sulfonylferrocenes 17 previously prepared, in order to compare their E1/2 values (Table 1). The measurements were performed in dry, oxygen-free dichloromethane, at a concentration of 1 mM, with nBu4NPF6 (0.1 M) as the supporting electrolyte. For all the experiments, the working electrode was a glassy carbon disk (diameter 1.5 mm) which was polished (5 μm grain size) with a slurry of alumina and ethanol and rinsed with dichloromethane before to use. The reference electrode was Ag/AgCl separated from the solution by a glass frit, while the counter electrode was a glassy carbon rod.

Table 1. Electrochemical data (in V). [a] Substrate R -X(Alkyl)1-2 -OiPr -NMe2

Compound Epa Epc E1/2 ipa/ipc [b] Epa Epc E1/2 ipa/ipc [b] H In all cases, cyclic voltammograms were recorded at a scan rate of 50 mV•s -1 . Determination of the half-wave (E1/2) potentials was carried out from a DPV (Differential Pulse Voltammetry) experiment which were recorded under the same reaction conditions. Thus, ferrocene (Fc-H) displayed a one-electron redox event at E1/2 = 0.45 V.

Similarly, all compounds exhibited a one-electron reversible process at a scan rate of 50 mV•s -1 with a peak separation below 100 mV range (see Supporting information). In all cases studied, the ferrocenesulfonates were characterized by slightly higher redox potentials than the corresponding sulfonamides. This trend is in line with the lower pKa values calculated for ferrocenesulfonates, indicating the better electron withdrawing properties of this group. When compared with the N,Ndimethylferrocenesulfonamides, the corresponding (Npyrrolidino)sulfonylferrocenes (see Table S3 of the Supporting information) have slightly lower redox potentials. As one would have expected, addition of trimethylsilyl group onto both ferrocenesulfonate and -sulfonamides had a limited impact onto the measured redox potential. However, addition of iodine led to derivatives with a ferrocene backbone of lower electron density, this effect being more pronounced on 3substituted than on 2-substituted ferrocenes (Figure 3).

We analyzed the impact of the substituent nature on the redox potential within linear free energy relationship methodology. It is known that half-wave potentials of relative compounds could be predicted by using Hammett constants of substituents. 36 In our case, the situation is tangled by multiple possible redox centers in ferrocenesulfonates and ferrocenesulfonamides.

The Hammett equation in its simple form was developed for six-membered rings, but not for five-membered or metallocene systems. Jaffé considered possible extensions of the Hammett equation, including 1) the effect of substituents in polysubstituted compounds can be expressed as the sum of the individual effects of the substituents in the corresponding monosubstituted compounds, and 2) for description of substituent effects in fivemembered systems, the constants could be 'mixed' with different weights. 36a,37 Earlier, we showed that a vicinal substituent in triazoles affects the reaction center similarly to that of a metagroup in a benzene ring. 38 Inspired by Ushijima et al. who proved half-wave potentials of (5-cyclopentadienyl)(1,2-ethylenedithiolato)cobalt(III) complexes to be linearly dependent on the sum of the constants of the substituents, 36d here we performed similar analysis for the sulfonates 1, 3-5, and their corresponding sulfonamides (the derivatives 6 and 8 were skipped due to the lack of  constant suitable for the 1,3-disubstitution case). By using the abovementioned approximations, we obtained the following best equations for the O-isopropylferrocenesulfonates: E1/2 = 0.87 + 0.34m (N = 4, r 2 = 0.997, rmse = 0.004), and for the N,N-dimethylferrocenesulfonamides: E1/2 = 0.77 + 0.35m (N = 4, r 2 = 0.939, rmse = 0.022).

All Hammett constant values were taken from the work of Hansch et al. 39 So, there is a correlation between the nature of the substituent (electron-donating/electron-withdrawing) and the half-wave potential change (see Figure S1 of the Supporting information). Also, one can admit that E is the same (-0.02 V) for the pairs of 1 and 4, 3 and 5, 8 and 6, proving additivity.

Specific solid-state structures of the ferrocenesulfonates

Although most of the ferrocenesulfonates were solids, only the compounds 5, 7 and 10 gave crystals suitable for X-ray diffraction 10.1002/ejic.202100448

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European Journal of Inorganic Chemistry analysis (Figures 456). At the solid state, although similar C-Si, C-S and C-I bond lengths were observed for the compounds 5 and 7, small differences were noticed in their geometry. Indeed, in 5, both S=O bonds of the sulfonate are directed towards the iron with similar angles while in 7, one oxygen is almost coplanar with the cyclopentadienyl (Cp) ring, the other one being more orientated towards the iron atom (47.7° angle). Furthermore, while one methyl attached to Si1 is coplanar with the Cp in both 5 and 7, one methyl linked to Si2 is rotated by 8.9(3) ° in compound 7, probably to reduce steric pressure on this tetrasubstituted ferrocene. Finally, an eclipsed conformation was observed for compound 5 (C10-Cg2•••Cg1-C3 = 9.22 °, Cg1 being the centroid of the C1-C2-C3-C4-C5 ring and Cg2 the one of the C6-C7-C8-C9-C10 ring) while staggered for compound 7 (C9-Cg2•••Cg1-C1 = 31.36 °). This differs from the previously reported ferrocenesulfonamide derivatives which were in an eclipsed conformation before and after the halogen 'dance' reaction. 14 Concerning the borylated ferrocene 10, the two S=O bonds of the sulfonate are directed toward the iron atom, so as the boron atom with a dip angle α* between the centre of gravity of the substituted Cp ring, the Cipso and the boron atom measured to 6.15 °. This lower value than the one observed for ferrocenylpinacolborane (8.55°) 40 suggests slightly increased interactions between the empty boron p orbital and the filled p orbital of the ipso carbon atom. Finally, due to the presence of both iodine and sulfonate on the compounds 5 and 7, halogen-chalcogen bonds were identified at the solid state (Figure 7). They result from the interaction between the σ-hole of the iodine atom and the lone pair of one oxygen of the sulfonate. 41

Accepted Manuscript

European Journal of Inorganic Chemistry Owing to the strong acidifying properties of alkyl sulfonate, we prepared original iodinated ferrocenesulfonates by using orthodeprotolithiation and "halogen dance" reactions. Further functionalization using cross-coupling or iodine/lithium exchange was also successfully demonstrated. Although the use of alkyllithium•diamine chelates was ineffective to deliver enantioenriched derivatives, we showed that chiral lithium amide, in combination with a suitable in situ trap, was able to give results close to those obtained by following a double asymmetric induction strategy. An electrochemical study of the most relevant compounds was further done, showing the better electronwithdrawing properties of the sulfonate when compared with the sulfonamides. Finally, as already observed in the ferrocenesulfonamide series, halogen bonds can also be present in iodinated derivatives of ferrocenesulfonates. Taking together, these results are expected to pave the way to the synthesis of polysubstituted derivatives tailored for applications.

Experimental Section

General Considerations. All reactions were performed under an argon atmosphere and by using anhydrous solvents in dried Schlenk tubes. THF was distilled on sodium-benzophenone prior to use. All organolithiated reagents were titrated before use. 43 Room temperature (r.t.) refers to 25 °C. 2,2,6,6-Tetramethylpiperidine was distilled on CaH2 under reduced pressure, and stored on KOH pellets. Bis[(R)-1-phenylethyl]amine was stored on KOH pellets 24 h before use. (R,R)-TMCDA was prepared according to Jacobsen 44 and Alexakis. 45 Column chromatography separations were achieved on silica gel (40-63 μm). Thin layer chromatographies were performed on aluminum-backed plates pre-coated with silica gel (Merck, silica gel 60 F254). They were visualized upon exposure to UV light. Melting points were measured on a Kofler bench. IR spectra were taken on a Perkin-Elmer Spectrum 100 spectrometer. 1 H and 13 C{1H} Nuclear Magnetic Resonance (NMR) spectra were recorded either (a) on a Bruker Avance III spectrometer at 300 MHz and 75.4 MHz, respectively, or (b) on a Bruker Avance III HD at 500 MHz and 126 MHz, respectively. 1 H chemical shifts (δ) are given in ppm relative to the solvent residual peak and 13 C chemical shifts are relative to the central peak of the solvent signal. 46 The numbering used in this experimental section is defined in Supporting information. Safety considerations. Due to its high pyrophoric character, tBuLi needs to be used only under inert conditions (anhydrous, nitrogen or argon atmosphere) and by people well trained to the manipulation of reactive organometallics.

Ferrocenesulfonyl chloride 17 and ZnCl2•TMEDA 47 were prepared as described previously. N,N-Dimethylferrocenesulfonamide, 17 2-iodo-N,Ndimethylferrocenesulfonamide, 14 N,N-dimethyl-2-(trimethylsilyl)ferrocenesulfonamide, 14 2-iodo-N,N-dimethyl-5-(trimethylsilyl)ferrocenesulfonamide, 14 4-iodo-N,N-dimethyl-2-(trimethylsilyl)ferrocenesulfonamide, 14 3-iodo-N,Ndimethylferrocenesulfonamide, 14 and (N-pyrrolidino)sulfonylferrocene 17 were prepared in previous studies. The synthesis of 1-(Npyrrolidino)sulfonyl-2-(trimethylsilyl)ferrocene is described in the experimental part.

Crystallography

The X-ray diffraction data were collected for the compound rac-5 on a D8 VENTURE Bruker AXS diffractometer equipped with a (CMOS) PHOTON 100 detector and for the compounds rac-7 and rac-10 on a APEXII Kappa-CCD Bruker AXS diffractometer equipped with CCD plate detector, by using Mo-K radiation ( = 0.71073 Å; multilayer monochromator for the D8 VENTURE Bruker AXS diffractometer and graphite monochromator for the APEXII Kappa-CCD Bruker AXS diffractometer). The structure of rac-5 was solved by direct methods using the SIR97 program, 48 and then refined with full-matrix least-squares methods based on F 2 (SHELXL). 49 The structures of rac-7 and rac-10 were solved by dual-space algorithm using the SHELXT program, 50 and then refined with full-matrix leastsquare methods based on F 2 (SHELXL). 49 All non-hydrogen atoms were refined with anisotropic atomic displacement parameters. Except hydrogen atoms linked to nitrogen atoms that were introduced in the structural model through Fourier difference maps analysis, H atoms were finally included in their calculated positions and treated as riding on their parent atom with constrained thermal parameters. The molecular diagrams were generated by MERCURY 2020.3.0.

