Maxime Julien
email: julien.m.aa@m.titech.ac.jp

Mathilde Liégeois

Patrick Höhener

Piotr Paneth

Gérald S Remaud

Intramolecular non-covalent isotope effects at natural abundance associated with the migration of paracetamol in solid matrices during liquid chromatography

Keywords:

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

Introduction

Liquid chromatography, commonly used to isolatee compounds from a complex mixture, is known to create isotope effects. The non-covalent interactions settled between the solid phase and the substrate during the migration are isotopically selective resulting in isotopic fractionation of the migrating compounds. The link between these interactions and the associated noncovalent isotope effects (NCIEs) remains poorly documented [START_REF] Tanaka | Isotope effects in hydrophobic binding measured by high-pressure liquid chromatography[END_REF][START_REF] Wade | Deuterium isotope effects on noncovalent interactions between molecules[END_REF][START_REF] Turowski | Deuterium Isotope Effects on Hydrophobic Interactions: The Importance of Dispersion Interactions in the Hydrophobic Phase[END_REF]. Understanding the isotopic fractionation processes associated with chromatography is of primary importance for correcting further isotopic measurements when used during the sample preparation and/or the direct coupling of isotope ratio monitoring by Mass Spectrometry (irm-MS, known also as IRMS: Isotope Ratio by Mass Spectrometry) with high-performance liquid chromatography (HPLC) [START_REF] Godin | Current applications and challenges for liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS)[END_REF] or gas chromatography (GC) [START_REF] Meier-Augenstein | Applied gas chromatography coupled to isotope ratio mass spectrometry[END_REF][START_REF] Benson | Forensic applications of isotope ratio mass spectrometry-A review[END_REF]. Moreover, liquid chromatography (and more generally sorption experiments) are used for modeling the migration of organic compounds in environment to determine the impact of this process on considered pollutants isotopic composition and to study their origins and fate [START_REF] Schmidt | Compoundspecific stable isotope analysis of organic contaminants in natural environments: a critical review of the state of the art, prospects, and future challenges[END_REF][START_REF] Elsner | Current challenges in compound-specific stable isotope analysis of environmental organic contaminants[END_REF].

It is difficult to study migration mechanisms and understand their associated isotope fractionations of a pollutant in environment studies, as example. Indeed, this phenomenon involves different processes such as advection, diffusion/dispersion and sorption. Advection refers to the transport of a compound depending on the ground relief or the presence of a stream (air, water) in the considered polluted site. It corresponds to the movement of a contaminant plume in the soil or in water) and does not depend on the chemical composition, so it is considered as inert in terms of isotope fractionation. Diffusion and dispersion define the way a contaminant will spread in the environment and may contribute to isotope fractionation.

However, these fractionations are considered to be small or non-significant in advectiondominated transport situations and for experiments with short durations [START_REF] Chiogna | Evidence of Compound-Dependent Hydrodynamic and Mechanical Transverse Dispersion by Multitracer Laboratory Experiments[END_REF][START_REF] Van Breukelen | Transverse Hydrodynamic Dispersion Effects on Isotope Signals in Groundwater Chlorinated Solvents' Plumes[END_REF][START_REF] Jin | Diffusive Fractionation of BTEX and Chlorinated Ethenes in Aqueous Solution: Quantification of Spatial Isotope Gradients[END_REF]. By contrast, the sorption/desorption processes occurring during the migration directly involve intermolecular non-covalent interactions, which have already been proven to be associated with intramolecular 13 C isotope effects, i.e. position-specific 13 C fractionation [START_REF] Botosoa | Evidence of 13 C non-covalent isotope effects obtained by quantitative 13 C nuclear magnetic resonance spectroscopy at natural abundance during normal phase liquid chromatography[END_REF]. Furthermore, different types of intermolecular interactions (hydrogen bonds, van der Waals, -stacking, halogen bonds) can be observed during migration depending on the molecular structures of both the contaminant and the solid matrix resulting in variable isotope effects.

The isotope fractionations and the calculation of the associated isotopic effects provide information on the intensity of interactions between the solute and the solid matrix. Most isotope studies are performed using irm-MS targeting mostly 13 C and 2 H in organic contaminants [START_REF] Elsner | Current challenges in compound-specific stable isotope analysis of environmental organic contaminants[END_REF][START_REF] Thullner | Quantification of organic pollutant degradation in contaminated aquifers using compound specific stable isotope analysis -Review of recent developments[END_REF] and moreover on enriched material for the chromatography investigations [START_REF] Tanaka | Isotope effects in hydrophobic binding measured by high-pressure liquid chromatography[END_REF][START_REF] Wade | Deuterium isotope effects on noncovalent interactions between molecules[END_REF][START_REF] Turowski | Deuterium Isotope Effects on Hydrophobic Interactions: The Importance of Dispersion Interactions in the Hydrophobic Phase[END_REF]. However, this method only provides a measurement of the average isotopic composition, which is the mean value of isotope ratios of all carbon or hydrogen positions within the considered molecule. Using this method, isotopologues (two molecules with a different number of heavy atoms, see section 3.1 for further definition) that react differently with the considered stationary phase will elute separately [START_REF] Julien | Position-Specific 13 C Fractionation during Liquid-Vapor Transition Correlated to the Strength of Intermolecular Interaction in the Liquid Phase[END_REF]. Fully deuterated compounds can be separated from their protiated counterparts by liquid [START_REF] Turowski | Deuterium Isotope Effects on Hydrophobic Interactions: The Importance of Dispersion Interactions in the Hydrophobic Phase[END_REF] or gas chromatography [START_REF] Morasch | Stable Hydrogen and Carbon Isotope Fractionation during Microbial Toluene Degradation: Mechanistic and Environmental Aspects | Applied and Environmental Microbiology[END_REF], for example. To access the intramolecular 13 C information without prior (bio)chemical degradation of the target compound, a small number of position-specific isotope analysis (PSIA) techniques have been developed such as isotope ratio monitoring by 13 C Nuclear Magnetic Resonance (irm- 13 C NMR) [START_REF] Caytan | Accurate Quantitative 13 C NMR Spectroscopy: Repeatability over Time of Site-Specific 13 C Isotope Ratio Determination[END_REF][START_REF] Gilbert | Intramolecular 13 C pattern in hexoses from autotrophic and heterotrophic C3 plant tissues[END_REF][START_REF] Jézéquel | The new face of isotopic NMR at natural abundance[END_REF], on-line pyrolysis coupled with irm-MS [START_REF] Corso | High-precision position-specific isotope analysis[END_REF][START_REF] Yamada | On-line measurement of intramolecular carbon isotope distribution of acetic acid by continuous-flow isotope ratio mass spectrometry[END_REF][START_REF] Gilbert | Accurate Method for the Determination of Intramolecular 13 C Isotope Composition of Ethanol from Aqueous Solutions[END_REF][START_REF] Gilbert | Measurement of position-specific 13 C isotopic composition of propane at the nanomole level[END_REF] and high-resolution irm-MS [START_REF] Piasecki | Analysis of the site-specific carbon isotope composition of propane by gas source isotope ratio mass spectrometer[END_REF][START_REF] Cesar | Isotope heterogeneity in ethyltoluenes from Australian condensates, and their stable carbon site-specific isotope analysis[END_REF]. All these techniques make it possible to identify which isotopomer (two molecules with the same amount of heavy atom but located on different position) preferentially interacts during the studied process [START_REF] Julien | Position-Specific 13 C Fractionation during Liquid-Vapor Transition Correlated to the Strength of Intermolecular Interaction in the Liquid Phase[END_REF]. PSIA using irm- 13 C/ 15 N NMR has been successfully used to reveal intramolecular NCIE during chromatographic elution on normal phase for 13 C [12,25] and 15 N [26] or inverse phase for 13 C [START_REF] Höhener | Analytical model for site-specific isotope fractionation in 13 C during sorption: Determination by isotopic 13 C NMR spectrometry with vanillin as model compound[END_REF]. Furthermore, this approach was able to found a possible link between the magnitude of fractionation and the strength of intermolecular interactions within a liquid during the liquid-vapor transition as evaporation of VOCs for 13 C [START_REF] Julien | Fractionation in position-specific isotope composition during vaporization of environmental pollutants measured with isotope ratio monitoring by 13 C nuclear magnetic resonance spectrometry[END_REF][START_REF] Julien | Insights into Mechanistic Models for Evaporation of Organic Liquids in the Environment Obtained by Position-Specific Carbon Isotope Analysis[END_REF] or distillation for 2 H and 13 C [START_REF] Julien | Position-Specific 13 C Fractionation during Liquid-Vapor Transition Correlated to the Strength of Intermolecular Interaction in the Liquid Phase[END_REF][START_REF] Zhang | Determination of Multiple Equilibrium Isotopic Fractionation Factors at Natural Abundance in Liquid-Vapor Transitions of Organic Molecules[END_REF].

