Lingaswamy Kadari

Thierry Roisnel

William Erb
email: william.erb@univ-rennes1.frprkgenius@iict.res.in

Radha Krishna

Florence Mongin

2-trimethylsilylferrocenecarboxamide: a new route toward 1,1'-disubstituted ferrocenes

Keywords: Ferrocene, 1,1'-Disubstitution, Carboxamide, Remote functionalization, Functional group manipulation

Remote deprotometalation-iodolysis of N,N-diisopropyl-

The discovery of ferrocene in 1952 has profoundly changed the organometallic chemistry landscape and this sandwich compound remains one of the most important organometallic scaffolds. 1 Among the various substitution patterns explored in ferrocene chemistry, the most popular is the 1,1'-disubstitution, with applications in catalysis, 2 material science [START_REF]Ferrocenes: Ligands, Materials and Biomolecules, Štěpnička[END_REF] and bioinorganic chemistry. 3a, 4 1,1'-Disubstituted ferrocenes with the same substituents can easily be obtained by two complementary approaches: (1) the ferrocene core assembly from the corresponding monosubstituted cyclopentadienes 5 and (2) the double deprotometalation-electrophilic trapping sequence from ferrocene. 6 However, while 1'-substituted iodoferrocenes represent an important family of precursors to unsymmetrical compounds, 7 they cannot be directly prepared by one of the above mentioned approaches. Therefore, the most popular synthetic routes currently rely on the halogen/Li or Sn/Li monoexchange of such 1,1'-disubstituted ferrocenes. Buttler initially reported the mono I/Li exchange from 1,1'-diiodoferrocene using nBuLi, followed by an electrophilic trapping step (Scheme 1, approach a, R = CO2H, SnBu3). 8 However, low yields were obtained (28-32%), linked with the highly sensitive nature of the reaction as expressed in a footnote. Similar low yields were recorded by Christmann, Sarkar and Heretsch using tosyl azide as the electrophile 9 while Lentz and later Nijhuis, both using tributyltin chloride, managed to isolate 1-iodo-1'-tributylstannylferrocene in good yields (77 and 84%, respectively). 10 The concentration was proposed as being an important reaction parameter in such reactions, 11 and the development of a flow-based approach partially solved this issue. 9 From 1,1'-bis(tributylstannyl)ferrocene, Wright reported a controlled Sn/Li mono-exchange by using nBuLi before interception with an electrophile. 12 However, iodine cannot be introduced this way due to a competitive Sn/I exchange leading to mixtures of products. Therefore, another substituent needs to be introduced first, before performing the final Sn/I exchange using iodine. Such approach was illustrated with success during the synthesis of 1'-iodoferrocenecarboxaldehyde (Scheme 1, approach b, R = CHO). 13 Finally, Dong developed a mono Li/Br exchange-electrophilic trapping sequence from 1,1'dibromoferrocene; 14 this was used by Ilyashenko as the key step in the synthesis of 1-bromo-1'-iodoferrocene (78% yield; Scheme 1, approach c, R = Br). 11 The other strategies toward 1'-substituted iodoferrocene start from monosubstituted ferrocenes and rely on functionalizations at the unsubstituted cyclopentadienyl (Cp) ring, remote from the substituent. The regioselective aromatic electrophilic substitution of iodoferrocene using acetyl chloride in the presence of iron trichloride was first attempted by Richards. 15 However, the expected 1,1'-disubstituted product was not obtained due to competitive deiodination observed under the reaction conditions. However, by using N-methylformanilide in the presence of phosphorus oxychloride, Yuan managed to isolate the corresponding 1'-iodoferrocenecarboxaldehyde in a moderate 61% yield (Scheme 1, approach d, R = CHO). 16 The use of directing groups able to reroute the classical orthodeprotometalation to the unsubstituted Cp ring represents the second approach in the remote functionalization strategy. Balavoine and Manoury reported the reaction of the lithium salt of N-methylpiperazine with ferrocenecarboxaldehyde, followed by a deprotolithiation-electrophilic trapping sequence toward 1'iodoferrocenecarboxaldehyde in a moderate 50% yield (Scheme 1, approach e, R = CHO). 17 The N-methylpiperazine acts as a protecting group for the aldehyde (temporary formation of the hemiaminal salt) and as the directing group with methylated nitrogen atom. More recently, Chong revealed a similar behaviour for N-Boc protected α-methylated aminomethylferrocene in deprotolithiation and managed to obtain the corresponding iodinated derivative in a 71% yield (Scheme 1, approach f, R = CH(Me)NHBoc). 18 A few other catalytic desymmetrizations of 1,1'-diiodoferrocene have been reported, based on Stille, 10a, 19 Sonogashira, 20 Suzuki-Miyaura 21 and Ulmann 22 cross-couplings and by aminocarbonylation. 23 However, the selectivity is often an issue in such transformations, leading to mixtures of products.

In the frame of ongoing studies dedicated to the synthesis of original ferrocene derivatives, 24 we required an access to different 1'-substituted iodoferrocene derivatives. We initially focused our work on the mono-exchange of 1,1'-diiodoferrocene by using nBuLi, as this approach has the potential to easily deliver many derivatives by varying the electrophile. 8 However, regardless of the reaction conditions, only traces of the desired products were invariably obtained while 1,1'-bis-functionalized derivatives and mixtures of 1,1'-diiodoferrocene, iodoferrocene and ferrocene were mainly obtained. We briefly evaluated the Manoury approach, 17 but failed to obtain significant amounts of the title products by using iodine as the electrophile. Moderate success was encountered in our attempts to achieve a mono Sn/Li exchange from 1,1'-bis(tributylstannyl)ferrocene 12 by using nBuLi and either methyl chloroformate or dimethylformamide as the electrophile (22 or 25% yield, respectively). However, as we recognized that the synthesis of the targeted compounds would require the manipulation of large quantities of toxic stannylated products, the need for another original approach emerged.

In his seminal paper on the enantioselective deprotometalation of N,N-diisopropylferrocenecarboxamide, 25 Snieckus reported that the introduction of a trimethylsilyl group at position 2 was able to reroute a further deprotometalation to the unsubstituted Cp ring. While a similar behavior was also noticed by Richards on ferrocene oxazolines, 26 this remote functionalization has never been considered as a valuable synthetic tool. Therefore, our plan was to use a silyl group to temporarily protect the position next to the carboxamide and favor the remote deprotometalation to introduce iodine onto the unsubstituted ring. Final deprotection of the silyl group would afford the targeted 1,1'-disubstituted ferrocenes.

We first deprotolithiated N,N-diisopropylferrocenecarboxamide 27 (1) by using the nBuLi•TMEDA (TMEDA: N,N,N′,N′-tetramethylethylenediamine) chelate before intercepting the lithiated intermediate with trimethylsilyl chloride toward the ferrocene rac-2, isolated in a 92% yield (Scheme 2). To reach the iodinated derivative rac-3, we initially applied the Snieckus reaction conditions by using sBuLi (1.2 equiv) in THF at cryogenic temperature (-80 to -78 °C) before addition of iodine as the electrophile. 25 However, the title product was isolated in a disappointing 50% yield together with recovered starting material. While recourse to the more reactive sBuLi•TMEDA chelate improved the conversion to 95%, formation of inseparable by-products was noticed. However, the use of 2 equivalents of sBuLi at cryogenic temperature (-80 to -78 °C) led to a complete conversion and the ferrocene rac-3 was isolated in a 95% yield on a 1 mmol scale. Pleasingly, the yield remained constant upon scaling up (97% yield on a 26 mmol scale). Although the trimethylsilyl group is usually removed upon treatment with fluorides, 28 we found that rac-3 was reluctant to desilylation under smooth reaction conditions (2 equivalents of tetrabutylammonium fluoride at room temperature) and that decomposition occurred upon heating. However, the use of a stoichiometric amount of potassium tert-butoxide in dimethylsulfoxide afforded 4 in a promising 70% yield after only 5 min. 29 Evaluation of other reaction conditions revealed a complete conversion with increased amount of potassium tertbutoxide, reaction time and concentration, delivering 4 in an 80% yield on a 1 mmol scale. Furthermore, it was possible to scale-up the reaction up to 25 mmol with a slightly better yield (88%, 10 g of product 4 made in a single batch).

Scheme 2 Remote functionalization strategy toward the 1,1'-disubstituted ferrocene 4. DMSO: dimethylsulfoxide.

Clues about the origin of this remote functionalization can be found in the solid-state structure of compound rac-2. Indeed, to release the steric pressure generated between the diisopropyl and trimethysilyl moieties, the C=O bond of the carboxamide needs to be almost perpendicular to the Cp ring (Scheme 3, right and S.I.). As shown by a variable temperature 1 H NMR study (see S.I.), rotation of the C=O to reach the conformation favorable to the ortho-deprotometalation (C=O parallel with the Cp ring, Scheme 3, left), is too energetic. Therefore, the only reaction observed is the remote deprotometalation, probably favored by the C=O bound pointing toward the unsubstituted Cp ring to guide the approach of the lithiated base (Scheme 3, right). With a reliable protocol toward 4 in hands, we next focused our attention onto the carboxamide transformation. While the borane-mediated reduction of similar substrates is well known, 25,30 transformation of the resulting diisopropylamine moiety did not receive much attention 31 until we recently documented the substitution of this bulky amine for an acetate. 32 Consequently, the carboxamide 4 was reduced by an excess of borane (generated in situ from sodium borohydride and iodine) in refluxing tetrahydrofuran to deliver 5 in a 95% yield (Scheme 4). The substitution step then occurred smoothly in neat acetic anhydride at 160 °C for 1 h and 6 was isolated in a 88% yield on a 20 mmol scale (6.7 g of compound in a single batch). During the reduction of 4 to 5, traces of a by-product, tentatively assigned to be 1-iodo-1'-methylferrocene (7), were noticed. To validate its structure, we engaged the acetate in another borane reduction and indeed isolated 7 in a 92% yield.

Scheme 4 Functional group manipulation toward the methylated derivative 7. THF: tetrahydrofuran.

Saponification of the acetate 6 with sodium hydroxide in a tetrahydrofuran-water mixture at 80 °C led to the isolation of the alcohol 8 in a 90% yield (Scheme 5). It should be noted that the use of methanol-water mixtures in such reaction should be avoided as variable amounts of the methyl ether 9 can be formed as a side-product. However, it is possible to obtain 9 in an almost quantitative yield by the deprotonation of the alcohol using sodium hydride followed by the interception of the alkoxide with methyl iodide. The known aldehyde 10 then was prepared by a ruthenium-mediated oxidation in the presence of Nmethylmorpholine oxide initially described by Sharpless. 33 Addition of phenylmagnesium bromide to the aldehyde afforded the alcohol 11 in a 97% yield, which was finally converted into the known ketone 12 in a 96% yield by following the same ruthenium-mediated oxidation protocol.