O-Isopropylferrocenesulfonate (1). The procedure was inspired by a previously reported protocol. 12 To a suspension of NaH (60% dispersion in oil, 2.70 g, 62.5 mmol, 2.50 equiv) in THF (100 mL) at 0 °C was added isopropanol (4.80 mL, 3.76 g, 62.5 mmol, 2.50 equiv) dropwise. After addition, the reaction mixture was warmed to room temperature and stirred for 15 min. A solution of ferrocenesulfonyl chloride (7.09 g, 20.0 mmol, 1.00 equiv) in THF (20 mL) was added dropwise and the reaction mixture was then stirred for 2 h at room temperature. Saturated aqueous NH4Cl (50 mL) was added, layers were separated, and the aqueous layer was extracted with AcOEt (2 x 50 mL). The combined organic layers were dried over MgSO4, filtrated, and concentrated under reduced pressure to give the crude product. This was purified by column chromatography over silica gel, eluting with petroleum ether-AcOEt 70:30 to give the title product 1 in 88% yield (6.80 g) as an orange solid: Rf (petroleum ether-AcOEt 90:10) = 0.31; mp 69-70 °C (lit. 12 General procedure 1. nBuLi (1.4 M in hexanes, 1.10 equiv) was added dropwise to a solution of the required substrate (1.00 equiv) in THF (0.15 M) at -80 °C and the reaction mixture was stirred at the same temperature for 1 h. The suitable electrophile (1.50 equiv), pure for liquids or in solution for solids, was added and the reaction was warmed to room temperature. Saturated aqueous Na2S2O3 when iodine is used as electrophile, or NH4Cl instead, was added. Layers were separated, and the aqueous layer was extracted with AcOEt. The combined organic layers were dried over MgSO4, filtrated, and concentrated under reduced pressure to give the crude product. This was purified by column chromatography over silica gel (eluent given in product description) to give the title product.

2-Iodo-O-isopropylferrocenesulfonate (3, racemic mixture).

It was prepared by following the general procedure 1, starting from Oisopropylferrocenesulfonate (1; 924 mg, 3.00 mmol) and using iodine (1.14 g) as the electrophile in THF ( 6 (4, racemic mixture). It was prepared by following the general procedure 1, starting from Oisopropylferrocenesulfonate (1; 2.15 g, 7.00 mmol) and using chlorotrimethylsilane (1.30 mL, 1.14 g) as the electrophile. It was isolated (eluent: petroleum ether-AcOEt 15:1) in a quantitative yield (2.60 g) as an 10.1002/ejic.202100448

O-Isopropyl-2-(trimethylsilyl)ferrocenesulfonate

Accepted Manuscript

European Journal of Inorganic Chemistry General procedure 2. nBuLi (1.4 M in hexanes, 1.10 equiv) was added dropwise to a solution of 2,2,6,6-tetramethylpiperidine (1.10 equiv) in THF (0.55 M) at -15 °C. After 15 min, the reaction mixture was cooled to -50 °C and 2-iodo-O-isopropyl-5-(trimethylsilyl)ferrocenesulfonate ( 5) was added in one portion. The reaction was stirred at the same temperature for 2 h before the desired electrophile was added. The reaction mixture was warmed to room temperature and aqueous HCl (1 M, 10 mL) was added. The reaction mixture was extracted with AcOEt (2 x 10 mL). The combined organic layers were dried over MgSO4, filtrated, and concentrated under reduced pressure to give the crude product. This was purified by column chromatography over silica gel (eluent given in product description) to give the title product.

4-Iodo-O-isopropyl-2-(trimethylsilyl)ferrocenesulfonate

(6, racemic mixture). It was prepared by following the general procedure 2, starting from 2-iodo-O-isopropyl-5-(trimethylsilyl)ferrocenesulfonate (5; 506 mg, 1.00 mmol) and using methanol in excess (1 mL) as the electrophile. It was isolated (eluent: petroleum ether-AcOEt 95:5) in 83% yield (421 mg) as an orange solid: Rf (petroleum ether-AcOEt 90:10) = 0.72; mp 64-65 °C; IR (ATR)  663, 693, 755, 825, 870, 910, 982, 1005, 1054, 1096, 1111, 1161, 1212, 1247, 1277, 1304, 1327, 1341, 1377, 1453, 1464, 1661, 2899, 2954, 2980, 3096 cm -1 ; 1 H NMR (CDCl3)  0.34 (s, 9H, SiMe3), 1.17 (7, racemic mixture). It was prepared by following the general procedure 2, starting from 2-iodo-O-isopropyl-5-(trimethylsilyl)ferrocenesulfonate (5; 380 mg, 0.75 mmol) and using chlorotrimethylsilane (105 µL, 89.6 mg, 0.82 mmol, 1.10 equiv) as the electrophile. It was isolated (eluent: petroleum ether- O-Isopropyl-2,5-bis(trimethylsilyl)ferrocenesulfonate (7'). This bis-silylated compound was also isolated in 8.5% yield (

3-Iodo-O-isopropyl-2,5-bis(trimethylsilyl)ferrocenesulfonate

3-Iodo-O-isopropylferrocenesulfonate

(8, racemic mixture). Tetrabutylammonium fluoride (1.0 M in THF, 1.60 mL, 1.60 mmol, 2.00 equiv) was added to a solution of 4-iodo-O-isopropyl-2-(trimethylsilyl)ferrocenesulfonate (6; 405 mg, 0.80 mmol, 1.00 equiv) in THF (2.5 mL) at room temperature and the reaction mixture was stirred for 30 min. Water (10 mL) was added and the reaction mixture was extracted with AcOEt (2 x 10 mL). The combined organic layers were dried over MgSO4, filtrated, and concentrated under reduced pressure to give the crude product. This was purified by column chromatography over silica gel, by using petroleum ether-AcOEt 95:5 as eluent, to give the title product 8 in 84% yield (293 mg) as an orange oil: Rf (petroleum ether-AcOEt 90:

4-(4-Fluorophenyl)-O-isopropyl-2-(trimethylsilyl)ferrocenesulfonate

(9, racemic mixture). 4-Iodo-O-isopropyl-2-(trimethylsilyl)ferrocenesulfonate (6; 126 mg, 0.25 mmol, 1.00 equiv), 4-fluorophenylboronic acid (140 mg, 1.00 mmol, 4.00 equiv), Pd(dba)2 (7.20 mg, 12.5 μmol, 5 mol%), SPhos (20.5 mg, 50.0 μmol, 20 mol%) and CsF (75.9 mg, 0.50 mmol, 2.00 equiv) were placed in a dried Schlenk tube, subjected to three cycles of vacuum/argon. Toluene (2.00 mL) was added, and the reaction mixture was stirred overnight at 110 °C (external temperature) in a pre-heated oil bath. The reaction mixture was cooled to room temperature and was diluted with water (10 mL). The reaction mixture was extracted with AcOEt (2 x 10 mL), the combined organic layers were dried over MgSO4, filtrated on cotton wool, and concentrated under reduced pressure on a rotary evaporator to give the crude product. This was purified by column chromatography over silica gel, eluting with petroleum ether-AcOEt 95:5 to give the title product 9 in 77% yield (92 mg) as an orange solid: Rf O-Isopropyl-3- [4,4,5,5-tetramethyl-2-(1,3,2dioxaborolyl)]ferrocenesulfonate (10, racemic mixture). tBuLi (1.6 M in pentane, 0.80 mL, 1.30 mmol, 2.00 equiv) was added to a solution of 3iodo-O-isopropylferrocenesulfonate (8; 282 mg, 0.65 mmol, 1.00 equiv) in THF (3.5 mL) at -85 °C. The reaction mixture was stirred at the same temperature for 5 min before triisopropylborate (450 µL, 367 mg, 1.95 mmol, 3.00 equiv) was added dropwise. The reaction mixture was stirred at -85 °C for 15 min before being warmed to room temperature. Methanol (2 mL) was added, and the reaction was stirred for 1 h. Volatiles were removed under reduced pressure, pinacol (154 mg, 1.30 mmol, 2.00 equiv) and THF (6.5 mL) were added, and the reaction mixture was stirred at room temperature for 15 min before volatiles were removed under reduced pressure. THF (6.5 mL) was added and the reaction mixture was stirred at room temperature for 14 h. Volatiles were removed under reduced pressure to give the crude product. This was purified by column chromatography over silica gel, eluting with petroleum ether-AcOEt 85:15, to give the title product 10 in 55% yield (156 mg) as an orange solid: Rf

1-(N-Pyrrolidino)sulfonyl-2-(trimethylsilyl)ferrocene (racemic mixture).

nBuLi (1.4 M in hexanes, 0.80 mL, 1.10 mmol, 1.10 equiv) and, 15 min later, a solution of (N-pyrrolidino)sulfonylferrocene (319 mg, 1.00 mmol, 1.00 equiv) in THF (6 mL) were added to a solution of N,N,N',N'tetramethylethylenediamine (0.16 mL, 128 mg, 1.10 mmol, 1.10 equiv) in THF (2 mL) at -80 °C. After 1 h at this temperature, chlorotrimethylsilane (0.19 mL, 163 mg, 1.50 mmol, 1.50 equiv) was added at -80 °C, and the mixture was stirred for 15 min. The reaction mixture was warmed to room temperature and aqueous HCl (1 M, 10 mL) was added. The reaction mixture was extracted with AcOEt (2 x 10 mL). The combined organic layers were dried over MgSO4, filtrated, and concentrated under pressure to give the crude product. This was purified by column chromatography over silica gel (eluent: petroleum ether-AcOEt 80:20; Rf = 0.47) to give the title product in 60% yield (230 mg) as an orange solid:

Deprotonation of O-isopropylferrocenesulfonate

(1) with sBuLi•TMCDA by using chlorotrimethylsilane as electrophile. sBuLi (1.1 M in cyclohexane-hexane 92:8, 1.10 mL, 1.20 mmol, 1.20 equiv) was added dropwise to a solution of (R,R)-TMCDA (204 mg, 1.20 mmol, 1.20 equiv) in Et2O (6 mL) at -80 °C. After 15 min at this temperature, a solution of O-isopropylferrocenesulfonate (1; 308 mg, 1.00 mmol, 1.00 equiv) in Et2O (4 mL) was added dropwise and the reaction mixture was stirred at the same temperature for 1 h. Chlorotrimethylsilane (152 µL, 130 mg, 1.20 mmol, 1.20 equiv) was added and the reaction mixture was warmed to room temperature. Aqueous HCl (1 M, 10 mL) was added, and the reaction mixture was extracted with AcOEt (2 x 10 mL). The combined organic layers were dried over MgSO4, filtrated, and concentrated under reduced pressure to give the crude product. This was purified by column chromatography, using petroleum ether-AcOEt 100:10 to 80:20 to give the title product 4 as an orange oil in 85% yield (323 mg). The ee (16%) was determined by HPLC analysis on a Chiralpak IC-3 column, hexaneisopropanol 98:2, 0.7 mL•min -1 , 20 °C, λ = 220 nm, t (major) = 17.03 min, t (minor) = 19.68 min. The bis-silylated product 7' was also obtained as an orange solid in 4% yield (21 mg).

Deprotonation of O-isopropylferrocenesulfonate

(1) with PEALi by using chlorotrimethylsilane as in situ trap. nBuLi (1.4 M in hexanes, 715 µL, 1.00 mmol, 1.00 equiv) was added dropwise to a solution of (R)-PEAH (228 µL, 225 mg, 1.00 mmol, 1.00 equiv) in THF (6 mL) at -15 °C. After 5 min at this temperature, this solution was cannulated onto a solution of O-isopropylferrocenesulfonate (1; 308 mg, 1.00 mmol, 1.00 equiv) and chlorotrimethylsilane (127 µL, 109 mg, 1.00 mmol, 1.00 equiv) in THF (6 mL) at -15 °C. After addition, the reaction mixture was warmed to -10 °C and stirred for 30 min. Aqueous HCl (1M, 10 mL) was added, and the reaction mixture was extracted with AcOEt (2 x 10 mL). The combined organic layers were dried over MgSO4, filtrated, and concentrated under reduced pressure to give the crude product. This was purified by column chromatography, using petroleum ether-AcOEt 90:10 to 80:20 to give the title product 4 as an orange oil in 70% yield (266 mg). The ee (4%) was determined by HPLC analysis on a Chiralpak IC-3 column, hexaneisopropanol 98:2, 0.7 mL•min -1 , 20 °C, λ = 220 nm, t (major) = 16.98 min, t (minor) = 19.60 min. The bis-silylated product 7' was also obtained as an orange solid in 11% yield (49 mg).

Deprotonation of O-isopropylferrocenesulfonate

(1) with PEALi by using (PEA)2Zn as in situ trap. nBuLi (1.4 M in hexanes, 1.43 mL, 2.00 mmol, 2.00 equiv) was added dropwise to a solution of (R)-PEAH (457 µL, 450 mg, 2.00 mmol, 2.00 equiv) in THF (6 mL) at -15 °C. After 5 min at this temperature, ZnCl2•TMEDA (252 mg, 1.00 mmol, 1.00 equiv) was added in one portion and the reaction mixture was stirred for 15 min. O-Isopropylferrocenesulfonate (1; 308 mg, 1.00 mmol, 1.00 equiv) was added in one portion at the same temperature, and the reaction mixture was stirred for another 15 min. A cooled (-70 °C) solution of (R)-PEALi (prepared by adding nBuLi (714 µL, 1.00 mmol, 1.00 equiv) dropwise to a solution of (R)-PEAH (229 µL, 225 mg, 1.00 mmol, 1.00 equiv) in THF (6 mL) at -15 °C and stirring for 5 min) was then added at -70 °C by cannula. After addition, the reaction mixture was slowly warmed to -10 °C over a period of 3 h. A solution of iodine (761 mg, 3.00 mmol, 3.00 equiv) in THF (4 mL) was added and the reaction mixture was stirred for 1 h at room temperature. A saturated aqueous solution of Na2S2O3 (10 mL) was added to the reaction mixture which was extracted with AcOEt (2 x 10 mL). The combined organic layers were dried over MgSO4, filtrated, and concentrated under reduced pressure to give the crude product. This was purified by column chromatography, using petroleum ether-AcOEt 90:10 to 80:20 to give an inseparable mixture (366 mg) of title product 3 and starting material 1 in a 29:71 ratio. The ee (64%) was determined by HPLC Computational details. All electronic structure calculations were performed by using Gaussian 09 package. [START_REF] Frisch | Gaussian 09, Revision A.02[END_REF] Full geometry optimization of the considered species was carried out by using the CAM-B3LYP hybrid functional. [START_REF] Yanai | [END_REF] Vibrational frequencies were calculated to establish the nature of the stationary points and to derive thermochemical corrections for enthalpies and free energies. The LANL2DZ basis set 53 with the effective core potential was used for Fe and I, while the 6-31G(d) basis set 54 was applied to the rest of the atoms. The latter was replaced by 6-311+G(d,p) set for single point energies. The pKa values were obtained from the Gibbs free energy of the homodesmic reaction. This approach was successfully applied in the ferrocene series earlier. 13e,14,20,23a The polarizable continuum model (IEF-PCM) 55 was used to account for solvation effects with a default parameters of THF to simulate the reaction conditions.

Figure 1 .

 1 Figure 1. Calculated pKa values of O-isopropylferrocenesulfonate (1), and comparison with N,N-dimethylferrocenesulfonamide14 and more commonly used N,N-diisopropylferrocenecarboxamide.20

  Figure 2. Calculated pKa values of 2-iodo-O-isopropyl-5-(trimethylsilyl)ferrocenesulfonate (5) and 4-iodo-O-isopropyl-2-(trimethylsilyl)ferrocenesulfonate (6), and comparison with 4-iodo-N,N-dimethyl-2-(trimethylsilyl)ferrocenesulfonamide and 1-fluoro-4-iodo-2-(trimethylsilyl)ferrocene.14

  Figure 3. Differential pulse voltammograms for N,Ndimethylferrocenesulfonamide (blue line), 1 (red line), 3 (green line), ferrocene (dotted blue line) and 8 (orange line).

Figure 4 .

 4 Figure 4. Molecular structure of compound rac-5 at the solid state. Thermal ellipsoids shown at the 30% probability level. Selected bond lengths [Å] and angles [°]: C9-Si1 = 1.884(4), C10-S1 = 1.744(3), C6-I1 = 2.091(4), C8-C9-Si1-C12 = -1.2(3), C6-C10-S1-O14 = 25.0(4), C9-C10-S1-O15 = -24.0(3).

Figure 5 .

 5 Figure 5. Molecular structure of compound rac-7 at the solid state. Thermal ellipsoids shown at the 30% probability level. Selected bond lengths [Å] and angles [°]: C8-Si1 = 1.891(3), C9-S1 = 1.758(3), C10-Si2 = 1.895(3), C6-I = 2.096(3), C7-C8-Si1-C13 = 0.7(2), C9-C10-Si2-C16 = 8.9(3), C8-C9-S1-O18 = -3.4(2), C10-C9-S1-O17 = -47.7(3).

Figure 6 .

 6 Figure 6. Molecular structure of compound rac-10 at the solid state. Thermal ellipsoids shown at the 30% probability level. Selected bond lengths [Å] and angles [°]: C7-S1 = 1.730 (4), C10-B21 = 1.553(3), C8-C7-S1-O12 = 16.5(4), C6-C7-S1-O11 = -28.2(4).

  However, due to the different substitution patterns, different networks are observed: a zig-zag chain in the case of 5 and a linear one for 7. Furthermore, as each enantiomer is involved into a different chain, two independent chains of halogen bonds exist for each compound. However, the bond lengths (I1•••O14 3.193(4) and I1•••O18 3.227(2) Å for 5 and 7, respectively) and angles (S1-O14•••I1 133.70(2) and O14•••I1-C6 167.85(12) for 5, S1-O18•••I1 148.99(11) and O18•••I1-C6 171.06(8) for 7) are within the range of classical values.14,42

Figure 7 .

 7 Figure 7. Halogen bonds network observed at the solid state for the compounds rac-5 (top) and rac-7 (bottom). Thermal ellipsoids shown at the 30% probability level.