In the present study, 13 C intramolecular isotope analysis using irm- 13 C NMR was employed to observe the position-specific isotope effects (PSIEs) associated with the chromatographic elution on several stationary phases. The potential connection between the magnitude of fractionation and strength of interaction between organic solute and solid phase is expected to provide insights about how different types of matrices could retain organic chemicals. Isotope fractionation associated with migration of organic contaminants were largely studied using deuterated compounds [START_REF] Tanaka | Isotope effects in hydrophobic binding measured by high-pressure liquid chromatography[END_REF][START_REF] Wade | Deuterium isotope effects on noncovalent interactions between molecules[END_REF][START_REF] Turowski | Deuterium Isotope Effects on Hydrophobic Interactions: The Importance of Dispersion Interactions in the Hydrophobic Phase[END_REF]. However, only few works looked at isotopes at natural abundance and the influence of migration on isotope composition is still debated. As an example, the migration of BTEX (Benzene, Toluene, Ethylbenzene, Xylenes) on reverse phase HPLC did not show any significant IE for 13 C at the global level [START_REF] Harrington | Carbon isotope systematics of monoaromatic hydrocarbons: vaporization and adsorption experiments[END_REF], nor did the migration of BTEX and MTBE on HPLC using humic acid as stationary phase [START_REF] Kopinke | Carbon Isotope Fractionation of Organic Contaminants Due to Retardation on Humic Substances: Implications for Natural Attenuation Studies in Aquifers[END_REF]. Some sorption experiments of BTEX and chlorinated solvents on activated charcoal also showed non-significant IEs for global 13 C and 2 H compositions [START_REF] Schüth | Carbon and hydrogen isotope effects during sorption of organic contaminants on carbonaceous materials[END_REF][START_REF] Slater | Carbon Isotope Effects Resulting from Equilibrium Sorption of Dissolved VOCs[END_REF]. Nevertheless, some other experiments highlighted the presence of 13 C and 2 H isotope fractionation during sorption/desorption cycles of BTEX on different adsorbing materials, still using average isotope parameters [START_REF] Imfeld | Carbon and hydrogen isotope fractionation of benzene and toluene during hydrophobic sorption in multistep batch experiments[END_REF], which encouraged us to undertake further experiments probing PSIA because the small detected IEs may be composed by counteractive normal and inverse IEs within the studied molecules resulting in the measurement of low or non-significant global isotope effects, i.e. using irm- 13 C MS. The concomitance of normal and inverse isotope effects within the same molecule was already detected during the migration of vanillin and derivatives on both normal phase silica gel (SG) and reverse phase (RP) [START_REF] Botosoa | Evidence of 13 C non-covalent isotope effects obtained by quantitative 13 C nuclear magnetic resonance spectroscopy at natural abundance during normal phase liquid chromatography[END_REF][START_REF] Botosoa | Unexpected fractionation in site-specific 13 C isotopic distribution detected by quantitative 13 C NMR at natural abundance[END_REF]. The results of these works were presented as a qualitative description of the 13 C intramolecular fractionations observed, with no enrichment factor calculated. Although, isotope fractionation of vanillin migrating on RP was further studied to develop a reactive transport capable of predicting the position-specific isotopic fractionation behavior of this compound in the environment [START_REF] Höhener | Analytical model for site-specific isotope fractionation in 13 C during sorption: Determination by isotopic 13 C NMR spectrometry with vanillin as model compound[END_REF].

As soil composition is very variable and not homogeneous, we have chosen to study the migration of paracetamol through different pure materials using liquid chromatography and to measure associated intramolecular 13 C and 15 N IEs. Four model materials with various chemical compositions were used as stationary phase in this study: silica gel normal phase (SG), cellulose (CE), activated charcoal (AC) and silica gel C8-reversed phase (RP). The choice of the solute and these solid matrices will be further rationalized in the section 3, as well the role of the eluent that is important in the selection of the isotopomers during the elution [START_REF] Tanaka | Isotope effects in hydrophobic binding measured by high-pressure liquid chromatography[END_REF][START_REF] Wade | Deuterium isotope effects on noncovalent interactions between molecules[END_REF][START_REF] Turowski | Deuterium Isotope Effects on Hydrophobic Interactions: The Importance of Dispersion Interactions in the Hydrophobic Phase[END_REF]. Thus, it is expected that different types of non-covalent intermolecular interactions contribute in the PSIF. The determination of the enrichment factor at each carbon position should help in the evaluation of the strength of these interactions. That is the reason why we have performed these calculations from previous published data of vanillin eluted through normal phase silica gel [START_REF] Botosoa | Evidence of 13 C non-covalent isotope effects obtained by quantitative 13 C nuclear magnetic resonance spectroscopy at natural abundance during normal phase liquid chromatography[END_REF] and reversed phase silica gel [START_REF] Höhener | Analytical model for site-specific isotope fractionation in 13 C during sorption: Determination by isotopic 13 C NMR spectrometry with vanillin as model compound[END_REF] to better highlight the role of non-covalent interactions in intramolecular isotope fractionations occurring during the migration. In addition, quantum mechanical calculations for the transition of paracetamol from diethyl ether (low polarity) to acetone (moderate polarity) or water (polar compound) were performed during this study. These theoretical data should help better understanding the effect of hydrogen-bonding and, more generally, the polarization on measured PSIEs associated with the migration.

Materials and Methods

Chemicals

Paracetamol (C8H9NO2, CAS-Number 103-90-2), silica gel high-purity grade, average pore size 60 Å (52-73 Å), 70-230 mesh, 63-200 μm, silica gel C8-reversed phase, and activated charcoal were purchased from Sigma Aldrich. Cellulose was obtained from Fluka-chemicals and DMSO-d6 was purchased from Eurisotop. Acetone and diethyl ether used as eluent were from Sigma Aldrich as HPLC quality, without further purification.

Chromatographic procedures

In order to determine both 13 C and 15 N isotope fractionation occurring during the migration of paracetamol through different materials, liquid chromatography experiments were performed using 4 different stationary phases (separately): normal phase silica gel (SG), silica gel C8-reversed phase (RP), cellulose (CE) and activated charcoal (AC). For each experiment the amount of material was adapted in order to obtain around 250 mm of stationary phase in a chromatography column (internal diameter 40 mm except for silica gel C8-reversed phase which was 30 mm). Then the protocol was the same for all types of stationary phase: (i) 2 g of pure paracetamol was introduced as a powder at the top of the column and gently mixed with the first 20 mm of the stationary phase to start the migration; (ii) The elution was performed using a mixture of 90% of diethyl ether and 10% acetone, as acetone solubilizes well paracetamol to some extent [START_REF] Granberg | Solubility of Paracetamol in Pure Solvents[END_REF], while it is almost totally non-soluble in ether that is used to decrease the polarity of acetone, avoiding thus, a too rapid elution of paracetamol and (iii) it was collected in 4 fractions (approx. 500 mg each). The exact amount of each fraction was determined by weighting the eluted paracetamol after evaporation of the solvents to dryness and the corresponding percentage of introduced paracetamol was calculated (see Table 2). The starting paracetamol and the 4 samples obtained from elution of each stationary phase chromatography were analyzed for their isotopic parameters. 13 C and 15 N isotope analysis Carbon ( 13 C/ 12 C) and nitrogen ( 15 N/ 14 N) global isotope ratio measurements, expressed in delta notation ( 13 Cg and  15 Ng (‰)), were determined by irm-MS using an Integra2 spectrometer (Sercon Instruments, Crewe, UK) linked to a Sercon elemental analyzer (EA) fitted with an autosampler (Sercon Instruments, Crewe, UK). About 1 mg of sample was sealed in a tin capsule.

Global

 13 Cg and  15 Ng of the resulting gases, CO2 and N2 were determined by reference to a working standard of glutamic acid standardized against calibrated international reference material (IAEA-CH6 and IAEA-CH7 for carbon isotope ratio and IAEA-N1 and IAEA-N2 for nitrogen isotope ratio).

The 13 C and 15 N global isotope compositions of the whole molecule were calculated from:

δ A X (‰) = ( 𝑅 𝑅 𝑆𝑡𝑑 -1) × 1000 eq. 1
Where A X stands for either 13 C or 15 N, R is the isotope ratio of the sample and Rstd is the isotope ratio of Vienna Pee Dee Belemnite reference standard (V-PDB) for 13 C (Rstd = 0.0112372) or atmospheric air for 15 N (Rstd = 0.003677).

Intramolecular 13 C isotope analysis by irm-13 C NMR

PSIA of paracetamol using irm-13 C NMR was described in a previous study [START_REF] Silvestre | Isotopic 13 C NMR spectrometry to assess counterfeiting of active pharmaceutical ingredients: Site-specific 13 C content of aspirin and paracetamol[END_REF]. The sample preparation consisted in the successive addition in a 4 mL vial of 250 mg of paracetamol and 600

L of DMSO-d6. The respective amount of each was adapted according to (i) the T1 values (longitudinal relaxation), (ii) the solubility in the deuterated solvent and (iii) the 13 C NMR spectrum: no peak overlapping (see Figure S3). Irm- 13 C NMR spectra were recorded using an AVANCE I 400 spectrometer (Bruker Biospin, Wissembourg, France), fitted with a 5 mm i.d. 1 H/ 13 C dual + probe, carefully tuned at the recording frequency of 100.61 MHz. The temperature of the probe was set to 303 K, without tube rotation. The exact NMR acquisition conditions are detailed in the SI. Isotope 13 C compositions were calculated from processed spectra as described previously [START_REF] Akoka | NMR-based isotopic and isotopomic analysis[END_REF]: further details are given in the SI (Table S2).