Scheme 5 Functional group manipulation toward the ketone 12. NMO: Nmethylmorpholine oxide.

While condensation of the aldehyde 10 with hydroxylamine followed by dehydration led to 1'-iodoferrocenecarbonitrile (13) in 91% yield (Scheme 6), 34 it is known that such substrates are difficult to oxidize under classical conditions. 35 However, reaction of 10 with iodine in the presence of potassium hydroxide 36 in methanol led to the ester 14 in a very good 97% yield. The acid 15 was easily made by saponification in 96% yield and it was further reacted with diphenylphosphoryl azide by following our recently developed protocol 37 toward the acyl azide 16, isolated in an 85% yield. Curtius rearrangement at 110 °C in the presence of tert-butanol to intercept the intermediate isocyanate allowed the isolation of the Boc-protected aminoferrocene 17 (85% yield). Removal of the protecting group was easily done by using an ethereal solution of HCl at room temperature toward 18, isolated in a 74% yield as its HCl salt. Although salts of aminoferrocene derivatives are known to be more stable when compared to the free base, storage of 18 in a closed vessel without specific protection led to decomposition. A double reductive amination 38 with paraformaldehyde in the presence of an excess of sodium cyanoborohydride was finally performed toward the sensitive dimethylaminoferrocene 19, isolated in a 40% yield. In conclusion, we have reported a new route toward 1'substituted iodoferrocenes based on a remote deprotometalation observed more than 20 years ago and never exploited. By following this methodology, grams of our key substrate, 1'-iodo-N,N-diisopropylferrocenecarboxamide, were prepared and further functionalized towards fifteen 1,1'-disubstituted ferrocenes, most of them being fully described for the first time.

General Considerations. Unless otherwise stated, all the reactions were performed under an argon atmosphere with anhydrous solvents using Schlenk technics. THF and Et2O were distilled over sodiumbenzophenone, DMSO and toluene were distilled over CaH2 and acetone and methanol were dried by prolonged contact over activated 3 Å molecular sieve. 39 Unless otherwise stated, all reagents were used without prior purification. All organolithiated reagents were titrated before use. 40 tBuOK (99.99% quality) was purchased from Sigma-Aldrich and used without further purification. Column chromatography separations were achieved on silica gel (40-63 μm). All Thin Layer Chromatographies (TLC) were performed on aluminum backed plates pre-coated with silica gel (Merck, Silica Gel 60 F254). They were visualized by 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 Nuclear Magnetic Resonance (NMR) spectra were recorded either (i) on a Bruker Avance III spectrometer at 300 MHz and 75.4 MHz, respectively, or (ii) on a Bruker Avance III at 400 MHz and 100 MHz, respectively or (iii) 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. Cp refers to the unsubstituted cyclopentadienyl ring of ferrocene.

Procedures

N,N-Diisopropyl-2-trimethylsilylferrocenecarboxamide (rac-2)

[CAS Reg. No. 173910-99-1] TMEDA (4.50 mL, 3.49 g, 30.0 mmol, 1.50 equiv, freshly distilled over CaCl2 and stored over KOH pellets) and anhydrous Et2O (90.0 mL) were introduced into a flame-dried round-bottom flask under argon. The reaction mixture was cooled between -80 and -78 °C (external temperature) in an acetone/liquid nitrogen bath. nBuLi (1.4 M, 21.4 mL, 30.0 mmol, 1.50 equiv) was then introduced dropwise by syringe. After addition, the reaction mixture was stirred at the same temperature for 15 min. Compound 1 (6.26 g, 20.0 mmol, 1.00 equiv) was introduced into a separate flame-dried round-bottom flask which was subjected to three cycles of vacuum/argon. Anhydrous Et2O (90.0 mL) was added and the solution was stirred until dissolution of all solids. The solution of 1 was transferred into the nBuLi•TMEDA chelate solution dropwise by cannula keeping the temperature between -80 and -78 °C. The flask was washed with 10.0 mL of anhydrous Et2O, also transferred by cannula. After addition, the reaction mixture was stirred at the same temperature for 1 h. Me3SiCl (5.10 mL, 4.34 g, 40.0 mmol, 2.00 equiv) was added dropwise by syringe keeping the temperature between -80 and -78 °C. The reaction mixture was then allowed to warm to -15 °C, keeping the flask into the bath. At -15 °C, the cooling bath was removed and the reaction mixture was warmed to rt. Water (100 mL) was added and the layers were separated. The aqueous layer was extracted with EtOAc (2 x 75 mL). The combined organic layers were washed with brine, dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography, using PET-EtOAc (80:20) to give the title product rac-2 as an orange solid (7.12 g, 92%). Analytical data analogous to those reported previously. 25 x CH3), 0.27 (s, 9H, Si(CH3)3). 13 C NMR (126 MHz, CDCl3): δ (ppm) 168.9 (C=O), 92.3 (C1), 73.7 (C2), 73.5 (C3), 69.9 (Cp + C5), 69.4 (C4), 50.1 (CH), 45.9 (CH), 21.1 (2 x CH3), 20.9 (2 x CH3), 0.7 (Si(CH3)3).

1'-Iodo-N,N-diisopropyl-2-trimethylsilylferrocenecarboxamide (rac-3)

sBuLi (1.2 M, 43.3 mL, 52.0 mmol, 2.00 equiv) was added dropwise to a solution of rac-2 (10.0 g, 26.0 mmol, 1.00 equiv) in anhydrous Et2O (300 mL) between -80 and -78 °C. The reaction mixture was stirred at the same temperature for 1 h after addition. Iodine (19.8 g, 78.0 mmol, 3.00 equiv) was introduced in a separate flame-dried round-bottomed flask under argon and was dissolved in anhydrous Et2O (300 mL). The iodine solution was transferred by cannula into the reaction mixture. The flask was washed with 10.0 mL of anhydrous Et2O, also transferred by cannula. After addition, the reaction was stirred at the same temperature for 30 min before being warmed to rt. A saturated aqueous solution of Na2S2O3 (100 mL) was added to the reaction mixture and the layers were separated. The aqueous layer was extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with brine, dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography, using PET-EtOAc (99:1) to give the title product rac-3 as an orange oil (12.9 g, 97%). Potassium tert-butoxide (5.72 g, 51.0 mmol, 2.00 equiv) was added to a solution of rac-3 (13.0 g, 25.5 mmol, 1.00 equiv) in DMSO (76.5 mL) and the reaction mixture was stirred for 10 min at rt. Cold water (200 mL) was added to the reaction mixture which was extracted with EtOAc (4 x 70 mL). The combined organic layers were washed with brine (4 x 50 mL), dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography, using PET-EtOAc (85:15) to give the title product 4 as an orange solid (9.92 g, 88%).

Mp 58-60 °C. νmax (film)/cm -1 2964, 1615, 1459, 1368, 1315, 1201, 1043, 1029, 862, 825, 804, 762. 1 H NMR (300 MHz, CDCl3): δ (ppm) 4.49 (t, J = 1.9 Hz, 2H, H2 + H5), 4.46 (br s, 1H, CH), 4.41 (t, J = 1.9 Hz, 2H, H2' + H5'), 4.26-4.24 (m, 4H, H3 + H4 + H3' + H4'), 3.41 (br s, 1H, CH), 1.47 (br s, 6H, 2 x CH3), 1.21 (br s, 6H, 2 x CH3). 13

1-(N,N-Diisopropylaminomethyl)-1'-iodoferrocene (5)

NaBH4 (2.84 g, 75.0 mmol, 5.00 equiv) was introduced into a flame-dried round-bottom flask equipped with a bubbler, and THF (75.0 mL) was added before the reaction mixture was cooled to 0 °C. Iodine (9.14 g, 36.0 mmol, 2.40 equiv) was introduced into a separate flame-dried roundbottom flask under argon and was dissolved into anhydrous THF (30.0 mL). The iodine solution was transferred into the NaBH4 suspension dropwise by cannula. Remark: vigorous evolution of hydrogen occurred during the addition. After addition, the reaction mixture was allowed to warm to rt out of the cooling bath and was stirred for 1 h, giving a colorless solution of BH3.THF. Compound 4 (6.60 g, 15.0 mmol, 1.00 equiv) was then added portionwise to the BH3.THF solution which was then stirred overnight at reflux Remark: the use of PTFE sleeve is strongly recommended to avoid blockage of the ground glass joints. The reaction mixture was cooled to 0 °C and a solution of NaOH (10%, 75 mL) was added dropwise. Caution: as a vigorous evolution of gas was noticed, the first drops of NaOH solution should be added slowly. After addition, the reaction mixture was stirred at reflux for 1 h. The reaction mixture was cooled to rt and the layers were separated. The aqueous layer was further extracted with EtOAc (2 x 50 mL). The combined organic layers were extracted with HCl (1.0 M aq., 3 x 50 mL). The combined aqueous layers were washed with Et2O (2 x 50 mL) and basified with solid K2CO3 until pH 8 was reached. The mixture was extracted with EtOAc (2 x 60 mL) and the combined organic layers were dried over MgSO4, filtrated on celite (washed with EtOAc until the filtrate was colorless), and concentrated under vacuum using a rotary evaporator to give the pure product 5 as an orange oil (6.08 g, 95%). νmax (film)/cm -1 3090, 2961, 1684, 1461, 1380, 1360, 1200, 1164, 1136, 1115, 1018, 861, 823, 807. 1 H NMR (300 MHz, CDCl3): δ (ppm) 4.31 (t, J = 1.6 Hz, 2H, H2' + H5'), 4.15 (t, J = 1.6 Hz, 2H, H2 + H5), 4.10 (d, J = 1.6 Hz, 2H, H3' + H4'), 4.09 (d, J = 1.6 Hz, 2H, H3 + H4), 3.47 (s, 2H, CH2), 3.04 (sept, J = 6.7 Hz, 2H, 2 x CH), 1.02 (d, J = 6.7 Hz, 12H, 4 x CH3). 13

1-(Acetoxymethyl)-1'-iodoferrocene (6)

Compound 5 (8.53 g, 20.0 mmol, 1.00 equiv) was dissolved into acetic anhydride (76.2 mL, 82.3 g, 800 mmol, 40.0 equiv) at rt and the resulting solution was stirred at 160 °C in a pre-heated bath for 1 h. The reaction mixture was cooled to 0 °C and EtOAc (150 mL) was added before pouring the reaction mixture onto ice (200 mL). Solid K2CO3 was added under stirring until pH 8 was reached. Caution: small portions of K2CO3 should be added each time as vigorous evolution of gas occurred. The layers were separated and the aqueous layer was extracted with EtOAc (1 x 75 mL). The combined organic layers were washed with water (1 x 75 mL), brine (1 x 50 mL), dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography over SiO2, using PET/EtOAc (95:5) to give the title product 6 as an orange solid (6.75 g, 88%).