  , 4.42 (t, 2H, J = 1.6 Hz, H3 and H4), 4.69 (sept, 1H, J = 6.2 Hz, CHMe2), 4.70 (t, 2H, J = 1.6 Hz, H2 and H5) ppm; 13 C{1H} NMR (CDCl3)  23.0 (2CH3), 69.2 (2CH, C2 and C5), 71.0 (2CH, C3 and C4), 71.1 (5CH, Cp), 76.5 (CH, CHMe2), 84.3 (C, C1, CSO3iPr) ppm. These data are as reported previously. 12

7 (

 7 mL). It was isolated (eluent: petroleum ether-AcOEt 10:1) in 84% yield (1.09 g) as an orange solid: Rf (petroleum ether-AcOEt 90:10) = 0.31; mp 82-85 °C; IR (ATR)  764, 829, 877, 913, 932, 1002, 1028, 1058, 1096, 1108, 1151, 1161, 1201, 1341, 1362, 1376, 1388, 1411, 1463, 1681, 2981, 3106 cm -1 ; 1 H NMR (CDCl3)  1.20 (d, 3H, J = 6.3 Hz, Me), 1.29 (d, 3H, J = 6.3 Hz, Me), 4.39 (s, 5H, Cp), 4.44 (t, 1H, J = 2.6 Hz, H4), 4.67 (dd, 1H, J = 2.5 and 1.5 Hz, H3), 4.78 (sept, 1H, J = 6.3 Hz, CHMe2), 4.81 (dd, 1H, J = 2.7 and 1.5 Hz, H5) ppm; 13 C{1H} NMR (CDCl3)  22.9 (CH3), 23.2 (CH3), 37.2 (C, C2, C-I), 71.0 (CH, C5), 71.CH, C4), 74.1 (5CH, Cp), 77.2 (CH, CHMe2), 79.6 (CH, C3), 86.0 (C, C1, CSO3iPr) ppm; MS (EI, 70 eV): 434 [M], 392 [M-iPr+H]. Anal. Calcd for C13H15FeIO3S (434.07): C, 35.97; H, 3.48; S, 7.39. Found: C, 36.10; H, 3.57; S, 7.10.

  orange oil: Rf (petroleum ether-AcOEt 90:10) = 0.50; IR (ATR)  660, 693, 762, 823, 874, 919, 960, 1004, 1039, 1069, 1099, 1143, 1161, 1199, 1246, 1284, 1346, 1411, 1467, 1681, 2900, 2956, 2982, 3107 cm -1 ; 1 H NMR (CDCl3)  0.35 (s, 9H, SiMe3), 1.13 (d, 3H, J = 6.2 Hz, CHMe2), 1.32 (d, 3H, J = 6.2 Hz, CHMe2), 4.36 (dd, 1H, J = 2.4 and 1.1 Hz, H3), 4.39 (s, 5H, Cp), 4.53 (t, 1H, J = 2.4 Hz, H4), 4.71 (sept, 1H, J = 6.2 Hz, CHMe2), 4.89 (dd, 1H, J = 2.4 and 1.1 Hz, H5) ppm; 13 C{1H} NMR (CDCl3)  0.7 (3CH3, SiMe3), 22.7 (CH3, CHMe2), 23.3 (CH3, CHMe2), 71.0 (5CH, Cp), 72.3 (CH, C4), 73.5 (C, C2, C-SiMe3), 73.9 (CH, C5), 75.9 (CH, CHMe2), 78.4 (CH, C3), 88.6 (C, C1, CSO3iPr) ppm. These data are as reported previously.12

37 .

 37 Crystal data for 5. C16H23FeIO3SSi, M = 506.24, T = 150(2) K, monoclinic P 1 21/a 1 (I.T.#14), a = 14.1732(19), b = 8.0466(9), c = 18.201(2) Å, β = 108.601(5) °, V = 1967.3(4) Å 3 , Z = 4, d = 1.709 g•cm - 3 , μ = 2.513 mm -1 . A final refinement on F 2 with 4509 unique intensities and 230 parameters converged at ωR(F 2 ) = 0.0919 (R(F) = 0.0394) for 3953 observed reflections with I > 2σ(I). CCDC 2084907.

  (d, 3H, J = 6.2 Hz, CHMe2), 1.34(d, 3H, J = 6.2 Hz, CHMe2), 4.40 (s, 5H, Cp), 4.56 (s, 1H, H3), 4.73 (sept, 1H, J = 6.2 Hz, CHMe2), 5.13 (s, 1H, H5) ppm; 13 C{1H} NMR (CDCl3)  0.5 (3CH3, SiMe3), 22.8 (CH3, CHMe2), 23.3 (CH3, CHMe2), 40.1 (C, C4, C-I), 74.0 (5CH, Cp), 75.4 (C, C2, C-SiMe3), 76.4 (CH, CHMe2), 79.3 (CH, C5), 84.6 (CH, C3), 89.3 (C, C1, CSO3iPr) ppm. Anal. Calcd for C16H23FeIO3SSi (506.25): C, 37.96; H, 4.58; S, 6.33. Found: C, 38.16; H, 4.71; S, 6.16.

  Et2O 98:2) in 46% yield (200 mg) as an orange solid: Rf (petroleum ether-AcOEt 90:10) = 0.78; mp 91-93 °C; IR (ATR)  669, 691, 761, 820, 829, 839, 878, 912, 940, 1002, 1097, 1167, 1209, 1244, 1310, 1325, 1345, 1376, 1388, 1413, 1469, 2899, 2958 cm -1 ; 1 H NMR (CDCl3)  0.36 (s, 9H, SiMe3), 0.56 (s, 9H, SiMe3), 1.21 (d, 3H, J = 6.3 Hz, CHMe2), 1.39 (d, 3H, J = 6.3 Hz, CHMe2), 4.38 (s, 5H, Cp), 4.75 (s, 1H, H4), 4.83 (sept, 1H, J = 6.3 Hz, CHMe2) ppm; 13 C{1H} NMR (CDCl3)  1.2 (3CH3, SiMe3), 2.8 (3CH3, SiMe3), 23.0 (CH3, CHMe2), 23.5 (CH3, CHMe2), 51.1 (C, C3, C-I), 74.1 (5CH, Cp), 75.9 (CH, CHMe2), 76.9 (C, C2, C-SiMe3), 80.7 (C, C5, C-SiMe3), 89.1 (CH, C4), 93.8 (C, C1, CSO3iPr) ppm. Anal. Calcd for C19H31FeIO3SSi2 (578.43): C, 39.45; H, 5.40; S, 5.54. Found: C, 39.13; H, 5.53; S, 5.49. Crystal data for 7. C19H31FeIO3SSi2, M = 578.43, T = 150(2) K, monoclinic P 21/n (I.T.#14), a = 11.0820(6), b = 17.2810(9), c = 13.2452(6) Å, β = 109.287(2) °, V = 2394.2(2) Å 3 , Z = 4, d = 1.605 g•cm -3 , μ = 2.123 mm -1 . A final refinement on F 2 with 5449 unique intensities and 252 parameters converged at ωR(F 2 ) = 0.0654 (R(F) = 0.0324) for 4385 observed reflections with I > 2σ(I). CCDC 2084908.

(

  petroleum ether-AcOEt 90:10) = 0.58; mp 104-105 °C; IR (ATR)  659, 695, 767, 822, 833, 878, 917, 964, 990, 1003, 1033, 1074, 1100, 1109, 10.1002/ejic.202100448 Accepted Manuscript European Journal of Inorganic Chemistry 1138, 1167, 1204, 1230, 1242, 1343, 1377, 1412, 1457, 1526, 1607, 2900, 2957, 2980, 3091 cm -1 ; 1 H NMR (CDCl3)  0.39 (s, 9H, SiMe3), 1.19 (d, 3H, J = 6.3 Hz, CHMe2), 1.36 (d, 3H, J = 6.3 Hz, CHMe2), 4.26 (s, 5H, Cp), 4.74 (d, 1H, J = 1.2 Hz, H3), 4.79 (sept, 1H, J = 6.3 Hz, CHMe2), 5.31 (d, 1H, J = 1.2 Hz, H5), 7.04 (t, 2H, J = 8.6 Hz, H3' and H5'), 7.48 (dd, 2H, J = 8.6 and 5.3 Hz, H2' and H6') ppm; 13 C{1H} NMR (CDCl3)  0.7 (3CH3, SiMe3), 22.9 (CH3, CHMe2), 23.4 (CH3, CHMe2), 71.5 (CH, C5), 72.7 (5CH, Cp), 74.5 (C, C2, C-SiMe3), 76.0 (CH, C3 or CHMe2), 76.1 (CH, C3 or CHMe2), 89.0 (C; C1, CSO3iPr or C4, C-C6H4-4-F), 89.1 (C; C1, CSO3iPr or C4, C-C6H4-4-F), 115.8 (2CH, J = 21.5 Hz, C3' and C5'), 127.8 (2CH, J = 8.0 Hz, C2' and C6'), 132.4 (C, J = 3.0 Hz, C1'), 162.1 (C, J = 246.9 Hz, C4') ppm; 19 F{1H} NMR (CDCl3)  -114.9 ppm. Anal. Calcd for C22H27FFeO3SSi (474.44): C, 55.70; H, 5.74; S, 6.76. Found: C, 55.74; H, 6.16; S, 6.63.