Calculation of enrichment factors () and associated expanded uncertainty (U)

Enrichment factors ( in ‰) is a common way used to express IEs. An isotope effect is considered as normal (light isotopologues respond preferentially) when  < 0 and inverse when  > 0.

Enrichment factors were determined using Rayleigh plots in which ln(f) is plotted on the x-axis (where f is the fraction of paracetamol remaining in the column) and ln(R/R0) on the y-axis (eq. 2 and 3, R is the isotope ratio of the sample and R0 the isotope ratio of paracetamol at t0) [START_REF] Elsner | Insight into Methyl tert-Butyl Ether (MTBE) Stable Isotope Fractionation from Abiotic Reference Experiments[END_REF][START_REF] Höhener | Rayleigh equation for evolution of stable isotope ratios in contaminant decay chains[END_REF].

ln ( R R 0 ) = (α -1) × ln(𝑓) eq. 2 ε = (𝛼 -1) × 1000 eq. 3
In Rayleigh plots, the slope of the trend line corresponds to the enrichment factor () and the associated root mean square (R²) gives a first indication of the quality of the linearity. However, in order to determine the significance threshold of the enrichment factor, the expanded uncertainty needs to be calculated. The determination of the expanded uncertainty associated with the enrichment factor determined using the Rayleigh-plot can be directly calculated using the function "LINEST" in Microsoft Excel™, as previously described [START_REF] Elsner | Insight into Methyl tert-Butyl Ether (MTBE) Stable Isotope Fractionation from Abiotic Reference Experiments[END_REF][START_REF] Julien | Expanded uncertainty associated with determination of isotope enrichment factors: Comparison of two point calculation and Rayleigh-plot[END_REF]. This function calculates both the slope and the standard deviation of the trend line (STDV slope). The expanded uncertainty can thus be calculated as follows (eq. 4):

U = k × STDV slope eq. 4
where k = 2 for a coverage factor at the 95% confidence level according to Student's t-distribution.

In this context, data are presented as  ± U and enrichment factors are considered as significant when|| > U.

Theoretical calculation of enrichment factors ()

We have used two models of the environment. The first one involved a SMD continuum solvent model [START_REF] Marenich | Universal Solvation Model Based on Solute Electron Density and on a Continuum Model of the Solvent Defined by the Bulk Dielectric Constant and Atomic Surface Tensions[END_REF], which uses bulk properties of the solvent to create a dielectric cavity in which the solute is immersed. In the second approach, explicit solvent molecules are used. Within this approach we have used 14 water molecules comprising the first solvation shell of paracetamol (see Figure 1) as a model of well-defined, strong hydrogen bonding network with availability of both proton donor and proton acceptor sites. Calculations were carried out at the DFT level of theory, using ωB97X-D functional [START_REF] Chai | Long-range corrected hybrid density functionals with damped atom-atom dispersion corrections[END_REF] expressed in the def2-TZVP basis set [START_REF] Weigend | Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: Design and assessment of accuracy[END_REF] as implemented in the Gaussian16 program [START_REF] Frisch | Revision C.01[END_REF], which proved successful in our recent studies on vapor pressure isotope effects of ethanol [START_REF] Klajman | Computational Investigations of Position-Specific Vapor Pressure Isotope Effects in Ethanol-Toward More Powerful Isotope Models for Food Forensics[END_REF]. We have carried out calculations for structures of paracetamol in the gas phase, continuum solvent models of diethyl ether and acetone, and surrounded by 14 explicitly treated water molecules. For the optimized structures frequency calculations were carried out to ensure they represent local minima, and for calculations of isotope effects (which were subsequently converted into isotopic enrichment factors) using the Isoeff program [START_REF] Anisimov | A program for studies of isotope effects using Hessian modifications[END_REF].

Results and Discussion

Design of the experiments: choices of solid phases and solute

The goal of the work being double: (i) relationship between the type of stationary phase and the NCIE for a given chemical in liquid chromatography; and (ii) contribution to modelling the isotope fractionation upon migration of organic chemicals in a soil, the choice of the solid matrices and of the solute should be further explained. The relationship between structural substitutions and NCIE during the chromatographic elution on normal phase silica gel of a group of phenolic compounds related to vanillin was previously investigated on qualitative basis [START_REF] Botosoa | Evidence of 13 C non-covalent isotope effects obtained by quantitative 13 C nuclear magnetic resonance spectroscopy at natural abundance during normal phase liquid chromatography[END_REF][START_REF] Botosoa | Unexpected fractionation in site-specific 13 C isotopic distribution detected by quantitative 13 C NMR at natural abundance[END_REF]. Herein one compound has been studied during elution on four stationary phases. This compound is paracetamol also called acetaminophen, it is one of the most widely used drugs in the world.

Although this painkiller is a prodrug, which means that it needs to be partially metabolized in the liver to become active [START_REF] Graham | Mechanism of Action of Paracetamol[END_REF], large quantities of this whole molecule are found in the environment.

Paracetamol can be converted into N-acetyl-p-benzoquinone imine and 1,4-benzoquinone in wastewater treatment plants by reacting with hypochlorite ions (ClO -) [START_REF] Bedner | Transformation of Acetaminophen by Chlorination Produces the Toxicants 1,4-Benzoquinone and N-Acetyl-p-benzoquinone Imine[END_REF]. These compounds are suspected to be genotoxic and mutagenic, so the monitoring of the fate of paracetamol is of primary importance [START_REF] Moore | The toxicity of acetaminophen and N-acetyl-p-benzoquinone imine in isolated hepatocytes is associated with thiol depletion and increased cytosolic Ca2+[END_REF][START_REF] Thijssen | Paracetamol (acetaminophen) warfarin interaction: NAPQI, the toxic metabolite of paracetamol, is an inhibitor of enzymes in the vitamin K cycle[END_REF][START_REF] Kalinec | Acetaminophen and NAPQI are toxic to auditory cells via oxidative and endoplasmic reticulum stress-dependent pathways[END_REF].

Because of a high complexity, the NCIE during migration of paracetamol into a soil would difficult to interpret as the different sources of interaction nor their strengths between the chemical and the solid phase could be described. We opted to a differentiation of each category of forces responsible of the retention (or not) during the elution. As such, normal phase silica gel (SG) is both polar and hydrogen bond provider (silanol groups), cellulose is much less polar but still provides hydrogen bonds (hydroxyl groups), the reverse C8 phase (RP) may be considered as nonpolar and will favor weak forces (van der Waals), and finally the charcoal is well known to sorb chemicals via -ring current interactions. This diversity of dominating interactions for the studies solid phases allowed us additionally to get a deeper insight into theoretical modelling of isotopic fractionations using quantum-mechanical calculations.

The nature of the eluent is recognized as contributing to the elution order of the isotopomers. All the studies of the isotope fractionation pointed out that the composition of solvents can change the interactions between the solute and the solid phase. For the present work, the eluent has been fixed at 10% of acetone and 90% of diethyl ether and the same for all experiments and therefore for the four stationary phase. Paracetamol is not very soluble in all organic solvents and is preferentially in alcohols. But the presence of strong hydrogen bonds via the hydroxyl groups could annihilate other weak interactions. It is then expected that the isotope fractionation due to weak interactions between paracetamol and the stationary phase could be observed. The mixture of acetone (10%) and diethyl ether (90%) is polar enough to allow a chromatographic experiment for the four solid phases with a gradual migration of paracetamol. Finally, being very volatile, these solvents can be easily removed for each collected fraction leaving dry paracetamol for 13 C NMR. 13 C NMR and repeatability of the isotope compositions

What is measured by irm-

Data obtained from isotopic NMR

Even if several papers are now available in the literature [START_REF] Jézéquel | The new face of isotopic NMR at natural abundance[END_REF][START_REF] Akoka | NMR-based isotopic and isotopomic analysis[END_REF][START_REF] Remaud | Isotope Ratio Monitoring by NMR. Part 1: Recent Advances[END_REF], we think that it is worthwhile to further describe the irm- 13 C NMR methodology and the data it provides. Any molecules containing isotopes are isotopologues, i.e. they have the same constitution and same configuration but differ in their isotope substitutions. Quantifying isotopologues having two or more heavy isotopes is not accessible for 13 C and 15 N, due their very low abundance, by current routine techniques as irm-MS or irm-NMR: the mono labelled, at natural abundance, molecules are considered. These molecules are named isotopomers, i.e. same chemical structure in which the heavy isotope differs as to its position in the structure. PSIA is therefore the approach consisting in the determination of the profile of isotopomers, as opposed to the quantitation of the mean ratio (bulk or global) of the 13 C isotopologues versus the light one ( 12 C). Irm- 13 C NMR resolves fractionation of natural isotope values in 13 C for every position in the target molecules.

For low isotopic abundance, as for natural-abundance 13 C (1.08%), molecules containing more than one 13 C are usually not detected by NMR. Therefore, each chemical shift (that is the peak in the spectrum, named i) corresponds to an isotopomer i (mono-labelled at natural abundance).