Mp 54-56 °C. νmax (film)/cm -1 3090, 2961, 1684, 1461, 1380, 1360, 1200, 1164, 1136, 1115, 1018, 861, 823, 807. 1 H NMR (500 MHz, CDCl3): δ (ppm) 4.91 (s, 2H, CH2), 4.39 (t, J = 1.7 Hz, 2H, H2' + H5'), 4.22 (m, 2H, H3 + H4), 4.21 (m, 2H, H2 + H5), 4.15 (t, J = 1.7 Hz, 2H, H34 + H4'), 2.05 (s, 3H, CH3). 13 C NMR (125 MHz, CDCl3): δ (ppm) 170.9 (C=O), 82.9 (C1), 75.3 (C2' + C5'), 72.6 (C3 + C4), 72.0 (C2 + C5), 69.5 (C3' + C4'), 62.2 (CH2), 39.9 (C1'), 21.1 (CH3).

Mass: 439 [M]

, 339 [M-NiPr2].

1-Iodo-1'-methylferrocene (7)

[CAS Reg. No. 31833-00-8] Compound 6 (384.0 mg, 1.0 mmol, 1.0 equiv) was added portionwise to a solution of BH3 in THF (1.0 M, 5.00 mL, 5.00 mmol, 5.00 equiv) at rt before the reaction mixture was heated at reflux overnight. Remark: the use of PTFE sleeve is strongly recommended to avoid blockage of the ground glass joints. The reaction mixture was cooled to 0 °C and a solution of NaOH (10%, 10 mL) was added dropwise. Caution: as a vigorous evolution of gas occurs, the first drops of NaOH solution should be added slowly. After addition, the reaction mixture was stirred at reflux for 1 h. The reaction mixture was cooled to rt and the layers were separated. The aqueous layer was extracted with EtOAc (1 x 10 mL). The combined organic layers were washed with water (1 x 10 mL), brine (1 x 10 mL), dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography over SiO2, using PET/EtOAc (95:5) to give the title product 7 as an orange oil (300 mg, 92%). NaOH (2.04 g, 51.0 mmol, 3.00 equiv) was dissolved in H2O (55.0 mL) and a solution of compound 6 (6.50 g, 17.0 mmol, 1.00 equiv) in THF (30.0 mL) was added at rt. The reaction mixture was heated at 80 °C for 2 h in a preheated bath. The reaction mixture was cooled to rt and layers were separated. The aqueous layer was extracted with EtOAc (2 x 20 mL). The combined organic layers were dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography over SiO2, using PET/EtOAc (95:5) to give the title product 8 as an orange solid (5.23 g, 90%). Analytical data analogous to those reported previously. 13 Mp 58-60 °C. νmax (film)/cm -1 3325, 3097, 2925, 2863, 1402, 1379, 1343, 1235, 1143, 1040, 996, 922, 862, 825, 738.

νmax (film)/cm

1-Iodo-1'-(methoxymethyl)ferrocene (9)

Sodium hydride (60% dispersion in oil, 120 mg, 3.00 mmol, 3.00 equiv) was added portionwise to a solution of compound 8 (342 mg, 1.00 mmol, 1.00 equiv) in THF (5.00 mL) at 0 °C. After addition, the cooling bath was removed and the reaction mixture was stirred for 1 h at rt. The reaction mixture was cooled to 0 °C and iodomethane (250 μL, 568 mg, 4.00 mmol, 4.00 equiv) was added dropwise. After addition, the cooling bath was removed and the reaction mixture was stirred for 1 h at rt. The reaction mixture was cooled to 0 °C and NH4Cl (aq. sat., 10 mL) was added dropwise. EtOAc (10 mL) was added and the layers were separated. The aqueous layer was extracted with EtOAc (10 mL) and the combined organic layers were dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography over SiO2, using PET/EtOAc (80:20) to give the title product 9 as an orange oil (346 mg, 97%). N-Methylmorpholine-N-oxide (NMO; 5.04 g, 43.0 mmol, 3.50 equiv) was placed in a Schlenk tube which was heated at 90 °C under high vacuum for 3 h before being cooled to rt. In a separate Schlenk tube, compound 8 (4.22 g, 12.3 mmol, 1.00 equiv) was dissolved in acetone (120 mL) and this solution was cannulated onto NMO. RuCl2(PPh3)3 (589 mg, 0.61 mmol, 0.05 equiv) was added in one portion and the reaction mixture was stirred at rt for 1 h, shielded from light. The reaction mixture was concentrated under vacuum using a rotary evaporator. HCl (1.0 M aq., 40 mL) was added and the reaction mixture was extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography over SiO2, using PET/EtOAc (80:20) with 1% of NEt3 to give the title product 10 as a red solid (3.57 g, 85%). Analytical data analogous to those reported previously. 13 Mp 31-33 °C. νmax (film)/cm -1 3096, 2801, 2761, 1673, 1658, 1455, 1368, 1343, 1242, 1026, 824, 740. 1 H NMR (300 MHz, CDCl3): δ (ppm) 10.00 (s, 1H, CHO), 4.77 (t, J = 2.0 Hz, 2H, H2 + H5), 4.59 (t, J = 2.0 Hz, 2H, H3 + H4), 4.50 (t, J = 1.9 Hz, 2H, H2' + H5'), 4.26 (t, J = 1.9 Hz, 2H, H3' + H4').

νmax (film)/cm

1-(Hydroxy(phenyl)methyl)-1'-iodoferrocene (11)

A solution of phenylmagnesium bromide in THF (0.90 M, 2.10 mL, 1.90 mmol, 0.95 equiv) was added dropwise to a solution of compound 10 (680 mg, 2.00 mmol, 1.00 equiv) in THF (20.0 mL) at -78 °C. Keeping the flask in the cooling bath, the reaction mixture was allowed to warm slowly to -40 °C and the progress of the reaction was monitored by TLC. After consumption of the starting material, NH4Cl (sat. aq., 20 mL) was added and the reaction mixture was warmed to rt before being extracted with EtOAc (2 x 20 mL). The combined organic layers were dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography over SiO2, using PET/EtOAc (80:20) to give the title product 11 as a red solid (812 mg, 97%). Splitting of some ferrocene peaks was noticed in both 1 H and 13 C NMR spectrum as previously observed in chiral ferrocenemethanol derivatives. 41 Mp 76-78 °C. νmax (film)/cm -1 3470, 1490, 1449, 1377, 1179, 1047, 1018, 806, 716. N-Methylmorpholine-N-oxide (NMO; 351 mg, 3.00 mmol, 3.00 equiv) was placed in a Schlenk tube which was heated at 90 °C under high vacuum for 3 h before being cooled to rt. In a separate Schlenk tube, compound 11 (418 mg, 1.00 mmol, 1.00 equiv) was dissolved in acetone (10.0 mL) and this solution was cannulated onto NMO. RuCl2(PPh3)3 (47.9 mg, 50.0 mol, 0.05 equiv) was added in one portion and the reaction mixture was stirred at rt for 1 h, shielded from light. Volatiles were removed under vacuum using a rotary evaporator. The reaction mixture was concentrated under vacuum using a rotary evaporator. HCl (1.0 M aq., 20 mL) was added and the reaction mixture was extracted with EtOAc (2 x 20 mL). The combined organic layers were dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography over SiO2, using PET/EtOAc (80:20) with 1% of NEt3 to give the title product 12 as a red oil (400 mg, 96%). Analytical data analogous to those reported previously. 10b νmax (film)/cm -1 1635, 1447, 1438, 1374, 1282, 1168, 1048, 1024, 852, 828, 797, 724. 1 H NMR (500 MHz, CDCl3): δ (ppm) 7.88 (d, J = 7.2 Hz, 2H, H2'' + H6''), 7.55 (t, J = 7.2 Hz, 1H, H4''), 7.47 (t, J = 7.6 Hz, 2H, H3'' + H5''), 4.90 (t, J = 1.9 Hz, 2H, H2 + H5), 4.55 (t, J = 1.9 Hz, 2H, H3 + H4), 4.41 (t, J = 1.8 Hz, 2H, H2' + H5'), 4.19 (t, J = 1.8 Hz, 2H, H3' + H4'). Compound 10 (0.68 g, 2.00 mmol, 1.00 equiv), hydroxylamine hydrochloride (181 mg, 2.60 mmol, 1.30 equiv), potassium iodide (0.53 g, 2.00 mmol, 1.00 equiv) and zinc oxide (0.16 mg, 2.00 mmol, 1.00 equiv) were introduced in a Schlenk tube which was subjected to three cycles of vacuum/argon. Acetonitrile (10.0 mL) was added and the reaction mixture was stirred at 80 °C for 2 h. The reaction mixture was cooled to rt and 5% Na2S2O3 (2.00 mL) was added and stirring was continued for 5 min. The reaction mixture was filtrated and the resulting solids were washed with EtOAc (10 mL). Water (10 mL) was added to the combined filtrates which were extracted with EtOAc (3 x 15 mL). The combined organic layers were washed with brine, dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography, using PET-EtOAc (90:10) to give the title product 13 as a dark red solid (0.61 g, 91%). Mp 39-41 °C. νmax (film)/cm -1 3101, 3087, 2224, 1406, 1381, 1346, 1233, 1023, 865, 845, 812. 1 H NMR (500 MHz, CDCl3): δ (ppm) 4.62 (t, J = 1.9 Hz, 2H, H2 + H5), 4.55 (t, J = 1.9 Hz, 2H, H2' + H5'), 4.39 (t, J = 1.9 Hz, 2H, H3 + H4), 4.34 (t, J = 1.9 Hz, 2H, H3' + H4'). Potassium hydroxide (2.53 g, 45.0 mmol, 6.00 equiv) was added to a solution of compound 10 (2.51 g, 7.50 mmol, 1.00 equiv) in methanol (98.0 mL) at 0 °C. Iodine (5.71 g, 22.5 mmol, 3.00 equiv) was added to the reaction mixture which was warmed to rt and stirred for 1 h. Volatiles were removed under vacuum and HCl (1 M, 30 mL) was added. The reaction mixture was extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with Na2S2O3 (sat. aq., 20 mL), water (10 mL), brine (10 mL), dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography over SiO2, using PET/EtOAc (90:10) to give the title product 14 as an orange solid (2.69 g, 97%).