(

  petroleum ether-AcOEt 80:20) = 0.39; mp 105-107 °C; IR (ATR)  671, 691, 707, 766, 827, 853, 874, 920, 964, 1003, 1058, 1096, 1108, 1141, 1159, 1198, 1279, 1334, 1347, 1479, 1500, 1678, 2943, 2986, 3091 cm -1 ; 1 H NMR (CDCl3)  1.21 (d, 3H, J = 6.3 Hz, CHMe2), 1.24 (d, 3H, J = 6.3 Hz, CHMe2), 1.34 (s, 12H, Me-pinacol), 4.38 (s, 5H, Cp), 4.63 (dd, 1H, J = 2.3 and 1.1 Hz, H4), 4.69 (sept, 1H, J = 6.3 Hz, CHMe2), 4.85 (dd, 1H, J = 2.3 and 1.1 Hz, H5), 4.96 (t, 1H, J = 1.1 Hz, H2) ppm; 13 C{1H} NMR (CDCl3)  22.9 (CH3, CHMe2), 23.0 (CH3, CHMe2), 24.9 (2CH3, CMe2-pinacol), 25.1 (2CH3, CMe2-pinacol), 71.6 (5CH, Cp), 72.6 (CH, C5), 74.9 (CH, C2), 76.3 (CH, C4), 76.6 (CH, CHMe2), 83.9 (2C, CMe2-pinacol), 87.5 (C, C1, CSO3iPr) ppm; 11 B NMR (CDCl3)  32.3 (br s) ppm. Anal. Calcd for C19H27BFeO5S (434.13): C, 52.57; H, 6.27; S, 7.38. Found: C, 52.65; H, 6.38; S, 7.30. Crystal data for 10. C19H27BFeO5S, M = 434.12, T = 150(2) K, orthorhombic, P 21 21 21 (I.T.#19), a = 7.4269(4), b = 12.2846(5), c = 22.7194(9) Å, V = 2072.84(16) Å 3 , Z = 4, d = 1.391 g•cm -3 , μ = 0.854 mm - 1 . A final refinement on F 2 with 4733 unique intensities and 251 parameters converged at ωR(F 2 ) = 0.0818 (R(F) = 0.0401) for 4213 observed reflections with I > 2σ(I). CCDC 2084909.

  mp 92 °C; IR (ATR)  655, 717, 702, 755, 814, 826, 954, 1007, 1047, 1076, 1108, 1135, 1108, 1185, 1222, 264, 1248, 1327, 1731, 2875, 2957, 3420 cm -1 ; 1 H NMR (CDCl3)  0.35 (s, 9H, SiMe3), 1.72-1.83 (m, 4H, NCH2CH2), 3.08-3.23 (m, 4H, NCH2), 4.29 (dd, 1H, J = 2.6 and 1.4 Hz, H3), 4.39 (s, 5H, Cp), 4.50 (t, 1H, J = 2.4 Hz, H4), 4.80 (dd, 1H, J = 2.3 and 1.4 Hz, H5) ppm; 13 C{1H} NMR (CDCl3)  1.1 (3CH3, SiMe3), 25.4 (2CH2, NCH2CH2), 47.7 (2CH2, NCH2), 70.8 (5CH, Cp), 72.4 (CH, C4), 72.7 (CH, C5), 73.5 (C, C2, C-SiMe3), 77.5 (CH, C3), 90.7 (C, C1, C-SO2-N-pyrrolidino) ppm. Anal. Calcd for C17H25FeNO2SSi (391.38): C, 52.17; H, 6.44; N, 3.58; S, 8.19. Found: C, 52.29; H, 6.64; S, 7.98.

  10.1002/ejic.202100448Accepted ManuscriptEuropean Journal of Inorganic Chemistry analysis on a Chiralpak IA-3 column, hexane-isopropanol 99:1, 0.4 mL•min -1 , 20 °C, λ = 220 nm, t (1) = 41.27 min, t (minor) = 31.46 min, t (major) = 36.97 min.Electrochemical measurements. Cyclic voltammetry was performed inCH2Cl2 containing nBu4NPF6 (0.1 M) as supporting electrolyte. An aqueous Ag/AgCl reference electrode, a Pt counter electrode and a glassy carbon working electrode were used.

  CSO3iPr) ppm. Anal. Calcd for C19H32FeO3SSi2 (452.54): C, 50.43; H, 7.13; S, 7.08. Found: C, 50.33; H, 7.11; S, 7.15.

	29 mg) as an orange solid: Rf
	(petroleum ether-AcOEt 90:10) = 0.72; mp 69-70 °C; IR (ATR)  753, 818,
	829, 872, 907, 1003, 1080, 1096, 1111, 1131, 1163, 1206, 1242, 1283,
	1320, 1386, 1411, 1452, 2901, 2954, 2987 cm -1 ; 1 H NMR (CDCl3)  0.37
	(s, 18H, SiMe3), 1.31 (d, 6H, J = 6.2 Hz, CHMe2), 4.37 (s, 5H, Cp), 4.48 (s,
	2H, H3 and H4), 4.94 (sept, 1H, J = 6.2 Hz, CHMe2) ppm; 13 C{1H} NMR
	(CDCl3)  1.1 (6CH3, SiMe3), 23.4 (2CH3, CHMe2), 71.1 (5CH, Cp), 74.8
	(CH, CHMe2), 77.9 (2C, C2 and C5, C-SiMe3), 80.3 (2CH, C3 and C4),
	93.1 (C, C1,

Acknowledgements

This work was supported by the Agence Nationale de la Recherche (ANR; Ferrodance program) and the Université de Rennes 1. William Erb thanks Rennes Métropole. We gratefully acknowledge BASF (generous gift of (R,R)-bis(1phenylethyl)amine), Thermofisher (generous gift of 2,2,6,6tetramethylpiperidine), and the Fonds Européen de Développement Régional (FEDER; D8 Venture Bruker AXS diffractometer).

Conflict of Interest

The authors declare no conflict of interest.

Entry for the Table of Contents

Original 2-or 3-substituted, 2,4-or 2,5-disubstituted, and 2,3,5-trisubstituted ferrocenesulfonates were obtained from Oisopropylferrocenesulfonate by applying deprotolithiation, "halogen dance" reaction, Suzuki-Miyaura cross-coupling and halogen/metal exchange to this series. Enantioselective deprotometalation was also attempted. Compared with N,Ndialkylferrocenesulfonamides, the corresponding O-isopropylferrocenesulfonates proved slightly more difficult to oxidize.

Institute and/or researcher Twitter usernames: @chimie_ISCR; @w_erb_chem 10.1002/ejic.202100448

Accepted Manuscript

European Journal of Inorganic Chemistry


domain: sdv.tox chim sde

text:
Cécile Bellanger Orcid

Apolline Chabenat

Thomas Knigge

Cécile Bellanger
email: cecile.bellanger@unicaen.fr

Colour change impacted by antidepressants in cuttlefish Antidepressants Modify Cryptic Behaviour in Juvenile Cuttlefish at Environmentally Realistic Concentrations

Keywords: fluoxetine, venlafaxine, camouflage, body colour changes, cephalopod, Sepia officinalis, development

Contamination of marine environment by antidepressants may affect neurophysiological processes in non-target organisms, such as the common cuttlefish, Sepia officinalis. The present study tested whether environmentally realistic concentrations of antidepressants, i.e., fluoxetine alone (5 ng•L -1 ) or cumulated with venlafaxine (2.5 or 5 ng•L -1 each), affect camouflage in newly hatched cuttlefish.

Results show that antidepressants improve uniform body patterns, while disruptive body patterns were not affected.

.

INTRODUCTION

For purposes of communication or camouflage, cuttlefish, Sepia officinalis, display various body patterns on their dorsal side of their mantle as well as their arms and head.

This rapid colour change in cuttlefish is effected by nervously controlled chromatophores embedded in the skin. The rapid relaxation of chromatophores is under the control of serotonin (5-hydroxytryptamine, 5-HT), while rapid contraction is brought about by glutamate [START_REF] Messenger | Cephalopod chromatophores: neurobiology and natural history[END_REF]. Chromatophore expansion and contraction are directly controlled by motoneurons from the chromatophore lobes of the brain and the optic lobes [START_REF] Messenger | Cephalopod chromatophores: neurobiology and natural history[END_REF], providing a point of action for antidepressants by changing the levels neurotransmitters.

Although newly-hatched cuttlefish are able to express complex body patterns, they learn to improve cryptic behaviours while their nervous system is maturing during the first month after birth [START_REF] Hanlon | Adaptive Coloration in Young Cuttlefish (Sepia Officinalis L.): The Morphology and Development of Body Patterns and Their Relation to Behaviour[END_REF][START_REF] Poirier | Early Experience and Postembryonic Maturation of Body Patterns in Cuttlefish (Sepia officinalis)[END_REF]. Cuttlefish can express uniform, mottle and disruptive body patterns to achieve camouflage on uniform as well as subtly heterogeneous or strongly contrasted backgrounds, respectively [START_REF] Hanlon | Cephalopod Behaviour[END_REF].

The highly efficient and dynamic camouflage allows the cuttlefish to blend into the environment within milliseconds [START_REF] Messenger | Cephalopod chromatophores: neurobiology and natural history[END_REF], and to stay hidden from