Paracetamol has 8 carbon atoms at 6 specific positions (then 6 13 C isotopomers numbered as described in Figure 2), and one nitrogen atom. shown are the molecular structures of paracetamol and vanillin with the carbon atoms numbered in relation to decreasing 13 C chemical shift in the 13 C NMR spectrum.

The area S of the corresponding signal in the NMR spectrum is directly proportional to the amount of this isotopomer. The relationship between the mean and the position-specific 13 C content is found at the isotopic abundance x level: xi = xg.fi/Fi (Table 1). Thus irm- 13 C NMR gives access to the relative intramolecular 13 C distribution, while irm-MS reports on the total amount of 13 C isotopes. Further details on the calculation of  13 Ci are given in the SI.

Fi

Statistical molar fraction for a carbon site i: molar fraction for the carbon site i in cases of homogeneous 13 C distribution within the molecule (example: F1 = 1/8 for paracetamol)

fi/Fi

Reduced molar fraction for a carbon i: any shift from 1 indicates an isotope fractionation

x Isotopic abundance g Enrichment factor calculated as described in the experimental section from  13 Cg i Enrichment factor calculated as described in the experimental section from  13 Ci Note: 15 N has only one possible position in paracetamol so only global analysis (irm-15 N MS) is done.

Precision of the isotopic compositions

The repeatability of both intramolecular and global isotope analysis was investigated. Data presented in Table 2 show a standard deviation (SD) of isotopic composition measurements comprised between 0.1 and 0.7‰. More interesting, global and intramolecular isotope compositions of material used as working reference were calculated using data from all collected fractions and the differences between these calculated values and direct measurements on the reference materials were calculated (see Table 2). These differences are comprised between 0.1 and 1.2‰ which confirms the repeatability and reproducibility of the isotope measurements (irm-EA/MS and irm-13 C NMR). Moreover, these data prove that differences in PSIEs discussed in this article are directly associated with the migration experiments and are not due to measurement error or a lack of recovery of the migrating material.

13 C and 15 N isotope effects

For each of the four migration experiments, global and 13 C intramolecular (carbon positions 1 to 6) data were plotted on the same graph in order to determine corresponding  values and nitrogen data are presented in a separated graph (see Figure 3). The Rayleigh equation and, by extension, Rayleigh plot are usually employed to follow the evolution of the isotope ratio of a compound as a function of a reaction's progress. This equation allows calculating the fractionation factor created by a considered process such as a (bio)chemical reaction [START_REF] Elsner | Insight into Methyl tert-Butyl Ether (MTBE) Stable Isotope Fractionation from Abiotic Reference Experiments[END_REF] or the migration during liquid chromatography in the present work. 3. As detailed in section 2.5, isotope effects are considered as normal (molecules containing light isotopes are preferentially eluted) when  < 0 and inverse (heavy isotopologues are less retained in the column) when  > 0. The link between observed IEs and the intermolecular interactions occurring between paracetamol and each considered stationary phase is discussed using their chemical structures described in Figure 2.In addition, previously published data obtained during the migration of vanillin through SG [START_REF] Botosoa | Evidence of 13 C non-covalent isotope effects obtained by quantitative 13 C nuclear magnetic resonance spectroscopy at natural abundance during normal phase liquid chromatography[END_REF] and RP [START_REF] Höhener | Analytical model for site-specific isotope fractionation in 13 C during sorption: Determination by isotopic 13 C NMR spectrometry with vanillin as model compound[END_REF] were treated using the method described in section 2.5 and summarized in Table 4 and were included in the discussion.

Normal phase silica gel (SG)

The migration of paracetamol in SG (Table 3) is associated with a small global 13 C normal isotope effect of -1.1 ± 0.5‰. This result agrees with previous data obtained during the migration of vanillin on this stationary phase (-0.9 ± 0.1‰, Table 4) where 13 C-depleted isotopologues are less retained in the stationary phase (preferentially eluted). The presence of 13 C within paracetamol molecules seems to result in a higher retention of heavy isotopologues. The PSIA revealed that this IE is not equitably distributed between the carbon positions with a maximum  located on C-1 (-3.3 ± 1.5‰). The presence of a strong PSIE on this carbon position can be explained by the presence of hydrogen-bonds between the oxygen of the acetamide function of paracetamol and acid hydrogens of silanols in SG (see Figure 2). The measurement of a smaller  on carbon 6 (-2.3 ± 1.6‰) could be due to a secondary PSIE associated with the described interaction. This deduction is reinforced by the presence of a strong normal 15 N (-4.7 ± 2.5‰) IE during the migration which could be associated with the interaction of hydrogens from silica gel and the electron lone pair of the nitrogen atom. Other normal PSIEs located on the aromatic cycle of paracetamol (carbon positions 4 and 5) may be due to interactions between  electrons and the stationary phase during the elution. Surprisingly, no significant PSIE is measured on the C-2 of paracetamol although interactions between this chemical function and silica gel were anticipated.

Intramolecular 13 C isotope data from migration of vanillin in SG are in accordance with these observations with the presence of a strong normal isotope effect on the carbon bearing the aldehyde function (-3.3 ± 0.6‰) and a smaller one on the methoxy group (-0.9 ± 0.1‰). The carbon of the aromatic cycle bearing the alcohol function (C-2) presents an inverse isotope effect (+1.5 ± 0.4‰) while no significant IE was observed on C-2 of paracetamol. Also, the amplitude of IEs within the aromatic cycle is higher in the case of vanillin with normal isotope effects with  values comprised between -1.5 ± 0.2 and -3,4 ± 0.2‰ and an important inverse IE on C-6 (+2.4 ±

0.3‰).

More generally, the migration of these compounds through a polar stationary phase is associated with a strong normal 13 C IE inequitably distributed between the carbon positions because of the presence of polar chemical functions (here acetamide, alkoxy, aldehyde and alcohol) and their likely contribution to hydrogen bonds set up with the stationary phase.

Cellulose (CE)

Paracetamol migration in CE does not show any significant 13 C g but PSIA revealed the contribution of both normal and inverse PSIEs on the different carbon positions of the molecule.

In contrast to the SG experiment, the migration of paracetamol in CE is associated with a small inverse PSIE located on the C-1 (+0.4 ± 0.3‰). Surprisingly, a normal PSIE is detected on the C-6 ( = -2.4 ± 0.2‰) which suggests that the presence of a 13 C on this carbon position may stabilize the interaction between the oxygen of the acetamide function of paracetamol and those from CE (Figure 3) resulting in a slower elution of the observed isotopomer. Nevertheless, no significant 15 N isotope fractionation is detected during this experiment. Other carbon positions (3 and 5) present positive  values demonstrating that the presence of 12 C in the aromatic cycle leads to less stable interactions between paracetamol and CE.

Activated charcoal (AC)

The migration through AC is associated with a small inverse global IE (+0.3 ± 0.0‰), which is due to the counteractive contribution of both normal and inverse PSIEs within the molecule. As explained in the introduction, only weak non-covalent interactions (i.e. van der Waals and stacking) between paracetamol and activated charcoal are expected. A few significant PSIEs are detected in this case such as a normal PSIE on the C-6 ( = -1.7 ± 0.6‰), which could be explained by the presence of a van der Waals interaction involving this methyl position of paracetamol. A normal PSIE here suggests that van der Waals interactions can be stabilized by the presence of 13 C, so isotopomers with a 12 C on C-6 will elute faster. Then, carbon positions 2, 3 and 4, located in the aromatic cycle, present inverse PSIEs of +1.0 ± 0.1‰, +1.6 ± 0.5‰ and +0.5 ± 0.3‰ respectively. These observed PSIEs may be due to the -stacking interactions between paracetamol and aromatic functions in activated charcoal (Figure 2). According to these data, the presence of 13 C on these carbon positions may stabilize the proposed intermolecular interaction and induce isotope fractionation during migration. A significant normal 15 N IE is also detected during the migration of paracetamol in activated charcoal ( = -2.7 ± 0.9‰), which could be due to the presence of van der Waals interaction between activated charcoal and this moiety of paracetamol.

Silica gel C8-reversed phase (RP)

No significant 13 C global IE was detected during the migration of paracetamol on RP and only small intramolecular PSIEs were identified. Previous data obtained from the migration of vanillin on RP also showed a non-significant global IE (see Table 4). A small inverse PSIE was measured on C-6 of paracetamol ( = +0.3 ± 0.2‰) which suggests that van der Waals interactions between this methyl position and C8 carbon chains from the stationary phase may be strengthened by the presence of a 12 C on C-6, so isotopomers with a 13 C on this carbon position are preferentially eluted. Conversely,  of -0.3 ± 0.1 ‰ and -0.5 ± 0.2‰ are measured on C-3 and C-4, respectively, so the presence of 13 C within the aromatic cycle of paracetamol seems to increase the retention of the considered isotopomers on this stationary phase. A few significant inverse IEs are observed within vanillin with +0.3 ± 0.1‰ on C-5, +0.7 ± 0.5‰ on C-3 and +1.1 ± 0.2‰ on C-2. This last isotope effect has a higher amplitude than those observed in paracetamol, but the experimental conditions used in the present study differed (reversed phase of type C8 instead of type C18, different eluents, different compounds studied).