Mp 75-77 °C. νmax (film)/cm -1 2942, 1706, 1465, 1375, 1344, 1273, 1191, 1139, 1030, 696, 865, 843, 819, 773. 1 H NMR (400 MHz, CDCl3): δ (ppm) 4.78 (t, J = 1.9 Hz, 2H, H2 + H5), 4.41 (t, J = 1.7 Hz, 2H, H2' + H5'), 4.38 (t, J = 1.9 Hz, 2H, H3 + H4), 4.17 (t, J = 1.7 Hz, 2H, H3' + H4'), 3.82 (s, 3H, CH3). NaOH (1.40 g, 35.0 mmol, 5.00 equiv) in H2O (42.0 mL) was added to a solution of compound 14 (2.60 g, 7.00 mmol, 1.00 equiv) in methanol (35.0 mL) at rt and the reaction mixture was heated at 80 °C in a preheated bath for 75 min. The reaction mixture was cooled to rt and methanol was removed under vacuum using a rotary evaporator. The resulting solution was cooled to 0 °C and HCl (35% aq.) was added dropwise until pH 1 was reached. The resulting solid was filtrated and washed with cooled (0 °C) water (2 x 5 mL) and pentane (10 mL). Drying the solid under high vacuum with a P2O5 trap afforded the title product 15 as a dark red solid (2.39 g, 96%).

Mp 153-155 °C. νmax (film)/cm -1 3108, 2857 (br), 2636, 2562, 1667, 1482, 1296, 1166, 1025, 840, 745. 1 H NMR (500 MHz, CDCl3): δ (ppm) 4.86 (s, 2H, H2 + H5), 4.48 (s, 2H, H2' + H5'), 4.47 (s, 2H, H3 + H4), 4.28 (s, 2H, H3' + H4'). Acid proton missing. 13 C NMR (125 MHz, CDCl3): δ (ppm) 176.9 (C=O), 76.5 (C2' + C5'), 75.4 (C3 + C4), 73.2 (C2 + C5), 72.1 (C1), 71.2 (C3' + C4'), 40.3 (C1').

1'-Iodoferrocenoyl azide (16)

Triethylamine (4.32 mL, 3.14 g, 31.0 mmol, 5.00 equiv) was added to a solution of compound 15 (2.20 g, 6.20 mmol, 1.00 equiv) in CH2Cl2 (12.0 mL) at 40 °C. Diphenylphosphoryl azide (1.47 mL, 1.88 g, 6.82 mmol, 1.10 equiv) was added dropwise to the reaction mixture which was then kept at the same temperature for 10 min. The reaction mixture was cooled to rt and HCl (1.0 M aq., 30 mL) was added. The reaction mixture was extracted with Et2O (2 x 30 mL) and the combined organic layers were dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography over SiO2, using pentane/Et2O (90:10) to give the title product 16 as a red solid (2.02 g, 85%).

Mp 102-104 °C. νmax (film)/cm -1 2146, 1673, 1452, 1372, 1261, 1191, 1052, 989, 829, 818. 1 H NMR (500 MHz, CDCl3): δ (ppm) 4.81 (t, J = 1.9 Hz, 2H, H2 + H5), 4.49 (t, J = 1.9 Hz, 2H, H3 + H4), 4.47 (t, J = 1.8 Hz, 2H, H2' + H5'), 4.24 (t, J = 1.8 Hz, 2H, H3' + H4').

1-(tert-Butoxycarbonyl)amino-1'-iodoferrocene (17)

tert-Butanol (2.40 mL, 1.85 g, 25.0 mmol, 5.00 equiv) was added to a solution of compound 16 (1.85 g, 5.00 mmol, 1.00 equiv) in toluene (31.0 mL) at rt and the reaction was heated at 100 °C for 1h in a pre-heated oil bath. The reaction mixture was cooled to rt and volatiles were removed under vacuum to give the crude product. This was purified by column chromatography, using PET-EtOAc (90:10) to give the title product 17 as an orange solid (1.81 g, 85%).

Mp 100-102 °C. νmax (film)/cm -1 3354, 2147, 1697, 1550, 1389, 1366, 1252, 1157, 1073, 1028, 867, 808. 1 H NMR (500 MHz, CDCl3): δ (ppm) 5.86 (br s, 1H, NH), 4.42 (br s, 2H, H2 + H5), 4.38 (s, 2H, H2' + H5'), 4.13 (s, 2H, H3' + H4'), 3.98 (s, 2H, H3 + H4), 1.51 (s, 9H, C(CH3)3). 13 C NMR (125 MHz, CDCl3): δ (ppm) 153.3 (C=O), 97.5 (C1), 80.3 (C(CH3)3), 75.6 (C2' + C5'), 69.7 (C3' + C4'), 67.1 (C3 + C4), 62.9 (C2 + C5), 42.6 (C1'), 28.5 (C(CH3)3).

1'-Iodoferrocenamine hydrochloride (18)

HCl in Et2O (≈ 5.00 M, 24.0 mL, 120 mmol, 40.0 equiv) was added to a solution of compound 17 (1.28 g, 3.00 mmol, 1.00 equiv) in Et2O (30.0 mL) and the reaction mixture was stirred at rt for 2 h. The resulting solid was quickly filtrated and washed with Et2O (2 x 10 mL) and pentane (10 mL) and dried under high vacuum to give the title product 18 as an orange solid (800 mg, 74%).

1-(Dimethylamino)-1'-iodoferrocene (19)

Sodium cyanoborohydride (754 mg, 12.0 mmol, 6.00 equiv) was added portionwise to a solution of compound 18 (727 mg, 2.00 mmol, 1.00 equiv) and paraformaldehyde (720 mg, 24.0 mmol, 12.0 equiv) in glacial acetic acid (6.00 mL) at rt. The reaction mixture was stirred for 4 h before paraformaldehyde (120 mg, 4.00 mmol, 2.00 equiv) and sodium cyanoborohydride (120 mg, 2.00 mmol, 1.00 equiv) were added to the reaction mixture which was stirred at rt overnight. EtOAc (20 mL) was added, followed by solid K2CO3 until pH 8 was reached. The layers were separated and the aqueous layer was extracted with EtOAc (2 x 10 mL). The combined organic layers were dried over MgSO4, filtrated over cotton wool and concentrated under vacuum using a rotary evaporator to give the crude product. This was purified by column chromatography over SiO2, using PET/EtOAc (80:20 to 70:30) to give the title product 19 as an orange solid (284 mg, 40%).

Mp 52-54 °C. νmax (film)/cm -1 3094, 2922, 1628, 1598, 1487, 1445, 1422, 1367, 1354, 1333, 1293, 1052, 1028, 1008, 814, 792, 726, 700. 1 H NMR (500 MHz, CDCl3): δ (ppm) 4.52 (s, 2H, H2' + H5'), 4.24 (s, 2H, H3' + H4'), 3.97 (s, 2H, H3 + H4), 3.71 (s, 2H, H2 + H5), 2.66 (br s, 6H, 2 x CH3). 13 C NMR (125 MHz, CDCl3): δ (ppm) 117.2 (C1), 73.1 (C2' + C5'), 67.8 (C3' + C4'), 66.9 (C3 + C4), 58.1 (C2 + C5), 42.4 (2 x CH3), 40.6 (C1').

Scheme 3

 3 Scheme 3 Rationalization of the origin of the remote deprotometalation.

Scheme 6

 6 Scheme 6 Functional group manipulation toward the dimethylaminoferrocene derivative 19.

  νmax (film)/cm -1 2961, 2866, 1626, 1442, 1368, 1326, 1274, 1244, 1150, 1034, 830, 809, 756. Mass: 511 [M], 496 [M-CH3]. 1'-Iodo-N,N-diisopropylferrocenecarboxamide (4) [CAS Reg. No. 2407448-99-9]

  C NMR (75 MHz, CDCl3): δ (ppm) 168.5 (C=O), 83.5 (C1), 76.5 (C2'+C5'), 73.1 (C2 + C5), 72.2 (C3 + C4), 71.3 (C3' + C4'), 49.8 (CH), 46.4 (CH), 40.1 (C1'), 21.3 (4 x CH3). Mass: 439 [M], 339 [M-NiPr2].

  C NMR (75 MHz, CDCl3): δ (ppm) 90.4 (C1), 75.1 (C2' + C5'), 72.9 (C2 + C5), 70.6 (C3 + C4), 69.4 (C3' + C4'), 47.7 (2 x CH), 43.4 (CH2), 41.0 (C1'), 21.0 (4 x CH3). Mass: 439 [M], 339 [M-NiPr2].

1 H

 1 NMR (500 MHz, CDCl3): δ (ppm) 4.31 (t, J = 1.6 Hz, 2H, H2 + H5), 4.11 (t, J = 1.6 Hz, 2H, H3 + H4), 4.09 (t, J = 1.7 Hz, 2H, H3' + H4'), 4.04 (t, J = 1.7 Hz, 2H, H2' + H5'), 2.01 (s, 3H, CH3).

13 C

 13 NMR (125 MHz, CDCl3): δ (ppm) 85.7 (C1'), 75.2 (C2 + C5), 72.4 (C2' + C5'), 70.3 (C3' + C4'), 69.5 (C3 + C4), 40.9 (C1), 14.2 (CH3). Mass: 326 [M].

1'-Iodoferrocenemethanol ( 8 )

 8 [CAS Reg. No. 224456-53-5]

1 H

 1 NMR (500 MHz, CDCl3): δ (ppm) 4.40-4.39 (m, 4H, H2' + H5' + CH2), 4.19 (s, 4H, H2 + H3 + H4 + H5), 4.16 (t, J = 1.8 Hz, 2H, H3' + H4'), 1.78 (br s, 1H, OH).

13 C

 13 NMR (125 MHz, CDCl3): δ (ppm) 89.6 (C1), 75.0 (C2' + C5'), 71.4 (C2 + C5 or C3 + C4), 70.9 (C2 + C5 or C3 + C4), 69.3 (C3' + C4'), 60.3 (CH2), 40.1 (C1'). Mass: 342 [M], 264.

13 C

 13 NMR (125 MHz, CDCl3): δ (ppm) 84.8 (C1'), 75.1 (C2 + C5), 72.4 (C2' + C5' or C3' + C4'), 71.7 (C2' + C5' or C3' + C4'), 70.2 (CH2), 69.4 (C3 + C4), 57.9 (CH3), 40.1 (C1). Mass: 356 [M].

1'-Iodoferrocenecarboxaldehyde ( 10 )

 10 [CAS Reg. No. 176100-20-2]

13 C

 13 NMR (125 MHz, CDCl3): δ (ppm) 193.4 (CHO), 80.6 (C1), 76.4 (C3 + C4), 76.3 (C2' + C5'), 72.4 (C2 + C5), 70.6 (C3' + C4'), 39.4 (C1').