Accepted Article

predators, but it can also be a strategy to ambush their prey. Thus, any modification of the colour change deriving from changes in the levels of neurotransmitters involved in the nervous control of chromatophores, such as 5-HT, for instance, can reduce or increase the chances for survival. Many pollutants detected in marine environments can potentially interfere with the serotoninergic system [START_REF] Mezzelani | Pharmaceuticals in the aquatic environments: Evidence of emerged threat and future challenges for marine organisms[END_REF], but one group of molecules is of particular interest, because it is designed to modify 5-HT levels: antidepressants of the class of selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs). Their increasing release in the environment derives from a constant rise of prescriptions (OECD Health Statistics 2019). Consequently, residues of antidepressants from human excretion or direct disposal of expired pills are continuously detected in concentrations ranging from a few ng•L -1 to several µg•L -1 in fresh-and marine waters [START_REF] Gonzalez-Rey | Occurrence of pharmaceutical compounds and pesticides in aquatic systems[END_REF][START_REF] Meador | Effect of antidepressants on circadian rhythms in fish: Insights and implications regarding the design of behavioural toxicity tests[END_REF][START_REF] Metcalfe | Antidepressants and their metabolites in municipal wastewater, and downstream exposure in an urban watershed[END_REF][START_REF] Klosterhaus | Method validation and reconnaissance of pharmaceuticals, personal care products, and alkylphenols in surface waters, sediments, and mussels in an urban estuary[END_REF][START_REF] Paíga | Presence of pharmaceuticals in the Lis river (Portugal): Sources, fate and seasonal variation[END_REF][START_REF] Schultz | Antidepressant Pharmaceuticals in Two U.S. Effluent-Impacted Streams: Occurrence and Fate in Water and Sediment, and Selective Uptake in Fish Neural Tissue[END_REF]. More specifically, [START_REF] Minguez | Toxicities of 48 pharmaceuticals and their freshwater and marine environmental assessment in northwestern France[END_REF] reported concentrations of 0.3 ng•L -1 of the SSRI fluoxetine (FLX) and 1.3 ng•L -1 of the SNRI venlafaxine (VEN) in the Orne estuary (Normandy, France), which is why, we focused on these two active molecules in the present study, which comprise some of most prescribed and most frequently detected antidepressant drugs [START_REF] Fong | The biological effects of antidepressants on the molluscs and crustaceans: A review[END_REF]. FLX was the first SSRI to be commercialised in 1988 [START_REF] Pe | Fluoxetine: a five-year review[END_REF] and still is one the most administered antidepressants. It has thus been extensively studied by ecotoxicologists [START_REF] Fong | The biological effects of antidepressants on the molluscs and crustaceans: A review[END_REF][START_REF] Sehonova | Effects of waterborne antidepressants on non-target animals living in the aquatic environment: A review[END_REF], which is why it served as reference antidepressant in this study. Nearly one decade later, the first SNRIs, with VEN, were commercialised (Zerjav et al., 2009). Both antidepressants are commonly detected in aquatic animals [START_REF] Fong | The biological effects of antidepressants on the molluscs and crustaceans: A review[END_REF][START_REF] Schultz | Antidepressant Pharmaceuticals in Two U.S. Effluent-Impacted Streams: Occurrence and Fate in Water and Sediment, and Selective Uptake in Fish Neural Tissue[END_REF][START_REF] Silva | Uptake, accumulation and metabolization of the antidepressant fluoxetine by Mytilus galloprovincialis[END_REF], even if their concentrations in the water are below detection limits [START_REF] Klosterhaus | Method validation and reconnaissance of pharmaceuticals, personal care products, and alkylphenols in surface waters, sediments, and mussels in an urban estuary[END_REF] . [START_REF] Meador | Effect of antidepressants on circadian rhythms in fish: Insights and implications regarding the design of behavioural toxicity tests[END_REF]. A number of studies revealed effects of antidepressants, such as FLX and VEN on different behaviours in various marine species: predator and antipredator behaviour [START_REF] Bisesi | Reprint of: Effects of the antidepressant venlafaxine on fish brain serotonin and predation behavior[END_REF][START_REF] Peters | Prozac in the water: Chronic fluoxetine exposure and predation risk interact to shape behaviors in an estuarine crab[END_REF], locomotion [START_REF] Fong | The antidepressants venlafaxine ("Effexor") and fluoxetine ("Prozac") produce different effects on locomotion in two species of marine snail, the oyster drill (Urosalpinx cinerea) and the starsnail (Lithopoma americanum)[END_REF][START_REF] Mesquita | Biochemical and locomotor responses of Carcinus maenas exposed to the serotonin reuptake inhibitor fluoxetine[END_REF], agonistic behaviour [START_REF] Tierney | Effects of fluoxetine on growth and behavior in the crayfish Orconectes rusticus[END_REF], photo-and geotaxis behaviour [START_REF] Guler | Anti-depressants make amphipods see the light[END_REF]. These studies used much higher concentrations than those detected in water bodies, and they tested single antidepressants only. Realistic environmental exposure scenarios are, however, characterised by low ng•L -1 concentrations of several antidepressants that may cumulate to concentrations in the µg•L -1 range [START_REF] Metcalfe | Antidepressants and their metabolites in municipal wastewater, and downstream exposure in an urban watershed[END_REF]. To date, few studies focused on the effects of environmentally realistic concentration of accumulated antidepressants such as FLX and VEN on different behavioural traits: predator behaviour [START_REF] Bisesi | Effects of an antidepressant mixture on the brain serotonin and predation behavior of hybrid striped bass[END_REF], predator avoidance [START_REF] Painter | Antidepressants at environmentally relevant concentrations affect predator avoidance behavior of larval fathead minnows (Pimephales promelas)[END_REF], reproductive behaviour [START_REF] Schultz | Selective uptake and biological consequences of environmentally relevant antidepressant pharmaceutical exposures on male fathead minnows[END_REF]), circadian rhythm (Melvin, 2017).

Accepted Article

Earlier studies from our laboratory demonstrated impacts of FLX and/or VEN at very low concentrations in the range of 1 to 100 ng•L -1 , on behaviour of cuttlefish: learning and memory processes [START_REF] Di Poi | Effects of perinatal exposure to waterborne fluoxetine on memory processing in the cuttlefish Sepia officinalis[END_REF], maturation and learning of sand-digging behaviour [START_REF] Chabenat | Hidden in the sand: Alteration of burying behaviour in shore crabs and cuttlefish by antidepressant exposure[END_REF], cryptic behaviour and colour change (Bidel et al., 2016a(Bidel et al., , 2016a;;[START_REF] Di Poi | Cryptic and biochemical responses of young cuttlefish Sepia officinalis exposed to environmentally relevant concentrations of fluoxetine[END_REF].

Juvenile Sepia officinalis are developing in coastal areas before starting seasonal offshore migrations. Continuous discharge of various pharmaceutical residues in the vicinity of estuarine outflows may, therefore, influence the physiological and behavioural development of juvenile cuttlefish. As antidepressants never occur as single compounds in the environment, this study tested the hypothesis that environmentally realistic concentrations of cumulated antidepressants present in the coastal environments affect the colour change of newly-hatched cuttlefish. To test this .

Accepted Article

hypothesis, the camouflage response was investigated on artificial backgrounds in S. officinalis exposed to two different concentrations of FLX alone or combined with VEN during the first month after hatching, which is essential for the cuttlefish's development.

Material and methods

Animal rearing and experimental conditions.

Clutches from wild animals (Sepia officinalis) were collected at various sites in the English Channel off the coast of Luc-sur-mer, France. Eggs from different clutches were combined and incubated in four 1000 L aquaria with natural seawater at the Centre de Recherches en Environnement Côtier (CREC) of the University of Caen Normandie at Luc-sur-mer, France. The aquaria were supplied by a flow-through system constantly renewing 50% of the water body (15±1°C). The photoperiod was following to natural summer conditions (~16h light: 8h dark).

At hatching, individual cuttlefish (n=14-16) were immediately isolated in glass beakers (Ø 11 cm, height 6 cm), henceforth designated as 'home tank', constituting a semi-static system with a 100% daily water renewal using 250 mL of activated charcoal filtered seawater (FSW) at a salinity of 35 PSU. Hatchlings were randomly assigned to either (1) FSW (control), or (2) 5 ng•L -1 FLX (FLX5) 1 , or (3) 2.5 ng•L -1 FLX and 2.5 ng•L -1 VEN in combination (FLXVEN2.5) as well as (4) 5 ng•L -1 FLX and 5 ng•L -1 VEN (FLXVEN5) and exposed over a period of 29 days post-hatching (dph). The concentrations of FLX and VEN were renewed together with the daily 1 No animal was exposed to VEN alone in the present experiment. The focus was on cumulating concentrations in order to approach a more environmentally realistic exposure scenario. For logistic limitation reasons, we could not include a VEN alone condition and opted for FLX alone as more experiments in the field have been conducted with FLX. For the testing of single antidepressants at different concentrations in Sepia officinalis, the reader is referred to Di [START_REF] Di Poi | Cryptic and biochemical responses of young cuttlefish Sepia officinalis exposed to environmentally relevant concentrations of fluoxetine[END_REF]Bidel et al. 2016." water changes, which also allowed maintaining appropriate O 2 -levels (≥80%) and low

Chemical contamination.

FLX and VEN concentrations were chosen to approximate surface water concentrations measured off the coast where the eggs where sampled [START_REF] Minguez | Toxicities of 48 pharmaceuticals and their freshwater and marine environmental assessment in northwestern France[END_REF]. Final concentrations of either 2.5 or 5 ng•L -1 were obtained from stock solutions of fluoxetine hydrochloride (CAS 56296-78-7, Sigma Aldrich, St. Louis, USA) or venlafaxine hydrochloride (CAS 99300-78-4, Sigma Aldrich) dissolved in filtered distilled water. Stock solutions were at 10 µg•L -1 of FLX and VEN, respectively, portioned in small aliquots of 0.5, 1 and 2 mL and kept at -80°C until use.

Behavioural tests.

Camouflage responses of the cuttlefish were assessed through the analysis of body patterns as described previously [START_REF] Barbosa | Disruptive coloration in cuttlefish: a visual perception mechanism that regulates ontogenetic adjustment of skin patterning[END_REF][START_REF] Di Poi | Cryptic and biochemical responses of young cuttlefish Sepia officinalis exposed to environmentally relevant concentrations of fluoxetine[END_REF]. Body patterns were displayed on two different backgrounds: uniform grey or black and white checkerboard. The test apparatus was a custom-made drop-shaped PVC compartment (length 11 cm, height 4 cm, max. width 5 cm). Eight independent compartments were concentrically arranged to allow for the simultaneous observation of eight individuals, i.e., two animals per exposure condition (Fig. 1). The inside walls and floors of the compartments were either bare medium grey PVC (Fig. 1A), or covered with laminated paper printed with a black and white checkerboard (Fig. 1B) in order to induce a uniform or disruptive pattern response, respectively. The square size of the checkerboards corresponded to 90% of the cuttlefish's dorsal white square [START_REF] Di Poi | Cryptic and biochemical responses of young cuttlefish Sepia officinalis exposed to environmentally relevant concentrations of fluoxetine[END_REF]. Each cuttlefish passed both behavioural tests at four different ages: at 7, 14, 21, 28 dph (n= 14 per group) for uniform background, and 8, 15, 22, 29 dph (n=16 per group) for checkerboard background.

Behavioural tests were conducted in the climate chamber where the cuttlefish were reared and exposed to antidepressants. The tests were performed in a custom-made black tent in order to avoid any external stimuli. A circular LED lamp (Neewer®, Guangdong, China) was positioned 60 cm above the test apparatus. To avoid direct light on cuttlefish, the lamp was placed in an oblique position and the inner surfaces of the tent were lined with white fabrics to diffuse light evenly. All the tests were recorded with a Legria HF R506 Camcorder (Canon, Tokyo, Japan).