However, the migration of such polar compounds through nonpolar stationary phases seems to be associated with only a few small inverse isotope effects.

Intramolecular  associated with non-covalent interactions

According to data obtained in the present study, the chemical composition of the solid medium traversed by an organic pollutant has an influence on the associated isotope fractionation. Note that the same eluent mixture is used during each chromatography experiments so the IEs potentially created by interactions between eluent and paracetamol are hidden. In this context, the  values measured for each matrix can be used to discuss the link between observed NCIEs and the chemical structure of the stationary phase. These results suggest that intermolecular interactions established between the substrate (here paracetamol and vanillin) and the soil (modeled by various stationary phases in this study) have a direct influence on the selectivity of isotopomers preferentially migrating. The PSIEs associated with the migration experiments allowed the identification of the isotopomers that preferentially interact with each of the tested stationary phases. The presence of small or non-significant global IEs (measured using irm-MS) is proved to be due to the counteractive contribution of both normal and inverse intramolecular PSIEs (measured by irm- 13 C NMR) for different carbon positions of the substrate.

The key point of the present study is the association of the non-covalent interactions between the stationary phase and the substrate with the resulting intramolecular PSIEs. As an example, both SG and CE are polar, so hydrogen-bonds between these stationary phases and paracetamol or vanillin can be made. The presence of PSIEs on C-1 associated with the migration through these two materials confirms the presence of hydrogen bonds between acidic hydrogens of the phases and the oxygen atom of the acetamide function (Table 3). Thus, it can be predicted that when a polar organic contaminant migrates in a soil containing polar phases, isotope fractionation on the carbon positions bearing a polar function (alcohol, ketone...) will occur. The presence of intramolecular IEs associated with the formation of hydrogen bonds has already been proven to be directly correlated in the case of evaporation of VOCs [START_REF] Julien | Position-Specific 13 C Fractionation during Liquid-Vapor Transition Correlated to the Strength of Intermolecular Interaction in the Liquid Phase[END_REF]. Conversely, no PSIEs should be observed on polar functions during the migration in non-polar medium such as AC or RP.

The presence of weak interactions, such as van de Waals forces or -stacking, can also induce PSIEs but it is much more difficult to discuss any direct implication in the distribution of PSIEs because they do not involve a specific chemical function of the substrate. The exact chemical structure of AC is not well defined, but the presence of aromatic cycles is expected in this material.

Thus, it is not surprising that significant PSIEs at carbon positions located in the aromatic cycle of paracetamol and vanillin (Tables 3 and4) are observed. However, isotope fractionation is also observed on carbon positions of the aromatic cycle during the migration of paracetamol through the other stationary phases which may be due to (i) interactions between the  electrons and acidic hydrogens from SG and CE or (ii) hydrophobic interaction with the C8 aliphatic chain of the RP. Similarly, it is hard fully to interpret the significant isotope effect observed on C-6 of paracetamol and C-8 of vanillin in all experiments (Table 2). This PSIEs could be considered as a secondary isotope effect due to the presence of hydrogen bonds on the C=O or the O of ether function for paracetamol and vanillin respectively (for SG and CE) or a direct van der Waals interaction in the case of the non-polar media (AC and RP). Further research is required to resolve these problems.

Moreover, these data directly demonstrate the value of intramolecular isotope analysis to study further the origin and fate of organic pollutants. As PSIEs are different depending on the traversed stationary phase, modeling the isotope fractionation occurring during migration in pure material associated with the chemical analysis of the contaminated soil could help tracing organic pollutants in the environment.

Theoretical calculations of the enrichment factors.

Theoretical prediction of heavy-atom isotope effects poses numerous problems since they are usually very small. Reported herein experimental results, obtained for different solid phases, provided a unique opportunity to evaluate quality of different theoretical models used in quantum-chemical calculations of isotope effects associated with changes in weak interactions.

The obtained results are collected in Table 5. The two first entries in Table 5 represent fractionations calculated for the process of going from diethyl ether solution either to very polar and hydrogen-bonded environment (aq14) or to the gas phase. As can be seen, except for the value for the C3 position, results obtained using aq14 model are in very good agreement with the experimental results for SG (normal phase silica gel).

We have tried to rationalize this single discrepancy, however, no clear indication of its source has been identified based on the geometrical features and electrostatic properties. The gas phase model, on the other hand, agrees well with those obtained for the other three solid phases although not with particular single one. We have, therefore, calculated a putative process of paracetamol changing phases by going from pure ethyl ether to acetone. These values might reflect preferential solvation and can be treated as uncertainty on the calculated values.

Results collected in Table 3 seem to implicate that the polarity effects dominate over specific hydrogen bonding interactions since the results obtained for cellulose support are much closer to those of activated carbon and reverse phase than to those obtained for silica gel. Thus the results obtained with the apparent solvent model indicate that the presence of the explicit hydrogen bonding between the paracetamol and solvent molecules of high polarity of the solvent shell environment results in strong polarization that affects the isotopic fractionation. On the contrary, when continuum models of solvent are used accounting for these polarization effects is much weaker leading to lower values of isotopic fractionation.

Conclusions

The data presented confirm IEs associated with the migration of organic compounds such as paracetamol, and that the extent of these depend on the matrix through which the considered chemical is migrating. These results also confirm that when the recovery of a target compound is not total during isolation by liquid chromatography, its isotope composition may be affected, especially when followed by intramolecular isotope analysis. Then, the corrections of the measured isotope data need to consider the chemical composition of both the stationary phase and the isolate molecule in addition to amount of lost compound (see Rayleigh plots). A better understanding of these "binding isotope effects" should help describing the processes involved in liquid chromatography and, more generally, in migration experiments. Hence, these results endorse the need to take the migration into account while studying the isotope composition of organic contaminants during their remediation. Furthermore, the use of intramolecular isotope analysis is potentially of considerable benefit for environmental forensic investigations, as they give indications of the type of matrix through which the pollutant has migrated. The identification of PSIEs associated with migration experiments should help in understanding the link between the transport of an organic contaminant and the chemical composition of the soil. This is the main objective of the present work. Understanding the mechanisms involved is essential to developing detailed transport models for the prediction of the intramolecular of isotope distribution within an organic contaminant and its evolution in the environment. As sorption is considered one of the most promising method to eliminate organic pollutants from water [START_REF] Rossner | MTBE adsorption on alternative adsorbents and packed bed adsorber performance[END_REF][START_REF] Rossner | Removal of emerging contaminants of concern by alternative adsorbents[END_REF] the determination of the PSIEs associated with this process should lead to a better understanding of the mechanisms involved, hence aid making contaminant monitoring more efficient. Thus, they provide further insight of value for the characterization of the source(s) and the detection/quantification of remediation mechanisms. Unfortunately, as NMR has a low sensitivity, large quantities of pure compound are required to perform PSIA on field samples. However, recent advances in NMR are making it possible to access the intramolecular 13 C isotope composition using much lower quantities [START_REF] Joubert | Full Spectrum Isotopic 13 C NMR Using Polarization Transfer for Position-Specific Isotope Analysis[END_REF], while other PSIA methods using on-line pyrolysis coupled with irm-MS [START_REF] Gilbert | Measurement of position-specific 13 C isotopic composition of propane at the nanomole level[END_REF][START_REF] Julien | Intramolecular 13 C isotope distributions of butane from natural gases[END_REF] or high resolution MS [START_REF] Gilbert | Measurement of position-specific 13 C isotopic composition of propane at the nanomole level[END_REF] are offering opportunities for similar detail to be obtained on much smaller samples still. Such approaches should make possible experiments in real soil conditions aimed at understanding and modeling the exact link between intermolecular non-covalent interactions and intramolecular isotope distributions within target molecules. Also, the great agreement between PSIEs measured by 13 C-NMR and those calculated using theoretical models demonstrate the great potential of quantum mechanics for predicting the evolution of intramolecular isotopic composition of organic environmental contaminants. Future developments of more sensitive intramolecular isotope analysis methods associated with theoretical calculations should provide excellent tools to trace the origin and fate of organic compounds detected in environment.

Supplementary material

Supporting information is added to this article, see "Supporting_Information.xlsx".