1 H

 1 NMR (500 MHz, CDCl3): δ (ppm) 7.40-7.28 (m, 2H, H2'' + H6''), 7.35-7.32 (m, 2H, H3'' + H5''), 7.28-7.26 (m, 1H, H4''), 5.59 (d, J = 3.2 Hz, 1H, CH), 4.46 (m, 1H, H2' or H5'), 4.45 (m, 1H, H2' or H5'), 4.27 (dd, J = 1.8, 3.3 Hz, 1H, H2 or H5), 4.21 (m, 2H, H3 + H4), 4.19 (m, 2H, H3' + H4'), 4.14 (dd, J = 1.8, 3.3 Hz, 1H; H2 or H5), 2.44 (d, J = 3.3 Hz, 1H, OH).

1 H

 1 NMR (500 MHz, DMSO-d6): δ (ppm) 7.38 (d, J = 7.7 Hz, 2H, H2'' + H6''), 7.31 (t, J = 7.7 Hz, 2H, H3'' + H5''), 7.22 (t, J = 7.7 Hz, 1H, H4''), 5.50-5.48 (m, 2H, OH + CH), 4.44 (m, 1H, FcCH), 4.40 (m, 1H, FcCH), 4.27 (m, 1H, FcCH), 4.18 (m, 2H, FcCH), 4.08 (m, 1H, FcCH), 4.06 (m, 1H, FcCH), 3.94 (m, 1H, FcCH).

13 C

 13 NMR (125 MHz, CDCl3): δ (ppm) 143.4 (C1''), 128.4 (C3'' + C5''), 127.7 (C4''), 126.4 (C2'' + C6''), 95.2 (C1), 75.3 (C2' or C5'), 75.2 (C2' or C5'), 71.9 (CH), 71.5 (C3 or C4), 71.3 (C3 or C4), 70.3 (C2 or C5), 69.6 (C3' or C4'), 69.5 (C3' or C4'), 68.9 (C2 or C5), 40.1 (C1').1-Benzoyl-1'-iodoferrocene (12)[CAS Reg. No. 1884149-19-2]

13 C

 13 NMR (125 MHz, CDCl3): δ (ppm) 198.2 (C=O), 139.6 (C1''), 131.8 (C4''), 128.4 (C3'' + C5''), 128.3 (C2'' + C6''), 79.7 (C1), 76.6 (C2' + C5'), 76.1 (C3 + C4), 74.0 (C2 + C5), 71.4 (C3' + C4'), 40.0 (C1').

1 '

 1 -Iodoferrocenecarbonitrile (13) [CAS Reg. No. 32876-20-3]

  13 C NMR (125 MHz, CDCl3): δ (ppm) 119.2 (C≡N), 77.0 (C2' + C5'), 74.4 (C2 + C5), 74.2 (C3 + C4), 71.7 (C3' + C4'), 54.1 (C1), 39.8 (C1').Mass: 337 [M], 210 [M-I], 183.Methyl 1'-iodoferrocenecarboxylate(14) [CAS Reg. No. 31869-23-5]

  13 C NMR (100 MHz, CDCl3): δ (ppm) 171.0 (C=O), 76.2 (C2' + C5'), 74.4 (C3 + C4=, 73.5 (C1), 72.7 (C2 + C5), 70.7 (C3' + C4'), 51.8 (CH3), 40.4 (C1'). Mass: 370 [M].

1 '-

 1 Iodoferrocenecarboxylic acid (15) [CAS Reg. No. 31832-98-1]

13 C

 13 NMR (125 MHz, CDCl3): δ (ppm) 176.0 (C=O), 76.6 (C2' + C5'), 75.7 (C3 + C4), 74.5 (C1), 72.8 (C2 + C5), 71.1 (C3' + C4'), 40.69 (C1').

  Mp 146-148 °C. νmax (film)/cm -1 2802, 2601, 2569, 1672, 1608, 1521, 1469, 1375, 1143, 1023, 855, 833, 818. 1 H NMR (500 MHz, CDCl3): δ (ppm) 9.84 (br s, 3H, NH3 + ), 4.61 (s, 2H, H2' + H5'), 4.43 (s, 2H, H2 + H5), 4.39 (s, 2H, H3' + H4'), 4.13 (s, 2H, H3 + H4).

  13 C NMR (125 MHz, CDCl3): δ (ppm) 89.2 (C1), 75.7 (C2' + C5'), 71.1 (C3' + C4'), 70.1 (C3 + C4), 65.7 (C2 + C5), 39.0 (C1').

  CCDC 2004798. H NMR (300 MHz, CDCl3): δ (ppm) 4.34 (dd, J = 1.2, 2.3 Hz, 1H, H5), 4.29 (t, J = 2.3 Hz, 1H, H4), 4.27 (s, 5H, Cp), 4.13 (dd, J = 1.2, 2.3 Hz, 1H, H3), 4.11 (br s, 1H, CH), 3.39 (br s, 1H, CH), 1.45 (br s, 6H, 2 x CH3), 1.11 (br s, 6H, 2

	Mp 108-110 °C.
	νmax (film)/cm -1 2966, 1628, 1441, 1368, 1328, 1278, 1243, 1148, 1035,
	833, 807, 753.

1

  -1 3087, 2918, 1476, 1453, 1403, 1382, 1368, 1342, 1227, 1144, 1039, 1022, 860, 822, 804.

  -1 3089, 2920, 2887, 2813, 1447, 1402, 1379, 1344, 1234, 1187, 1175, 1085, 1038, 1021, 899, 862, 824, 809. H NMR (500 MHz, CDCl3): δ (ppm) 4.36 (s, 2H, H2 + H5), 4.25 (s, 2H, CH2), 4.19 (s, 4H, H2' + H3' + H4' + H5'), 4.13 (s, 2H, H3 + H4), 3.34 (s, 3H, CH3).

1

H NMR (500 MHz, CDCl3): δ (ppm) 4.51 (s, 1H, FcH), 4.40 (s, 1H, FcH), 4.38 (s, 1H, FcH), 4.29 (s, 1H, H5), 4.28 (s, 1H, FcH), 4.25 (s, 1H, H4), 4.08 (s, 1H, H3), 3.98 (br s, 1H, CH), 3.38 (br s, 1H, CH), 1.46 (br s, 6H,

x CH3), 1.07 (br s, 6H, 2 x CH3), 0.26 (s, 9H, Si(CH3)3). 13 C NMR (126 MHz, CDCl3): δ (ppm) 168.2 (C=O), 93.5 (C1), 77.4 (C3), 76.7 (FcCH), 75.6 (C2), 75.3 (FcCH), 73.9 (C4), 73.6 (C5), 72.1 (FcCH), 71.6 (FcCH), 50.3 (CH), 45.8 (CH), 40.4 (C1'), 21.2 (2 x CH3), 20.8 (2 x CH3), 0.7 (Si(CH3)3).

Acknowledgment

The authors would like to acknowledge Région Bretagne for a postdoctoral fellowship grant (L. K.). This work was supported by the Université de Rennes 1 and CNRS. We gratefully acknowledge the Fonds Européen de Développement Régional (FEDER; D8 VENTURE Bruker AXS diffractometer) and Thermofisher (generous gift of 2,2,6,6tetramethylpiperidine). This research has been performed as part of the Indo-French 'Joint Laboratory for Natural Products and Synthesis towards Affordable Health'. The authors would like to acknowledge Mehdi Tazi for growing crystals of compound rac-2.


F Levy

Karine Badonnel

A Bertin

F Cornilleau

D Durieux

M Meurisse

Raymond Nowak

C Parias

M A Persuy

Christine Baly

F Lévy

Artificial milk preference of newborn lambs is prenatally influenced by transfer of the flavor from the maternal diet to the amniotic fluid

Keywords: Prenatal environment, Olfaction, Flavor, Sheep, Artificial rearing, GC-MS analysis

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In vertebrates, the capacity of fetuses to memorize sensory information from the prenatal environment is widespread and thought to pre-adapt the future neonate to its postnatal environment [START_REF] Bertin | In Ovo Olfactory Experience Influences Post-hatch Feeding Behaviour in Young Chickens[END_REF][START_REF] Wells | Prenatal olfactory learning in the domestic dog[END_REF][START_REF] Hepper | Prenatal and Early Sucking Influences on Dietary Preference in Newborn, Weaning, and Young Adult Cats[END_REF][START_REF] Schaal | Olfactory preferences in newborn lambs: Possible influence of prenatal experience[END_REF][START_REF] Coureaud | Transnatal olfactory continuity in the rabbit: behavioral evidence and short-term consequence of its disruption[END_REF][START_REF] Gruest | Emergence of long-term memory for conditioned aversion in the rat fetus[END_REF]. Particularly, maternally-derived flavor components promote locomotion, suckling, and dietary preferences at birth or later. Commonly, neonates show a positive attraction towards chemosensory (olfactory/gustatory) compounds present in the maternal environment and this adaptive mechanism drives neonates towards presupposed safe chemosensory cues.

In mammals, amniotic fluid (AF) plays an important role in the development of chemosensory preferences. For instance, mouse pups [START_REF] Logan | Learned recognition of maternal signature odors mediates the first suckling episode in mice[END_REF][START_REF] Kodama | Preference for amniotic fluid in newborn mice[END_REF], rat pups [START_REF] Teicher | First suckling response of the newborn albino rat: the roles of olfaction and amniotic fluid[END_REF][START_REF] Mendez-Gallardo | Odor-induced crawling locomotion in the newborn rat: Effects of amniotic fluid and milk[END_REF], piglets [START_REF] Parfet | Attraction of newborn piglets to auditory, visual, olfactory and tactile stimuli[END_REF] and lambs [START_REF] Schaal | Olfactory preferences in newborn lambs: Possible influence of prenatal experience[END_REF] show an attraction to the odor of AF at birth. In rodents, attraction to the nest area, the mother and initiation of suckling can be driven by volatile olfactory cues released from the AF [START_REF] Logan | Learned recognition of maternal signature odors mediates the first suckling episode in mice[END_REF]. The odor of milk also has highly powerful effects on the newborn of rodents, lagomorphs and humans, inducing searching and grasping the nipples [for review : 12]. These two perinatal substrates share some identical olfactory information with similar motivational characteristics leading to the notion of transnatal chemosensory continuity that appears evolutionary conserved in various mammals [START_REF] Coureaud | Transnatal olfactory continuity in the rabbit: behavioral evidence and short-term consequence of its disruption[END_REF][START_REF] L'olfaction | Organes des sens l'olfaction[END_REF][START_REF] Schaal | Olfaction in utero: Can the rodent model be generalized?[END_REF]. As a result, rabbit [START_REF] Coureaud | Transnatal olfactory continuity in the rabbit: behavioral evidence and short-term consequence of its disruption[END_REF], porcine [START_REF] Rohde Parfet | Attraction on newborn piglets to auditory, visual, olfactory and tactile stimuli[END_REF] and human infants [START_REF] Marlier | Neonatal responsiveness to the odor of amniotic and lacteal fluids: a test of perinatal chemosensory continuity[END_REF] fail to show a preference between odors of AF and colostrum at birth. Further evidence of transnatal chemosensory continuity is supported by the ability of newborns to prefer milk containing odorants that resemble those transferred from the maternal diet to the AF during gestation. Addition of artificial odors to the maternal diet could alter the odor of AF and milk in various species, eliciting subtle modifications of the maternal chemical signature that are perceived by newborns. These initially irrelevant odors once experienced in utero initiate suckling at birth [START_REF] Logan | Learned recognition of maternal signature odors mediates the first suckling episode in mice[END_REF] and trigger behavioral attraction [2, 17-Physiology & Behavior 227 (2020) 113166 -https://doi.org/10.1016/j.physbeh.2020. 1131 20]. For instance, newborn rabbits born to females fed with a cumin-added diet during pregnancy are attracted to colostrum from cumin-fed females [START_REF] Coureaud | Transnatal olfactory continuity in the rabbit: behavioral evidence and short-term consequence of its disruption[END_REF].