Each cuttlefish was gently transferred from its home tank into one of the eight compartments previously filled with 50 mL of water from its own home tank to avoid additional stress. For each test session, two cuttlefish per exposure condition (FSW; FLX5; FLXVEN2.5 and FLXVEN5) of exactly the same age were randomly placed in the eight compartments of the experimental apparatus.

The animals were videorecorded during 30 min to evaluate the stability of the body patterns over time.

Data analysis.

For each video-recording, two still photographs were analysed: one at the beginning of the test, as soon as the cuttlefish was transferred into the compartment and stopped

Accepted Article

moving (T0 ± 1min), and one at the end of the test period (T30 ± 1min). Insofar as cuttlefish hatchlings hardly express a true uniform body pattern, we choose to use the disruptive score on both uniform and checkerboard background to evaluate background matching, rather than the mean gray value or the heterogeneity index (Bidel et al., 2016a;[START_REF] Di Poi | Cryptic and biochemical responses of young cuttlefish Sepia officinalis exposed to environmentally relevant concentrations of fluoxetine[END_REF]. According to [START_REF] Barbosa | Disruptive coloration in cuttlefish: a visual perception mechanism that regulates ontogenetic adjustment of skin patterning[END_REF], juvenile cuttlefish can express up to 11 body components, all of which may be more or less expressed and, consequently, graded from 0 (not expressed) to 3 (strongly expressed).

A final disruptive score ranging between 0 (perfect uniform body pattern, i.e., no component expressed) to 33 (perfect disruptive body pattern, i.e., the 11 components strongly expressed) was assigned to each cuttlefish at T0 (beginning of the test) and at T30 (end of the test, 30 min). The evolution of the calculated disruptive scores according to the developmental stages (from 7 to 28 dph) and the comparison between the exposure conditions (control; FLX5; FLXVEN2.5 and FLXVEN5) were assessed for both, uniform and checkerboard backgrounds. A lower disruptive score on uniform background and a higher disruptive score on checkerboard indicate a more efficient background matching.

All statistical tests were performed using R Studio (Version 1.1.453, RStudio Inc., Boston, USA, http://www.r-project.org). As data did not meet the assumptions of normality and homoscedasticity, we used non-parametric permutation test analyses of data from factorial experiments (function ezPerm; "ez" package). The number of iterations was set to 1000. We used mixed within-and-between Ss designs (i.e., repeated factors: time, age; independent factor: experimental groups) to evaluate uniform and disruptive body pattern of juvenile cuttlefish at T0 and T30 between groups and ages. If the null hypothesis was rejected (p ≤ 0.05), post-hoc permutation tests were performed to highlight any differences between experimental groups and Accepted Article between ages, using the "RVAidememoire" package. The false discovery rate was adjusted using the Benjamini-Hochberg procedure [START_REF] Benjamini | Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing[END_REF].

Results

Body pattern on uniform background (Fig. 2)

The disruptive score was significantly different between treatment groups (p = 0.001) and between t0 and t30 min (p = 0.001), but there was no difference between ages (p = 0.479) (Fig. 2). Pairwise comparisons showed a significant difference between each of the exposed groups and the controls (post-hoc permutation test: p = 0.004 for each of the treatment groups vs. control, respectively). All groups exposed to antidepressants exhibited a better background matching than the control group, i.e., from 7 dph to 28 dph they expressed a lower disruptive score than the control group. Moreover, the score exhibited by the FLXVEN5 group was also significantly different from the two other treatment groups (post-hoc permutation test: FLXVEN5-FLXVEN2.5, p = 0.027; FLXVEN5-FLX5, p = 0.038). This means that the FLXVEN5 group expressed an even better background matching than the FLXVEN2.5 and FLX5 groups.

In addition, there was a significant interaction between groups and time (p = 0.016), i.e., the difference of disruptive score at the beginning and the end of the test was different depending on the group. Pairwise comparisons revealed that no difference of the score over time existed in the control group (post-hoc permutation test: control: control:t0-control:t30, p = 0.343), that a trend towards a difference over time was found in the FLX5 group (post-hoc permutation test: FLX5:t0-FLX5:t30, p = 0.065), and a significant difference over time existed in both FLXVEN2.5 and FLXVEN5 exposures (post-hoc permutation test: FLXVEN2.5:t0-FLXVEN2.5:t30, p = 0.004; FLXVEN5:t0-FLXVEN5:t30, p = 0.036). While the control group exhibited a fairly stable uniform pattern over time, FLXVEN2.5, FLXVEN5 and most probably FLX5 .

Accepted Article

displayed a more dynamic evolution of their body patterns. Cuttlefish from the exposed groups improved their body pattern over a 30 min period of time towards a more uniform pattern.

Body pattern on checkerboard background (Fig. 3)

The disruptive body pattern was neither significantly different between groups (p = 0.768), nor between age (p = 0.109), or over the 30 min test period (p = 0.767).

Overall, juvenile cuttlefish, whether exposed to antidepressants or not, expressed on average the same disruptive score over a 30 min test period during their first month.

Nevertheless, it may be noticed that the disruptive score of cuttlefish of 8 dph was generally lower than that of 15, 22 and 29 dph, although no statistically significant difference emerged because of the high intragroup variability (p = 0.109). In addition, there was a significant interaction between groups and time (p = 0.004) and between ages and time (p = 0.007). Yet, pairwise comparisons failed to reveal the statistical differences in both interactions. Again, this may be due to the high intragroup variability. However, regarding the means, at 8 dph, cuttlefish from all groups followed the same tendency of a decreasing disruptive score after 30 min (as for the uniform body pattern, see above). At 15 dph, all cuttlefish exhibited this pattern of decreased disruptive scores after 30 min, except those from FLXVEN2.5 group, which increased their disruptive score. At 22 dph, the control and FLXVEN5 groups slightly decreased their disruptive score after 30 min, but not the FLX5 and FLXVEN2.5 group. Finally, at 29 dph, all cuttlefish, whether exposed or not, displayed an increased disruptive score after 30 min.

DISCUSSION

Like many invertebrate species, cuttlefish hatchlings receive no parental care and must learn on their own how to adapt their behaviours in response to environmental .

Accepted Article

changes or to threats [START_REF] Dickel | Behavioral and Neural maturation in the cuttlefish[END_REF]. Although background matching through chromatic colouration is visible on the cuttlefish's body as soon as it hatches, this primary defence is still immature, i.e., hatchlings and early juvenile cuttlefish often display an 'inappropriate' body pattern [START_REF] Hanlon | Adaptive Coloration in Young Cuttlefish (Sepia Officinalis L.): The Morphology and Development of Body Patterns and Their Relation to Behaviour[END_REF]. Notably, they are more likely to express a disruptive colouration when placed on uniform substrates than older animals [START_REF] Hanlon | Adaptive Coloration in Young Cuttlefish (Sepia Officinalis L.): The Morphology and Development of Body Patterns and Their Relation to Behaviour[END_REF]. Thus, expressing a uniform pattern for brightness matching is more challenging for cuttlefish hatchlings than producing a disruptive pattern on a contrasted background. The ability to express a uniform pattern improves, however, in the course of the first two months of life according to their early experience (i.e., rearing environment) [START_REF] Poirier | Early Experience and Postembryonic Maturation of Body Patterns in Cuttlefish (Sepia officinalis)[END_REF]. The disruptive scores obtained in our control group confirm this difficulty for hatchlings to express a uniform pattern on uniform background. The unusual lack of improvement with age may be related to the impoverished rearing conditions, which were constrained in our study by the antidepressant exposure, as compared to the richer natural environment. Indeed, minimised rearing conditions have been shown to slow down the maturation of uniform camouflage abilities in hatchling cuttlefish [START_REF] Poirier | Early Experience and Postembryonic Maturation of Body Patterns in Cuttlefish (Sepia officinalis)[END_REF]. Our results, therefore, confirm that is useful to evaluate the efficiency of both, uniform and disruptive pattern by the disruptive score, as juvenile cuttlefish did not express uniform body pattern easily.

Cuttlefish exposed to antidepressants at very low environmentally realistic concentrations displayed a less disruptive pattern on a uniform background, which is indicative of a better background matching compared to the control group. This holds particularly true for the highest concentrations of FLX and VEN at 5 ng•L -1 each, i.e., 10 ng•L -1 of accumulated antidepressants in total. Moreover, cuttlefish exposed to FLX and VEN distinctly improved this behaviour over the 30 min test period.

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Cuttlefish exposed to FLX alone followed this trend, albeit less evidently. On the contrary, the control group displayed a fairly stable, much less dynamic evolution of their body pattern. Whether VEN alone would have equally improved background matching in the present experimental setting cannot be told. Di [START_REF] Di Poi | Cryptic and biochemical responses of young cuttlefish Sepia officinalis exposed to environmentally relevant concentrations of fluoxetine[END_REF] found that two weeks-old cuttlefish exposed to FLX at 1 and 100 ng•L -1 improved their uniform body pattern over a 30 min test period, whereas controls did not. This was, however, not the case in newly-hatched cuttlefish exposed to 1 ng•L -1 of FLX until two weeks of age, which displayed a decreased efficiency to express uniform body patterns [START_REF] Di Poi | Cryptic and biochemical responses of young cuttlefish Sepia officinalis exposed to environmentally relevant concentrations of fluoxetine[END_REF]. Furthermore, cuttlefish exposed to VEN for 19 days to 100 ng•L -1 demonstrated a lower quality of the uniform body pattern compared to the controls, while no difference emerged in cuttlefish exposed to 5 ng VEN •L -1 for 19 days (Bidel et al., 2016). Finally, embryonic exposure at higher concentrations of FLX, i.e., 1 and 10 µg•L -1 , did not induce any noticeable modification of uniform body pattern. Overall, on a 30 min period, concentrations of FLX had an impact on uniform body pattern, whereas VEN did not. In addition, our results show significant effects of the combination of both FLX and VEN towards a better uniform body pattern on medium grey background. In summary, the results of the present and earlier experiments produced inconclusive responses with respect to the effects of antidepressants on achieving uniform body patterns. Those studies differed slightly in their experimental design and concentrations used, but also in the way of assessing camouflage efficiency. Indeed, Bidel et al. (2016a, 2016b[START_REF] Di Poi | Cryptic and biochemical responses of young cuttlefish Sepia officinalis exposed to environmentally relevant concentrations of fluoxetine[END_REF] used the Mean Gray Value and the Heterogeneity Index from ImageJ © to evaluate the efficiency of uniform pattern of cuttlefish despite the fact that juvenile cuttlefish more easily display disruptive components and struggle to display a perfect uniform body pattern. Those parameters may be more relevant for adult cuttlefish that master the uniform body pattern, while the disruptive score may be more appropriate for young cuttlefish. In addition, a growing body of evidence points to a non-monotonic doseresponse for antidepressants with greater effects at concentrations in the lower range [START_REF] Fong | The biological effects of antidepressants on the molluscs and crustaceans: A review[END_REF][START_REF] Vandenberg | Hormones and Endocrine-Disrupting Chemicals: Low-Dose Effects and Nonmonotonic Dose Responses[END_REF]. This might explain the variability of responses between different studies.