Figure 1 :

 1 Figure 1: Solvent network around paracetamol molecule.

Figure 2 :

 2 Figure 2: General chemical structure of the stationary phases employed in the present study. Also

Figure 3 :)

 3 Figure 3: Rayleigh plots drawn using global and intramolecular (C-1 to C-6) 13 C experimental data

( 5 CE 2 AC

 52 position-specific, C-1 to C-6) upon migration of paracetamol through the different solid phases as described in experimental section (SG = silica gel; CE = cellulose; AC = activated charcoal and RP = silica gel C8-reversed). The values shown after "±" are the expanded uncertainties of (U in ‰). 1 ± 0.5 -3.3 ± 1.5 +0.1 ± 0.3 +0.1 ± 0.1 -1.0 ± 0.2 -0.6 ± 0.1 -2.3 ± 1.6 -4.7 ± 2.+0.1 ± 0.1 +0.4 ± 0.3 -0.3 ± 0.5 +0.4 ± 0.3 +0.3 ± 0.3 +1.1 ± 0.1 -2.4 ± 0.2 -0.1 ± 0.+0.3 ± 0.0 +0.2 ± 0.6 +1.0 ± 0.1 +1.6 ± 0.5 +0.5 ± 0.3 +0.1 ± 0.2 -1.7 ± 0.6 -2.7 ± 0.9 RP -0.1 ± 0.1 -0.1 ± 0.5 +0.2 ± 0.5 -0.3 ± 0.1 -0.5 ± 0.2 -0.1 ± 0.1 +0.3 ± 0.2 -0.5 ± 0.2

8 SG 1 RP

 81 -0.9 ± 0.1 -3.3 ± 0.6 +1.5 ± 0.4 -1.5 ± 0.2 -3.4 ± 0.2 -2.5 ± 0.4 +2.4 ± 0.3 -1.3 ± 0.2 -0.9 ± 0.+0.3 ± 0.3 +0.6 ± 0.6 +1.1 ± 0.2 +0.7 ± 0.5 +0.4 ± 0.8 +0.3 ± 0.1 +0.3 ± 0.4 +0.2 ± 0.4 +0.3 ± 0.3

Table 1 :

 1 Symbols and their definitions used in the present work.

	Symbol	Definition
	 13 C	Carbon isotope composition: carbon isotopic ratio of the molecule relative to the international standard (Vienna Pee Dee Belemnite V-PDB)
	 13 Cg
	 13 Ci	13 C isotopic composition of the carbon position i measured by PSIA (as irm-13 C NMR in this work)
	 13 Cg	Difference between final  13 Cgand initial  13 Cg
	 13 Ci	Difference between final  13 Ci and initial  13 Ci
		Molar fraction for a carbon site i measured by irm-13 C NMR = area S of the peak
	fi	corresponding to the carbon position i (Si) divided by the sum of all the carbon S i sites of the molecule: 𝑓 i = ∑ S i

13

C mean isotopic composition of a whole molecule measured by irm-MS: global

13

C content. Also found as 

13

Cb for bulk value, or  13 CT for total value n .

Table 2 :

 2 Repeatability study of 13 Cg, 15 Ng by irm-MS and intramolecular13 C distribution measurements in paracetamol by irm-13 C NMR,  13 Ci (SD = standard deviation), expressed in ‰.

	Also shown, the isotopic composition of material used as working reference calculated from
	values measured on the different fractions (see calculated) and the absolute difference with the
	direct measurement of paracetamol (see |difference|).
					 13 C (‰)				 15 Ng
	Experiment global	C-1	C-2	C-3	C-4	C-5	C-6	(‰)
	1	-26.9	-32.4	-27.9	-34.9	-22.8	-21.6	-31.1	-3.7
	2	-26.9	-31.8	-26.3	-35.2	-23.1	-22.3	-31.2	-3.7
	3	-26.9	-31.6	-27.6	-34.1	-22.8	-22.0	-32.4	-3.8
	4	-26.9	-31.1	-26.9	-35.0	-22.9	-22.2	-32.1	-3.7
	5	-27.0	-32.3	-26.5	-34.5	-22.7	-22.7	-31.2	-3.7
	mean	-26.9	-31.9	-27.0	-34.7	-22.9	-22.2	-31.6	-3.7
	SD	0.1	0.5	0.7	0.4	0.2	0.4	0.6	0.1
	calculated	-27.6	-32.9	-28.1	-35.7	-23.7	-22.7	-30.9	-3.9
	SG
	|difference|	0.6	0.4	0.2	0.8	0.9	1.1	0.3	0.2
	calculated	-26.9	-32.4	-27.6	-34.7	-22.2	-22.3	-31.7	-3.9
	CE
	|difference|	0.0	0.0	0.3	0.2	0.6	0.6	0.5	0.1
	calculated	-27.1	-31.8	-27.6	-35.0	-23.2	-22.5	-31.3	-3.2
	AC
	|difference|	0.2	0.7	0.3	0.1	0.4	0.8	0.2	0.5
	calculated	-26.9	-32.0	-27.4	-34.0	-23.3	-22.5	-29.9	-3.6
	RP
	|difference|	0.0	0.5	0.5	0.9	0.5	0.9	1.2	0.1

Table 3 :

 3 Enrichment factors ( in ‰) obtained from irm-EA/MS (bulk) and from irm-13 C NMR

Table 4 :

 4 Enrichment factors ( in ‰) obtained from irm-EA/MS (bulk) and from irm-13 C NMR (position-specific, C-1 to C-8) upon migration of vanillin through silica gel (SG,[START_REF] Botosoa | Evidence of 13 C non-covalent isotope effects obtained by quantitative 13 C nuclear magnetic resonance spectroscopy at natural abundance during normal phase liquid chromatography[END_REF] and reversed phase silica gel RP-18 (RP,[START_REF] Höhener | Analytical model for site-specific isotope fractionation in 13 C during sorption: Determination by isotopic 13 C NMR spectrometry with vanillin as model compound[END_REF]). The values shown after "±" are the expanded uncertainties of (U in ‰), at 95% confident level.

Table 5 :

 5 Enrichment factors ( in ‰) obtained from quantum mechanical calculations for transition from diethyl ether to aqueous solution (14aq), gas phase, and acetone.

	 (‰)

Acknowledgements

M.J. thanks the ANR for funding his PhD bursary through the ISOTO-POL project and Kiban S for funding his postdoctoral fellowship (Grant-in-Aid for Scientific Research S, 23224013, MEXT, Japan). We thank Dr. Richard J. Robins for his help with language correction of this article.

Funding

This work is funded by the French National Research Agency ANR, project ISOTO-POL funded by the program CESA (no. 009 01). The access to PL-Grid computing resources at Cyfronet is gratefully acknowledged.


Quentin Bonnin

Sook-Yen Wong

Cédric Balan

Virginie Comte

Raluca Malacea

Marie-José Penouilh

Philippe Richard

Gerald Kehr

Adrien T Normand

Gerhard Erker

Pierre Le Gendre

A new Route toward (Aminomethyl)cyclopentadienide Ligands and their Group 4 Metal Complexes

Keywords: titanium, zirconium, cyclopentadienyl ligands, amines

A new synthetic method is described, by which Nfunctionalized cyclopentadienide ligands are synthesized, via the nucleophilic substitution of sodium cyclopentadienide onto Katritzky's (benzotriazolyl) compounds. This route enables the introduction of bulky and basic amines separated from the cyclopentadienyl ring by a methylene spacer, and thus represents a valuable alternative to the general fulvene route. The resulting ligands have been coordinated to titanium and zirconium, producing a series of discrete metallocene derivatives with a pendant amine arm. These complexes are extremely sensitive towards moisture, but they can easily be converted into benchtop-stable ammonium chloride derivatives. The ligands and their complexes have been fully characterized by NMR and IR spectroscopy, elemental analysis and/or high resolution ESI-MS. The solid state structures of ten complexes have been established by single crystal X-ray diffraction analysis.

Introduction

Over the last decades, amino-and amido-functionalized cyclopentadienyl early metal complexes have attracted increased interest for their use in polymerization catalysis, [1,2] organic catalysis, [3] and also for therapeutic purposes. [4] The incorporation of amino or amido functions on the cyclopentadienyl ring allows to tailor the properties of these complexes and, consequently, to achieve optimal performance. One drawback of this approach is that the synthesis of these complexes is not straightforward. Unlike ferrocene, [5] lithiation and subsequently functionalization of early metallocenes is usually not feasible. [6] Instead, the synthesis of a functionalized cyclopentadienyl ligand is required before metalation. Several synthetic routes already exist towards aminofunctionalized cyclopentadienyl ligands. [7] In the case of (aminomethyl)cyclopentadienyl ligand, fulvenes and aminofulvenes appear as ubiquitous intermediates. [8] The carbolithiation of the 6-(dimethylamino)fulvene using alkyl, aryl or heteroaryl lithium species leads to a variety of dimethylamino-functionalized cyclopentadienides (Scheme 1, eq. 1). [4e,9] In addition, it has been shown that 6-(amino)fulvene can undergo exchange reaction with morpholine or (methoxymethyl)pyrrolidine to furnish the corresponding fulvenes derivatives which can be further reduced to amino-functionalized cyclopentadienide ligands (eq. 2). [10] Scheme 1. Synthetic routes towards (aminomethyl)cyclopentadienyl ligand.

The introduction of an amino function to the cyclopentadienyl moiety can also be realized by adding Li-amido reagents at the C6 carbon atom of an aliphatic fulvene but this reaction is limited to non "enolizable" fulvenes such as 6-t-butylpentafulvene (eq. 3) or tetramethylpentafulvene because of the basic character of the Li-amido reagents. [2f,11] Aware of these limitations and with the aim of introducing bulky and basic tertiary amines close to the metal center, we envisioned another synthetic strategy based on the addition of the cyclopentadienide anion to aminomethylbenzotriazole derivatives (eq.4). In solution, these compounds -developed mainly by Katritzky -exist in equilibrium with highly electrophilic iminium salts and, therefore, represent valuable candidates for our purpose. [12] Herein we describe the synthesis of (aminomethyl)cyclopentadienide ligands using this new benzotriazole route. Preliminary metalation studies with early metal precursors (Ti, Zr) are reported.