In sheep, data on the early postnatal period are scarce. It has been shown that newborns display preference for AF paired with citral after prenatal exposure to this odorant, but that the behavioral response induced in lambs by maternal aromas appears to be weak [START_REF] Schaal | Olfactory preferences in newborn lambs: Possible influence of prenatal experience[END_REF] relatively to the behavioral effect of prenatal odorant exposure in the rat [START_REF] Pedersen | Prenatal and postnatal determinants of the 1st suckling episode in albino rats[END_REF]. Some studies investigating the role of flavor prenatal exposure in feeding preferences after weaning reported a decreased [START_REF] Villalba | Does experience with sagebrush in utero and early in life influence use of sagebrush by sheep?[END_REF] or an increased preference [START_REF] Simitzis | Feeding preferences in lambs influenced by prenatal flavour exposure[END_REF] for that flavor in solid feeds. However, no data is available concerning the impact of prenatal flavor exposure on the short-term preference for milk containing that flavor or some of its components, as it was reported for rabbit pups [START_REF] Coureaud | Transnatal olfactory continuity in the rabbit: behavioral evidence and short-term consequence of its disruption[END_REF]. Because lambs belong to precocial species with fully functional sensory systems, it could be thought that they are less sensitive to prenatal chemosensory learning compared to altricial neonates (most rodents, lagomorphs, carnivores) which only rely on somesthesis and chemoreception. In addition, in dairy-farm systems, or when ewe's milk is not available in sufficient amount or of poor sanitary conditions, newborns are separated from their mothers after birth and fed a milk replacer from a feeder. However, they are not able to suck on their own and training is time-consuming [START_REF] Belanche | Short-and long-term effects of conventional and artificial rearing strategies on the health and performance of growing lambs[END_REF]. One explanation for the unwillingness to drink artificial milk could be found in the chemosensory characteristics of the substitute as it is not made of ewe's milk, breaking the transnatal continuity. A more rapid acceptance of the artificial teat is reported when lambs were fed ewes milk instead of milk replacer [START_REF] Sevi | The effect of type of milk on sucking response of artificially reared lambs[END_REF]. Thus, testing the existence of prenatal chemosensory learning through a flavored diet given to the pregnant mothers may facilitate adaptation of lambs to artificial feeding, should the same flavors be added to the artificial milk. If this is the case, such methodology could then be adapted to suit farming conditions. To this end, cumin was added as the source of flavors to the maternal diet in the last month of gestation. Cumin was chosen as a source of flavors because a previous report, as well as our pilot study, showed that it was easily and readily accepted by ewes [START_REF] Desage | Gas chromatographic-mass spectrometric method to characterise the transfer of dietary odorous compounds into plasma and milk[END_REF]. Some of the odorant compounds, p-cymene, β-pinene, γ-terpinene and cuminaldehyde are transferred to milk [START_REF] Desage | Gas chromatographic-mass spectrometric method to characterise the transfer of dietary odorous compounds into plasma and milk[END_REF]. It is also devoid of any fetal toxicity in sheep [START_REF] Schaal | Transfer of the aromas of the pregnant and lactating mothers diet to the fetal and neonatal environments in the sheep[END_REF] as reported in humans [START_REF] Hauser | Peculiar odours in newborns and maternal prenatal ingestion of spicy food[END_REF] and rabbits [START_REF] Coureaud | Transnatal olfactory continuity in the rabbit: behavioral evidence and short-term consequence of its disruption[END_REF]. At birth, preference of lambs born to odorized and non-odorized mothers was tested using a p-cymene flavored milk. Indeed, p-cymene is one of the two chemosensory compounds of cumin transferred into milk in quantifiable amounts, and the only one to be detected in both maternal plasma and milk [START_REF] Desage | Gas chromatographic-mass spectrometric method to characterise the transfer of dietary odorous compounds into plasma and milk[END_REF]. However, as the presence of this compound in AF is uncertain [START_REF] Schaal | Transfer of the aromas of the pregnant and lactating mothers diet to the fetal and neonatal environments in the sheep[END_REF], AF was also collected from cumin-fed and control mothers and its aromatic profile was analyzed by gas chromatography/tandem mass spectrometry (GC-MS/MS), which provides a high separation capability, sensitivity and specificity. According to the literature, our hypothesis was that the p-cymene compound would be transferred from the maternal diet to AF and, that lambs born from the cumin-fed ewes would exhibit an attraction towards p-cymene-flavored artificial milk, compared to an unflavored milk.

Material and methods

Animals, housing and flavor exposure

Experiments were conducted on 22 multiparous Ile de France ewes (3 years-old) from the INRAE experimental unit UEPAO Val de Loire (Indre et Loire, France). Animals were permanently housed indoors, with free access to water and were fed daily 600 g of concentrated pelleted feed mainly composed of wheat (60%), sunflower meal, dehydrated alfalfa granules, maize, molasses, 1% mineral-vitamin complement (Axereal Elevage, St Germain de Salles, France) and straw. Reproduction was fully synchronized by using vaginal sponges impregnated with fluorogestone acetate for 14 days followed by an intra-muscular injection of pregnant-mare-stimulating gonadotropins to induce ovulation.

Thirty-seven days before parturition, all ewes were acclimated to their housing conditions (4x2 m individual pens) and fed with similar troughs so that ingestion of the dietary rations could be measured. During the last 30 days of gestation, 9 out of the 22 ewes randomly chosen, received a similar daily diet added with 250 g of cumin seeds (African ecotype, purchased at La Bovida, Paris, France). Each of the 9 ewes consumed 80% of their food during the first 4 days and 100% of the ration the following 26 days. The cumin-fed ewes were housed in a different room from that of the control ewes (n=13) to avoid any odorant cross-contamination.

Parturition was induced by an injection of 16 mg of dexamethasone (Dexadreson, Intervet France) on day 144 of gestation. Thus, lambing occurred within a period of 24-36 h, starting 36h after dexamethasone injection. Fifteen lambs from the 9 cumin-fed ewes composed the Cumin group (4 single, 8 twin and 3 triplet lambs).

Fifteen lambs from the 13 ewes fed with the unflavored diet composed the Control group (11 single and 4 twin lambs). Each group consisted of 7 females and 8 males.

No difference in body weight at birth was detected between groups (Control group: 4.11±0.7 kg; Cumin group: 3.69±0.9 kg, p=0.2, t test).

Immediately after expulsion, lambs were separated from their mother to avoid any postnatal contact with some cumin compounds via suckling. They were dried, eartagged, weighed and assigned to a straw-bedded pen (1x2m), 3 meters apart so that any visual or direct contact between the two groups was possible. Two hours after birth, lambs were bottle-fed twice with 100 ml of bovine colostrum (from our local experimental farm) on their first day of life to ensure passive immunological protection.

Bovine colostrum might contain p-cymene [START_REF] Coppa | Effect of a Hay-Based Diet or Different Upland Grazing Systems on Milk Volatile Compounds[END_REF][START_REF] Tornambe | Changes in terpene content in milk from pasture-fed cows[END_REF] and one cannot exclude that lambs of both groups have been exposed to this compound 2 hours after birth. However, pcymene is specifically found in milk of cows grazing natural diversified upland pasture, which was not the case for the cows reared in our research center.

Behavioral tests

Eight hours after birth, testing for milk preference started. Each lamb was transferred to an adjacent pen in which 2 rubber teats, 10 cm apart, fitted on a partition of the pen were connected to two graduated cylinders. One experimenter filled both cylinders with 300 ml of milk replacer (23% protein, 25% fat, 8% ash, 3.5% water, 0.15% cellulose, 2.1% minerals, 200 g/L freshly prepared, 30°C; Agnodor Tradition plus, Soreal Nutrition Animale, Vonnas, France), one being flavored with 10 -6 M pcymene. This concentration was chosen from a GC-MS/MS pilot analysis of AF from five cumin-fed ewes (not shown). P-cymene at 10 -6 M could not be detected by an experimenter (not shown). At that age, lambs were not yet able to feed on their own and milk preference was determined as follows. Another experimenter, different from the one preparing the flavored or unflavored artificial milk, gently introduced one teat into the lamb's mouth, let it suck for 20 s and proceeded similarly with the other teat for 20 s. If the lamb rejected the teat, the experimenter reintroduced the teat in the lamb's mouth. The experimenter was not aware of which teat delivered the flavored or unflavored artificial milk and could not detected any odor difference. This procedure was performed 3 times so that a maximum of 60 s of suckling per teat was achieved.

The experimenter who had prepared the unflavored and flavored milk recorded the volumes consumed during the three repetitions of the procedure by measuring the leftovers in each cylinder. He was different from the experimenter holding animals, but

Physiology & Behavior 227 (2020) 113166 -https://doi.org/10.1016/j.physbeh.2020.1131 was the same who recorded the volume of unflavored and flavored milk consumed during the three repetitions of the procedure. A session consisted of the three repetitions for each teat that delivered the flavored or unflavored milk. This session was repeated 4 times at 6-hour intervals, at 8, 14, 20, 26 hours of age. To control for side biases, the position of the teats was randomly assigned for the first session, then switched between two sessions. At the start of each session, milk replacement was freshly prepared at constant concentration (200 g/L) and temperature (30°C). To avoid cross-contamination, a plastic cylinder was designated to be filled with the flavored milk or with the unflavored milk. In addition, before each session both cylinders were washed with water.

Preference for the flavored milk on day 1 was calculated within each group as the total volume of flavored milk consumed during the four sessions divided by the total volume of both flavored and unflavored milk consumed.