Unlike uniform pattern, disruptive patterns are easily expressed, by hatchlings and juvenile cuttlefish, even if they may not be perfect. This provides them with an efficient camouflage when their environment comprises contrasted components. No distinct effects of antidepressants on the disruptive body pattern of newly-hatched cuttlefish placed in a checkerboard visual environment emerged from the present study. Previous studies from Bidel et al. (2016a, 2016b[START_REF] Di Poi | Cryptic and biochemical responses of young cuttlefish Sepia officinalis exposed to environmentally relevant concentrations of fluoxetine[END_REF] are in line with these findings. Indeed, when exposed to either FLX at 1 and 100 ng•L -1 or 1 and 10 µg•L -1 , or VEN at 5 and 100 ng•L -1 , juvenile cuttlefish did not show any modification of the disruptive pattern on a checkerboard background. Despite the fact that its complexity, i.e., the number of chromatic component expressed, can be modulated by environmental conditions [START_REF] Poirier | Early Experience and Postembryonic Maturation of Body Patterns in Cuttlefish (Sepia officinalis)[END_REF] and, therefore, is subject to maturation as well, the disruptive pattern seems to be a far more robust and insensitive to waterborne antidepressants, even at the highest concentrations tested or when different antidepressants were combined.

The modification of background matching towards a greater efficiency after an exposure to a SSRI and/or SNRI may be explained by their action on the chromatophores. Both antidepressants used in this study are designed to modulate the levels of 5-HT, which is involved in the retraction of chromatophores muscles [START_REF] Messenger | Cephalopod chromatophores: neurobiology and natural history[END_REF]. If FLX and/or VEN increase the synaptic concentrations of 5-HT, the exposure to such antidepressants may cause a relaxation of the chromatophore's

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muscle crown leading to pigment concentration. This reduces disruptive colour patterning and brings forward the uniform coloration by the leucophores.

Accordingly, [START_REF] Florey | Electrical and mechanical responses of chromatophore muscle fibers of the squid, Loligo opalescens, to nerve stimulation and drugs[END_REF] demonstrated that 5-HT-application to the skin of Loligo opalescens caused a paling due to rapid chromatophore retraction. [START_REF] Loi | Peptidergic regulation of chromatophore function in the European cuttlefish Sepia officinalis[END_REF] obtained similar results with 5-HT causing a more pronounced retraction than in normal state in an in-vivo bioassay with cuttlefish skin. Accordingly, such a direct action on chromatophores may explain the effect of FLX alone as well as in combination with VEN in promoting an improved background matching on uniform substrate due to a retraction of all the chromatophores resulting in a decrease in contrasts between the different skin chromatic components. But, if so, a decrease of the expression of the disruptive pattern should have been observed on the checkerboard background as well, which was not the case, neither for the disrupting score in this study, nor for the heterogeneity index in previous studies (Bidel et al., 2016b(Bidel et al., , 2016a;;[START_REF] Di Poi | Cryptic and biochemical responses of young cuttlefish Sepia officinalis exposed to environmentally relevant concentrations of fluoxetine[END_REF]. Furthermore, both Bidel et al. (2016a, 2016b[START_REF] Di Poi | Cryptic and biochemical responses of young cuttlefish Sepia officinalis exposed to environmentally relevant concentrations of fluoxetine[END_REF] suggested that FLX and VEN influences the dopaminergic and noradrenergic system, respectively, rather than the serotoninergic system. Some studies also pointed to the effect of antidepressants on glutamatergic system, in particular by modulating N-methyl-D-aspartate (NMDA) receptors [START_REF] Musazzi | The Action of Antidepressants on the Glutamate System: Regulation of Glutamate Release and Glutamate Receptors[END_REF][START_REF] Musazzi | Stress, glucocorticoids and glutamate release: Effects of antidepressant drugs[END_REF][START_REF] Wong | an antidepressant drug: Twenty years[END_REF]. Bidel et al. (2016a) investigated if VEN could modulate binding sites of NMDA-like receptors in brain of cuttlefish. Indeed, exposure to either 5 or 100 ng•L -1 of VEN for 20 days post-hatching decreased the number of NMDA-like receptor binding sites. Thus, VEN may have an impact on chromatophore contraction in neuro-muscular junctions, enhancing a uniform body pattern. Yet, this modulation should exert a similar effect on a disruptive background.

As stated before, however, no effect could be measured in this condition.

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Accepted Article

Notwithstanding, the modulation of glutamatergic system may have an effect in brain structures, such as optic lobes in cuttlefish. For instance, in Sepia pharaonis, visual development has been linked with the expression of NMDA receptors in optic lobes [START_REF] Lee | Early visual experience of background contrast affects the expression of NMDA-like glutamate receptors in the optic lobe of cuttlefish, Sepia pharaonis[END_REF]. Exposure to either FLX or VEN has been linked with decreased cell proliferation in the optic lobes of juvenile cuttlefish (Bidel et al., 2016b(Bidel et al., , 2016a)). Both antidepressants may, putatively, modulate brain development and functions, such as visual perception or motor control of chromatic skin components and/or inhibition of the disruptive pattern that cuttlefish hatchlings readily express. The possible effects of FLX and VEN on neurotransmitter levels and structural changes in brain may lead to a different processing of visual information and, consequently, to modifications in the expression of uniform body pattern.

Our study reveals an effect of antidepressants on colour change, specifically when a uniform body pattern is required. The expression of uniform body pattern, immature at hatching [START_REF] Poirier | Early Experience and Postembryonic Maturation of Body Patterns in Cuttlefish (Sepia officinalis)[END_REF], seemed to be the more sensitive aspect of cryptic colouration. Because its maturation was more pronounced, it was more prone to modification by antidepressants. Maturation in cuttlefish appeared to be affected by antidepressants in an earlier study [START_REF] Chabenat | Hidden in the sand: Alteration of burying behaviour in shore crabs and cuttlefish by antidepressant exposure[END_REF]. In that study, the sand digging behaviour -which is part of the cryptic behaviour-decreased, whilst homochromy at about 80% remained largely unaltered when S. officinalis was exposed to concentrations as low as 2.5 ng•L -1 FLX and VEN combined. This demonstrates that while certain aspects of camouflage may be improved by antidepressants, others may be deteriorated, with the potential to impair overall camouflage abilities. Whether improving uniform body patterns, which is a priori a positive effect, could have negative repercussions in other parameters of camouflage is difficult to predict. What becomes evident, however, is that relatively low

Accepted Article

concentrations of antidepressants can affect the maturation of camouflage at early periods of cuttlefish development in various, yet unpredictable manners. This implies the possibility that SSRIs/SNRIs at environmental concentrations may also impair complex behavioural traits, such as cryptic behaviour, or that positive effects on one parameter may entail negative trade-offs on other parameters. and FLX + VEN at 2.5 ng•L -1 each) at four different time points (8, 15, 22, 29 days post-hatching [dph]) and over a 30 min test period each (i.e., from T0 to T30). Results are expressed as means ± SEM of disruptive scores/33, n=16.

  Pretests demonstrated that the actual concentrations never fell below 75% of nominal concentrations within 24 h, with VEN being more stable than FLX. Animals were fed daily with one sand shrimp (Crangon crangon) until the end of the experiments. Animal rearing and animal experiments were carried out in accordance with the guidelines of the EU Directive 2010/63/EU for animal experiments (Protocol approved by the French National Ethical Committee for Animal Experimentation, Agreement number: 035121.02).

Figure 1 .

 1 FIGURE CAPTIONS

Figure 2 .

 2 Figure2. Evolution of disruptive score on a uniform medium-grey background in four treatment groups of juvenile cuttlefish (control, FLX at 5 ng•L -1 , FLX + VEN at 2.5 ng•L -1 each, and FLX + VEN at 5 ng•L -1 each), at four different ages(7, 14, 21, 28

Figure 3 .

 3 Figure 3. Evolution of disruptive score on checkerboard background in four treatment groups of juvenile cuttlefish (control, FLX at 5 ng•L -1 , FLX + VEN at 2.5 ng•L -1 each,

Acknowledgment-This research work was supported by a doctoral grant from Normandy Region provided by the Research Federation CNRS 3730 SCALE (SCiences Appliquées à L'Environnement). The authors wish to thank the staff of the Centre de Recherches en Environnement Côtier for egg collecting and Celine Thomasse and Nadège Villain-Naud. The authors gratefully acknowledge Christelle Jozet-Alves for her help with R and statistical analyses. Disclaimer-The authors declare no competing interest.

Data availability statement-Data, associated metadata, and calculation tools are available from the corresponding author (cecile.bellanger@unicaen.fr).