We thus first synthesized several benzotriazole adducts according to the procedures described in the literature. [13] The reaction of formaldehyde with N-methylbenzylamine, pyrrolidine, diisopropylamine, dibenzylamine or tetramethylpiperidine and one equivalent of benzotriazole produced 1-(aminomethyl)benzotriazole substrates. We next reacted these compounds with sodium cyclopentadienide in ether (Scheme 2), and monitored the reaction by TLC and/or GCMS. Once the 1-(aminomethyl)benzotriazole was fully consumed, the reaction mixture was successively filtered, hydrolyzed, dried over magnesium sulfate and evaporated to dryness. Addition of a toluene solution of KHMDS to the mixture of cyclopentadiene isomers re-solubilized in diethyl ether led to the target potassium amino-functionalised cyclopentadienides (1-5) in 39-86% yields. The anions were characterized by 1 H, 13 C NMR spectroscopy and high resolution ESI-MS. The 1 H NMR spectra of compounds 1-5 show a similar pattern, two multiplets (AA'BB' spin system) in the range of 5.2-5.4 ppm and one singlet between 3.3-3.5 ppm for the bridging CH2 protons.

N N N N R 2 R 1 N M Cl Cl K + 1-5 N R 3 R 3 R 3 R 3 R 3 N Ph N N i Pr i Pr N Ph Ph N N R 2 R 1
1 (39 %) 2 (74 %) 3 (67 %) 4 (60 %) 5 (86 %) With these anions in hands, we next focused on the synthesis of early metallocene complexes with one pendant amine function and proceeded as follow: the amino-functionalised cyclopentadienyl anion in THF solution was added dropwise at -30 °C to a toluene solution containing one equivalent of CpTiCl3 or Cp*TiCl3. The reaction was allowed to warm to room temperature and stirred at this temperature for 12 h. The resulting dark red solution was evaporated, then the residue was solubilized in CH2Cl2 and the solution was filtered. The target complexes 6-11 were then obtained by precipitation with pentane as highly moisture sensitive red powders in 46-80% yields. Using a similar procedure, the tetramethylpiperidine-functionalized zirconocene complex 12 was obtained in 54% yield from the potassium cyclopentadienide salt 5 and CpZrCl3. Complexes 6-12 were characterized by 1 H, 13 C, 15 N NMR spectroscopy, IR spectroscopy, elemental analysis and high resolution ESI-MS (see Supporting Information). The 1 H NMR spectra of complexes 6-12 show downfield shifted signals for the Cp' ring and bridging CH2 protons (Δδ = 0.1-1.4 ppm) compared to the anionic ligands. The 15 N NMR spectra of complexes 6-12 display single resonance between δ = -339 and δ = -309 ppm, a range comparable to that observed for other amino-functionalised group 4 organometallic complexes. [14] Single crystals of complexes 9-12 suitable for X-ray diffraction analysis were obtained by slow diffusion of pentane into saturated CH2Cl2 solutions of the complexes. ORTEP views of these complexes are presented in Figure 1. Table 1. Relevant bond distances (Å) and angles (°) in compounds 9-12. 9 [a] 10 11 [a] [a] Structural parameters of one of the two independent molecules present in the cell are given.

M = Zr R 3 = H R 2 R 1 R 2 R 1 i, ii, iii iv 1-4 g scale R 3 = H R 3 = Me M = Ti
The geometries of the three titanium complexes 9-11 are typically pseudo-tetrahedral with similar structural parameters (Table 1). The zirconium complex 12 shows similar geometry to complexes 9-11 with slightly longer M-Cl bonds lengths and M-Ct distances, consistent with the longer covalent radius of Zr vs Ti (Ti = 1.60( 8) Å; Zr = 1.75(7) Å). [15] Despite these metric similarities, complexes 9-12 show startling orientational differences which are dependent on the steric properties of the amine moiety. These differences can be described by the torsion angles ω and ϕ, which indicate the position of the amine moiety with respect to the Cl ligands (ω) and the Cp' ring (ϕ) (see Figure 1 for definitions). Thus, in 9 and 10, the CH2 bridge is located to the open side of the bent metallocene, and lies above the TiCl2 fragment (torsion angle ω = -1.51(6)° and -5.35(7)°, respectively). In contrast, for compounds 11 and 12, the bulky TMP substituent is rotated from the bisecting position, leading to ω values of -81.35 (18)° in 11 and 68.61(8)° in 12. The position of the N atom with respect to the Cp' plane is also particularly noteworthy in complexes 11 and 12 compared to complexes 9 and 10. Indeed whereas the C1CCH2N plane is more or less orthogonal to the Cp' ring in 9 and 10 (ϕ = 147.67(3)° and 179.88(15)°, respectively), this plane is tilted toward the Cp' ring due to the steric hindrance of the bulky tetramethylpiperidine (ϕ = -115.4(3)° and 118.72(16)°, respectively). It has been previously shown that the instability of amino titanocene toward moisture can be attenuated by protonating the amine function. [7j] We conducted the protonation of complexes 6-8, 10 and 11 in hydrochloric acid-saturated solution of toluene and obtained the corresponding ammonium salts 13-17 in 52-73% yields (Scheme 3). These turned out to be air and moisture-stable, and even water soluble. The protonation of the amine function has a profound impact on the shape of the 1 H NMR spectra of these complexes, by changing stereotopic relationships between atoms and/or groups. Thus the 1 H NMR spectrum of the chiral ammonium salt 13 shows four quartets centered at δ = 7.07/6.97, 6.60/6.57 ppm for the pairs of diastereotopic Cp' protons, and four doublets of doublets centered at δ = 4.38/4.04 and 4.37/4.13 ppm for the pairs of diastereotopic methylene protons. For the other ammonium salts, diastereotopic effects are also observed for the methylene protons of the pyrrolidine ring in 14, for the methyl protons of the isopropyl groups in 15, for the methylene protons of the benzyl groups in 16 and for both methyl and methylene protons of the tetramethylpyperidine in 17.

13-17

Scheme 3. Synthesis of the ammonium-functionalised titanocene complexes.

X-ray diffraction studies have been performed on single crystals of 14, 15 and 17, and allowed to confirm their structures (Figure 2). The ammonium moieties, in complexes 14 and 15 adopt a bisecting position, above the Cl ligands like in the neutral complexes 9 and 10 (ω = -0.99(18)°, 8.55(3)°, respectively), and they also sit above the Cp' ring (ϕ = 176.1(4)°, -148.28(7)°, respectively). The crystal structure of 17 exhibits a disorder over two positions, this lead to two different conformations where the TMP moiety is either located in a bisecting position (ω = 16.43( 15)) or to the side (ω = 86.6(2)). These results suggest that the peculiar orientation of the TMP moiety in the solid-state structures of 11 and 12 is the result of subtle crystal packing effects and is not fixed in solution. Further support for this hypothesis comes from the 1 H NMR spectra of these complexes, which do not show any evidence for restricted rotation in solution.

We also attempted the synthesis of 1,1'-bis(aminomethyl) group 4 metallocenes. The addition of the potassium cyclopentadienide 4 to TiCl4(THF)2 in 2:1 ratio in Et2O gave the target complex 18, albeit in 15% yield (Scheme 4). In the zirconium complexes, the reaction of two equivalents of 4 with ZrCl4 gave the target bisaminozirconocene complex 19 in 31% yield (Scheme 4). A similar reaction with the cyclopentadienide salt 3 afforded complex 20 in 76% yield.

M Cl Cl N N MCl 4 (THF) 2 2 N K + 4-5 R 2 R 1 R 2 R 1 R 1 R 2 N i Pr i Pr N Ph Ph N R 2 R 1 18 (15 %) 19 (31 %) 20 (76 %) M = Ti M = Zr

18-20

Scheme 4. Reagents and conditions: Et2O (M = Ti) or THF (M = Zr), -30 °C (30') to r.t. (12 h).

The 1 H NMR spectrum of 18 shows the expected pattern for the Ph group at δ = 7.36 (ortho), 7.32 (meta) and 7.24 (para) ppm, two triplet-like signals for the Cp' protons at δ = 6.35 and 6.25 ppm (AA'BB' spin system), and two broad signals at δ = 3.53 (8H) and 3.52 (4H) ppm corresponding to the both methylene protons. The 1 H 15 N HMBC spectrum of 18 reveals that the N atom resonates at almost the same chemical shift as that of 10 (δ = -327 ppm for 18 vs δ = -328 ppm for 10). Unsurprisingly, the 1 H NMR spectrum of zirconium complex 19 shows a set of signals comparable to those of its Ti analogue 18, and the 15 N chemical shift determined by 1 H 15 N HMBC is almost identical (δ = -328 ppm). In the case of zirconium complex 20, the signals of the protons from the Cp' ring appear as two multiplets at δ = 6.35 and 6.33 ppm in the 1 H NMR spectrum, while the bridging CH2 shows a singlet at δ = 3.66 ppm.