At 36 hours of age (day 2), because lambs were able to feed on their own, they were allowed to choose to suck freely between the two rubber teats for 3 min (choice test). Before the start of the choice test, the experimenter held the lamb and made it sniff both teats for 5 s and then positioned the lamb on the midline between the teats.

The volume of unflavored and flavored milk consumed was recorded at the end of the test. This choice test was performed 4 times at 6-hr intervals, at 36, 42, 48, 54 hours of age. Localization of the flavored milk was randomly assigned at the first choice test, and then systematically switched between two subsequent tests by the experimenter who had prepared both liquid feeds. Preference for the flavored milk on day 2 was calculated within each experimental group as the total volume of that milk consumed during the four choice tests divided by the total volume of both flavored and unflavored milk consumed. During the whole testing procedure lambs did not show any sign of fear toward the experimenter and they were highly motivated to suck. To avoid any odor contamination, Control and Cumin groups were tested in different rooms.

Consequently, the experimenter who handled the lambs was aware of the allocation of the lambs to a group.

Sampling procedure and GC-MS/MS analyses

To evaluate the presence of volatile compounds at birth, AF samples from Control group (n= 3) and Cumin group (n=11) lambs were collected from their coat at birth in glass vials and stored at -20°C until analysis.

For calibration purpose and identification of the main volatile components of cumin, a standard made of cumin essential oil (5 µl of a 10 -5 diluted essential oil of Cuminum cyminum; AROMA-ZONE, France) was extracted by HeadSpace Solid-Phase Microextraction (HS-SPME) using a 2 cm DVB/CAR/PDMS fiber. It was incubated at 60°C for 30 min (agitation cycle 10 sec on /10 sec off) and the fiber (Triplus with SPME module; Thermo Scientific) was introduced after 15 min of incubation. The fiber was then placed into the injector port of the GC (trace 1300, Thermo Scientific) for 1 min at 250°C in splitless mode (held 0.5 min; 1.2 ml/min). Separation of the volatile components was performed on a DB-FFAP column (60 mx0.32 mm i.d., 1 mm film thickness; Agilent Technology). Helium was used as the carrier gas at a constant flow of 1.2 ml/min. The volatiles components were desorbed using a GC oven temperature program starting at 40°C, ramping to 240°C at a rate of 10°C x min -1 , with a final step at 240°C for 15 min. The mass spectrometer (TSQ 8000, Thermo Scientific) was operated in the electron impact (EI) ionization positive mode (70 eV). The temperatures of the transfer line and the ion source were set at 250°C and 260°C, respectively. Argon was used as collision gas. The parent ion characteristic (mass to charge ratio, m/z) of Physiology & Behavior 227 (2020) 113166 -https://doi.org/10.1016/j.physbeh.2020.1131 each of the eight prominent volatile compounds was split into three specific product ions peaks as given (Table S1).

Then, GC-MS/MS analyses were carried out on crude extracts of AF in the same conditions. The abundance of each of the eight main compounds was calculated by summing areas under the curves (AUC) of the three product ions peaks in each AF sample. The relative abundance of each compound in individual AF for each lamb numbered from 1 to 2 (twins) or 3 (triplets) born to each mother (M) was calculated as the percentage of each AUC relative to the sum of AUC of all compounds in the same AF sample.

Statistical analysis

Due to lack of normality, preference for the flavored milk was analyzed with nonparametric tests. Inter-group comparisons were analyzed using two-tailed Mann-Whitney tests, and intra-group comparisons were analyzed using Wilcoxon tests. The preference was also compared to chance (0.5) using Wilcoxon tests. Statistical analyses were performed using the statistical package Statistica 10 and the level of statistical significance was set at p≤0.05. All behavioral data are represented as median and interquartile ranges. (N° CE19 -2015-01). All efforts were made to minimize the number of animals (15 animals per group).

Results

GC-MS/MS identified p-cymene as one of the major compounds detectable in AF from flavored ewes

The GC-MS/MS analyses of AF of randomly selected Cumin lambs (n=11) and Control lambs (n=3) at birth was focused on the eight prominent volatile compounds of Cuminum Cyminum (Fig. 1). Individual variations were noticed in AF of Cumin lambs, but none of these compounds was found in detectable levels in AF of Control lambs.

While -pinene, limonene, cuminaldehyde and 2-caren-10-al were almost undetectable in all AF samples, -pinene, -terpinene, p-cymene and p-cymenene were detected above the basal noise in AF of Cumin lambs. pinene, p-cymene and -terpinene were the most abundant compounds, although their abundance displays large variations among AF samples. When expressed as a percentage of total compounds, the relative proportion of seven of the cumin compounds in AF was roughly similar to that we found in Cuminum cyminum essential oil (Table 1). Only the cuminaldehyde that represents 24% of the eight selected volatile compounds in cumin essential oil was almost undetectable in all AF. Large qualitative variations were observed in the aromatic profile between AF of lambs from siblings (Table 1).

Newborn cumin-enriched lambs exhibit preference for flavored milk

Analysis of the preference for the flavored milk, test by test, did not reveal any significant difference between both groups or within a group across time either on day 1 or on day 2 (data not shown, p>0.05 for the eight comparisons). However, when pooling the four sessions on day 1, preference for the flavored milk was significantly higher in the Cumin than in the Control group (Z=2.53, p=0.009, Fig. 2). The total volume of flavored and unflavored milk ingested during the four sessions did not significantly differ between both groups (Control vs Cumin: 345 ml [285-415] vs 300 ml [220-370], Z=1.36, p=0.17). Intra-group comparisons revealed that the preference for the flavored milk in the Control group was significantly lower than by chance (Z=2.83, p=0.004), whereas it did not differ from chance in the Cumin group (Z=0.9, p=0.36).

On day 2, preference for the flavored milk was significantly higher in the Cumin than in the Control group (Z=2.32, p=0.018, Fig. 2). The total quantity of flavored and unflavored milk ingested during the four choice tests did not significantly differ between both groups (Control vs Cumin: 500 ml [320-820] vs 425 ml [235-560], Z=1.24, p=0.21).

Intra-group comparisons revealed that the preference for the flavored milk in the Cumin group was significantly higher than by chance (Z=2.83, p=0.004) whereas it did not differ from chance in the Control group (Z=0.11, p=0.9).

No statistical difference in the preference for the flavored milk was observed between day 1 and day 2 in the Control group (Z=1.36, p=0.17), while the preference increased on day 2 in the Cumin group (Z=1.98, p=0.04). The total volume of flavored and unflavored milk ingested during the two days of testing did not significantly differ between both groups (Control vs Cumin: 980 ml [605-1260] vs 755 ml [620-900], Z=1.24, p=0.21). There were no significant differences in the preference for the flavored milk between males and females either at day 1 (Z=1.22, p=0.2) or at day 2 (Z=0. 5, p=0.5) or between single lambs and twin/triplet lambs either at day 1 (Z=1.41, p=0.16) or at day 2 (Z=0.5, p=0.5).

Discussion

The study shows that prenatal experience with complex flavors influences behavioral feeding-related responses to one of its components when added to artificial milk. Specifically, this is the first evidence in lambs that a chemosensory compound, p-cymene, is transmitted from the mother's diet to AF and then drives a preference for it when added in artificial milk within 2 days after birth.

Cumin was chosen as a source of flavors because it was easily and readily accepted by ewes [START_REF] Desage | Gas chromatographic-mass spectrometric method to characterise the transfer of dietary odorous compounds into plasma and milk[END_REF] and devoid of any fetal toxicity [START_REF] Schaal | Transfer of the aromas of the pregnant and lactating mothers diet to the fetal and neonatal environments in the sheep[END_REF]. We extend here the observation that a longer period of cumin consumption affected neither diet consumption of mothers nor fetal growth of lambs. Another characteristic of cumin is that, some of its flavor components were already shown to be transferred into milk and plasma in ewes after a single intake of cumin seeds [START_REF] Desage | Gas chromatographic-mass spectrometric method to characterise the transfer of dietary odorous compounds into plasma and milk[END_REF]. Mainly p-cymene, β-pinene, and to a lesser extent γ-terpinene and cuminaldehyde, were identified in milk and, only p-cymene was detected in plasma. The present study clearly shows that p-cymene is also transferred into AF. This compound, together with p-cymenene, β-pinene and γterpinene was found in almost all samples of AF of cumin-fed lambs in quantifiable amounts, albeit remaining low for γ-terpinene. The fact that these compounds were not detectable in AF in the study by Schaal et al. [START_REF] Schaal | Transfer of the aromas of the pregnant and lactating mothers diet to the fetal and neonatal environments in the sheep[END_REF] can be explained by a difference in sample preparation and extraction, in the use of a tandem mass spectrometry procedure, 10 times more sensitive than a single MS, or in a longer time exposure window to cumin diet during gestation. However, in both studies, limonene and cuminaldehyde remained undetectable, indicating that some components of cumin aromas cannot be transferred into the fetal compartment and are probably metabolized. Such qualitative variations in the efficiency of transfer of individual aromatic compounds, in or between samples have been described in AF [START_REF] Nolte | Garlic in the ovine fetal environment[END_REF] and lacteal matrix for other compounds [START_REF] Hausner | Differential transfer of dietary flavour compounds into human breast milk[END_REF][START_REF] Kirsch | Time-dependent aroma changes in breast milk after oral intake of a pharmacological preparation containing 1,8-cineole[END_REF][START_REF] Kirsch | Characterisation of the metabolites of 1,8-cineole transferred into human milk: concentrations and ratio of enantiomers[END_REF][START_REF] Scheffler | Detection of Volatile Metabolites of Garlic in Human Breast Milk[END_REF]. A chemosensory qualitative and/or

Physiology & Behavior 227 (2020) 113166 -https://doi.org/10.1016/j.physbeh.2020.1131 quantitative difference between the maternal diet flavor and the conveyed compounds in AF could participate to equivocal postnatal behavioral responses to compounds that are partly transferred in maternal fluids [START_REF] Schaal | Olfactory preferences in newborn lambs: Possible influence of prenatal experience[END_REF][START_REF] Pedersen | Prenatal and postnatal determinants of the 1st suckling episode in albino rats[END_REF][START_REF] Nehring | Impacts of in utero and early infant taste experiences on later taste acceptance: a systematic review[END_REF].

The postnatal behavioral response associated with the presence of p-cymene in AF indicates that lambs from cumin-fed mothers developed a clear preference for the artificial milk containing this compound within 48 hr. On day 1, the proportion of flavored milk ingested by Control lambs was less than that what was expected by chance, indicating a slight aversion to p-cymene. Cumin lambs, on the other hand, did not show such aversion since the proportion of flavored milk they ingested did not differ from random. This indicates that the two milks were discriminated based on olfactory/gustatory cues by both groups and that the behavioral differences relied on prenatal experience. Behavior of lambs on day 1 is unlikely to result from a difference in the handling procedure during the test. Helping lambs to feed is a mandatory step, as lambs must learn how to suck artificial teats. Since the experimenters were not aware of which teat delivered the flavored or unflavored artificial milk they could not bias the lamb's choice. On day 2, while aversion weakened in Control lambs, the preference for the flavored milk emerged in Cumin lambs, the proportion of flavored milk ingested being higher in comparison to chance. In addition, on both days, this proportion was significantly higher in the Cumin group. It is unlikely that differences in food motivation accounts for these behavioral effects since the total amount of milk ingested during the tests did not differ between both groups. Among other parameters that may have contributed to the observed effect, we cannot exclude that the supplementation in cumin seed has provided a caloric increase beneficial to the mothers. However, such enrichment did not modify lamb's weight by the time of birth nor the total amount of milk ingested.

Physiology & Behavior 227 (2020) 113166 -https://doi.org/10.1016/j.physbeh.2020.1131

Whether the development of a preference for the flavored artificial milk is specific to p-cymene or could expand to other flavors was not the aim of the present study but it would have been interesting to test the preference of Cumin lambs for other odorants or to add another experimental group fed another flavored mother's diet.

Hesitancy to consume unfamiliar foods usually referred as 'food neophobia' is widespread throughout the animal kingdom including sheep [START_REF] Bertin | In Ovo Olfactory Experience Influences Post-hatch Feeding Behaviour in Young Chickens[END_REF][START_REF] Forrest | Pollinator experience, neophobia and the evolution of flowering time[END_REF][START_REF] Aldridge | The role of familiarity in dietary development[END_REF][START_REF] Van Tien | Grass odor and flavor overcome feed neophobia in sheep[END_REF][START_REF] Launchbaugh | Overcoming food neophobia in domestic ruminants through addition of a familiar flavor and repeated exposure to novel foods[END_REF] and p-cymene avoidance in control lambs on day 1 could be explained as such. The lack of avoidance expressed by Cumin lambs indicates that the chemosensory cues had been memorized and were perceived as familiar. Similar attenuation of food neophobia was reported in newborn mice exposed to an aversive flavor during their fetal life [START_REF] Nolte | Maternal ingestion of ortho-aminoacetophenone during gestation affects intake by offspring[END_REF].

On day 1, the behavioral effect induced in Cumin lambs by the presence of pcymene in the artificial milk was unnoticeable since no clear preference was observed, even though the GC-MS/MS analysis indicated that p-cymene is found in quantifiable amount in AF. Likewise, newborn lambs exposed to citral in utero do not express a preference for that odorant relatively to AF odor [START_REF] Schaal | Olfactory preferences in newborn lambs: Possible influence of prenatal experience[END_REF]. It may be possible that the concentration of p-cymene added to the artificial milk was inadequate to induce a preference. The weak effect observed in Cumin lambs on day 1 could also be due to the fact that p-cymene on its own, like citral, is not an appropriate stimulus and that using a mix of compounds might have induced an immediate postnatal preference. Indeed, a blend of compounds representative of the cumin aromas transferable to AF, including -pinene, γ-terpinene, and p-cymenene in addition to p-cymene, as our GC-MS/MS analysis suggested, would have been more efficient to induce a behavioral response.

The slight qualitative variations observed in the aromatic profile between AF of lambs suggest a possible differential impregnation of animals through maternal diet Because lambs were submitted to repetitive sessions on day 1, it is conceivable that the gustatory and post-ingestive effects of the flavored milk could have become more prevalent to establish a preference than its sole chemosensory properties. However, avoidance of p-cymene in Control lambs could not be due to negative post-ingestive consequences since it vanished on day 2 and was never seen in Cumin lambs.

Our findings that p-cymene induced a clear preference on day 2 in Cumin lambs points out the remarkable ability of newborn lambs to detect and memorize a single flavor from the composite aroma of AF, as shown in rabbit pups [START_REF] Coureaud | Transnatal olfactory continuity in the rabbit: behavioral evidence and short-term consequence of its disruption[END_REF], in rat pups [START_REF] Gruest | Emergence of long-term memory for conditioned aversion in the rat fetus[END_REF][START_REF] Sreng | Chemical Identification of "Maternal Signature Odors[END_REF] and in babies [START_REF] Schaal | Human foetuses learn odours from their pregnant mother's diet[END_REF]. The present study supports earlier research reporting that newborn lambs display preference for their own AF compared to AF of unfamiliar ewes [START_REF] Schaal | Olfactory preferences in newborn lambs: Possible influence of prenatal experience[END_REF] despite very similar chromatographic profiles [START_REF] Viviers | Characterization of amniotic fluid of Dohne Merino ewes (Ovis aries) and its possible role in neonatal recognition[END_REF]. Thus, it appears that precocial neonates possess selective perception and learning of the chemosensory properties of their fetal environment, similarly to altricial newborns. This prenatal chemosensory The current study supports the hypothesis that flavor preference displayed at birth by lambs can be shaped by prenatal chemosensory experience through maternal diet. In sheep dairy farming systems, or when mother's milk is not sufficient to feed the whole litter, newborns are separated soon after birth and fed a milk replacer. Training lamb to suck is time-consuming and lamb mortality can be high [START_REF] Bouvier | Rapport technique élevage ovin[END_REF]. Flavor exposure during the last month of gestation could be a beneficial method to adapt artificially reared lambs to their new diet by adding the same flavor to milk replacement. Thus, both the welfare and performance of artificially reared lambs may be improved, but some precautions should be required. In young mammals, graded concentrations of odors can elicit graded responses ranging from attraction to avoidance [START_REF] Coureaud | Newborn rabbit responsiveness to the mammary pheromone is concentration-dependent[END_REF]. Because both trigeminal and olfactory receptors are present within the nasal cavity, adding flavors in the maternal diet may also have negative perceptual consequences.

Installing olfactory continuity between the prenatal and post-natal environmentwith a moderate intensity of chemosensory signalsmay be the best option to help young lambs to orient positively towards artificial milks. Further studies are required to better characterize the most efficient blend of cumin aroma and concentration to facilitate suckling at an automatic feeder.

Table S1: MS parameters and energy transitions monitored for the eight main

Cuminum cyminum volatile compounds analysed by GC-MS/MS. m/z: mass to charge ratio.

  Physiology & Behavior 227 (2020) 113166 -https://doi.org/10.1016/j.physbeh.2020.1131

  Physiology & Behavior 227 (2020) 113166 -https://doi.org/10.1016/j.physbeh.2020.1131

  Physiology & Behavior 227 (2020) 113166 -https://doi.org/10.1016/j.physbeh.2020.1131

2. 5 .

 5 Ethics statement Animal care and experimental treatments complied with the guidelines of the French Ministry of Agriculture for animal experimentation and European regulations on animal experimentation (86/609/EEC). They were performed in accordance with the local animal regulation (authorization N° 006352 of the French Ministry of Agriculture in accordance with EEC directive) and with the ethical committee Val de Loire N°19 Physiology & Behavior 227 (2020) 113166 -https://doi.org/10.1016/j.physbeh.2020.1131

  Physiology & Behavior 227 (2020) 113166 -https://doi.org/10.1016/j.physbeh.2020.1131 that could participate in the diversity of behavioral postnatal responses to p-cymene.However, in absence of internal standards or calibration curve, the GC-MS/MS prevents us to calculate the abundance of each compound. Whether the relative pcymene abundance as measured by GC-MS/MS at birth is directly correlated to the in utero exposure remains to be demonstrated. Indeed, several parameters could have participated in both intra-and inter-variability. Physiologically, the olfactory impregnation depends on the elapsed time between maternal food consumption and AF sampling, on the heterogeneity of transfer between compounds and on the metabolic kinetics. Methodologically, conditions of sample collection (volume, viscosity), elapsed time between collection and analyses, might have also interfered with the GC-MS/MS.

  Physiology & Behavior 227 (2020) 113166 -https://doi.org/10.1016/j.physbeh.2020.1131 experience may play a key role in shaping the development of postnatal feeding behaviors by orienting the newborn towards familiar chemosensory compounds.

Figure 1 :

 1 Figure 1: Abundance of the eight main Cuminum cyminum volatile compounds in

Figure 2 .

 2 Figure 2. Preference for p-cymene flavored artificial milk in lambs born to ewes that

Table 1 :

 1 Relative abundance of each compound in individual amniotic fluid of lambs born to cumin-fed mothers. Heat map showing the relative abundance of each compound in individual amniotic fluid expressed as a percentage of total aromatic compounds. M: mother; Cumin Oil: Cuminum cyminum essential oil, dilution factor 10 -5 .

Figure 2 Physiology&

 2 Figure1

Table 1 suppl

 1

	Compound	retention time (min)	parent ion (m/z)	product ion (m/z) collision energy (eV)
	 -pinene	8.68	92.8	50.7	22.00
			92.8	77.1	12.00
			92.8	91.1	6.00
	-pinene	10.22	93	51	22.00
			93	77.1	10.00
			93	91.1	6.00
	Limonene	11.58	68.2	39.5	18.00
			68.2	53.1	10.00
			68.2	67.1	4.00
	-terpinene	12.33	136.2	77.1	26.00
			136.2	93.1	10.00
			136.2	121	8.00
	p-cymene	12.79	119.1	77.2	20.00
			119.1	91	12.00
			119.1	116.8	8.00
	p-cymenene	15.25	132.2	91.1	24.00
			132.2	115.1	20.00
			132.2	117.1	12.00
	Cuminaldehyde	20.05	148	105.1	16.00
			148	119.1	6.00
			148	133.1	8.00
	2-caren-10-al	20.32	106.9	51	32.00
			106.9	77	18.00
			106.9	79.1	8.00

Physiology & Behavior 227 (2020) 113166 -https://doi.org/10.1016/j.physbeh.2020.1131

Acknowledgements

We particularly thank (1) the staff of the UEPAO of INRAE Centre Nouzilly (https://doi.org/10.15454/1.5573896321728955E12) for animal breeding and daily care; (2) Edith Pajot (INRAE), director of NBO research unit for her constant support; (3) Angélique Favreau-Peigné (INRAE) for fruitful discussions on animal preferences; (4) Isabelle Pellerin (UPMC, Paris VI) for her work on the GC-MS/MS analyses; (5) Melody Morisse for experimental assistance.

Funding

The authors would like to acknowledge the financial support from the PHASE department, INRAE, through an incentive program awarded to C. Baly.

Declaration of Competing Interest

The authors have no financial or non-financial competing interests to declare.