The four equivalent isopropyl groups exhibit a set of two signals at δ = 2.97 and 1.01 ppm as an septet and a doublet, respectively. The X-ray structures of 18 and 19 are presented in Figure 3, showing a C2 symmetric conformation (half a molecule is present in each asymmetric unit). The two amine moieties of 18 are located close to the bisecting plane of the bent metallocene (ω = 0.93(4)°) and oriented away from each other in an antiperiplanar-conformation (ϕ = 170.4(1)°).

A slight difference in the position of the amine moiety is noted for

19 (ω = -9.91(5)°), ϕ = -160.34(11)°).
Having succeeded in installing tertiary amines close to the metallocenyl moiety, we next envisioned analogous halfsandwich titanium complexes. Indeed, we wondered whether these would show an interaction between the titanium center and the amine. All attempts at forming such complexes failed except for the synthesis of TMP complex 22 (Scheme 5). In this case, metalation was carried out with TiCl4 on the mixture of silylated fulvenes 21, readily prepared from 5 and TMSCl. Complex 22 was obtained in 74% yield and fully characterized. The 1 H NMR spectrum has roughly the same aspect as the one from 5. The Cp' signals are shifted downfield to 6.40 and 6.15 ppm. The signal of the bridging CH2 appears as a singlet at 3.75 ppm, while the CH2 groups of the TMP ring resonate as multiplets centered at δ = 1.26 and 1.36 ppm. The methyl groups of the TMP moiety resonate as a singlet at δ = 0.82 ppm, which argues against the presence of a Ti-N interaction in solution. Consistently, the N atom of 22 resonates at -308 ppm (established by 1 H 15 N HMBC), a value which is very close to the one observed for complex 11. [14] Ti Cl The solid-state structure of 22 (Figure 4) shows a typical pseudotetrahedral geometry for the titanium center (piano stool-like structure), and corroborated the absence of Ti-N interaction previously inferred from solution 1 H NMR spectroscopy. The torsion angle ϕ defined above is equal to 107.03(11)° indicating that the tetramethylpiperidine moiety remains tilted toward the Cp' ring as in the structures of 11 and 12. To the best of our knowledge, this is the first example of a trichlorido aminomethylcyclopentadienyl half-sandwich titanium complex. Precedents with longer alkyl spacers and less bulky amines have been described. [1a,3c,7g,16] In most of these complexes, intra-or intermolecular coordination of the amine to Ti was observed.

Cl

Conclusions

In summary, we have described the synthesis of new (aminomethyl)cyclopentadienide salts via the nucleophilic substitution of sodium cyclopentadienide on Katritzky's (benzotriazolyl) derivatives. These ligands can be coordinated to Ti and Zr, yielding early metallocene complexes with a pendant tertiary amine arms. The absence of interactions between the Lewis acidic metal center and the Lewis basic N atom in all of the prepared complexes suggests that these new ligands could be used in organometallic frustrated Lewis pairs. [17] Experimental Section General considerations. All reactions were carried out under an atmosphere of Ar using Schlenk techniques or an Ar glovebox. Alumina (Brockman grade II) and 3Å molecular sieves were activated by heating for at least 6 hours above 230°C under vacuum (< 10 -1 mbar), and stored in the glovebox in order to be used as drying agents (see below). Dichloromethane, diethyl ether, pentane, tetrahydrofuran and toluene were dried using a solvent purification system (MBraun SPS-800) whilst acetonitrile, benzonitrile, bromobenzene, chlorobenzene, and heptane were distilled over CaH2 and stored over activate 3Å molecular sieves under Ar. Deuterated solvents were dried by passage through a short column of activated neutral alumina and stored over activated molecular sieves in the glovebox, either at room temperature (C6D6, C6D5Br, d6-DMSO) or at -18°C (d8-THF, CD2Cl2, CDCl3, CD3CN). Chlorotrimethylsilane was distilled under atmospheric pressure before use. The following chemicals CpNa, [18] TiCl4(THF)2, [19] ZrCl4(THF)2, [19] CpTiCl3, [20] Cp*TiCl3, [21] and the N-benzotriazolylmethyl-amine derivatives [13] were synthesized according to literature procedures. CpZrCl3 was sublimed (1.9x10 -2 mbar, 170°C). All other reagents were commercially available and used as received or purified according to the purification guide of laboratory chemicals if required. [START_REF] Armarego | Purification of laboratory chemicals 6th ed[END_REF] All of the analyses were performed at the "Plateforme X-Ray experimental procedure. Suitable crystals for X-ray analysis were selected and mounted on a mylar loop with oil on either a Bruker APEX-II CCD or on a Bruker D8 VENTURE diffractometer. Crystals were kept at 115 K or at 100K during data collections. The structures were solved by direct methods using SIR2004, [START_REF] Burla | [END_REF] ShelXS [24] or ShelXT [25] structure solution programs and refined with the ShelXL [26] refinement package using Least Squares minimization against |F| 2 with the aid of Olex2 program. [27] The structure of 9 was refined as a 2-component twin with the twin law (-1.0, 0.0, 0.0, 0.0, -1.0, 0.0 1.0, 0.0, 1.0), BASF 0.3909 (5) in P21/c. The structure of 14 is slightly disordered: two positions for the CHCl3 solvate molecule, the pyrrolidine ring and the Cp ring were refined (Cp ring as rigid group). The structure of 17 is also disordered, the TiCl2Cp fragment occupies two positions with a multiplicities of 0.5/0.5. Two different conformations are thus obtained. The disordered Cp rings were also refined as rigid groups.

CCDC 1825458-1825467 contain the Supporting crystallographic data for this paper. These data can be obtained free of charge via www.ccdc. Representative procedure for the synthesis of 1-5: To a suspension of CpNa (2.6 g, 0.024 mol, 1.2 eq.) in 50 mL of Et2O, was added dropwise by cannulation at -15°C, an etheral solution of N-benzotriazolylmethyl-amine (0.02 mol, 1 eq.). The resulting solution was left to warm up to room temperature and then stirred for two additional hours (refluxed overnight for compounds 1 and 4). The white precipitate formed during the reaction was filtered off and extracted with 15 mL of Et2O. The yellow filtrate was washed twice with 30 mL of deionized water. The combined organic phases were washed with a solution of saturated brine (30 mL) and dried over MgSO4. After filtration and careful evaporation of the solvent, the yellow oil was dissolved in pentane (40 mL) and left 10 min during which time the residual starting material precipitated. After filtration and careful (25°C, mild depression) evaporation the corresponding diene was obtained as yellow oil. This unstable compound was directly used after dissolving in 50 mL of Et2O. The resulting solution was cooled down to 0°C, and a freshly-prepared solution in toluene of KHMDS (0.95 eq. vs isolated diene, 20 mL) was added dropwise. The cold bath was left to warm to room temperature over 5 h. The precipitate formed during this time was filtered off, washed with 10 mL of cold Et2O, 2 x 15 mL of pentane, yielding the desired compound.

Synthesis of 1 (beige powder, 1.83 g, yield: 39 %). [START_REF]high airsensitivity of the compounds and / or the presence of residual byproducts[END_REF] Amount of substrates/reactants: CpNa (2.09 g, 0.0238 mol, 1.2 eq.); Nbenzotriazolylmethyl-N-methyl-1-phenylmethanamine (5.00 g, 0.0198 mol, 1.0 eq.); KHMDS ( 2.82 g, 0.0141 mol, 0.95 eq.). Synthesis of 2 (beige powder, 6.80 g, yield: 74 %). [START_REF]high airsensitivity of the compounds and / or the presence of residual byproducts[END_REF] Amount of substrates/reactants: CpNa (5.30 g, 0.0602 mol, 1.2 eq.); Nbenzotriazolylmethyl-pyrrolidine (10.0 g, 0.0494 mol, 1 eq.); KHMDS (7.60 g, 0.0381 mol, 0.98 eq.). Synthesis of 3 (beige powder, 4.80 g, yield 67%). [START_REF]high airsensitivity of the compounds and / or the presence of residual byproducts[END_REF] Amount of substrates/reactants: CpNa (3.80 g, 0.0431 mol, 1.3 eq.); Nbenzotriazolylmethyl-N,N-diisopropylamine (7.70 g, 0.0331 mol, 1.0 eq.). KHMDS (4. Synthesis of 4 (brown powder, 8.52 g, overall yield: 60%). [START_REF]high airsensitivity of the compounds and / or the presence of residual byproducts[END_REF] Amount of substrates/reactants: CpNa (6.00 g, 0.0547 mol, 1.2 eq.); Nbenzotriazolylmethyl-N-benzyl-1-phenylmethanamine (15 g, 0.0457 mol, 1.0 eq.); KHMDS (5.77 g, 0.0289 mol, 0.90 eq.). Synthesis of 5 (beige powder, 12.0 g, yield 86%). [START_REF]high airsensitivity of the compounds and / or the presence of residual byproducts[END_REF] Amount of substrates/reactants: CpNa (0.0682 mol, 1.36 eq.); Nbenzotriazolylmethyl-2,2,6,6-tetramethylpiperidine (13.8 g, 0.0507 mol, 1.0 eq.); KHMDS (8.85 g, 0.0444 mol, 0.99 eq.). HMRS (ESI negative mode, dimethylsulfoxide): calcd. General procedure for the synthesis of 6-11: A solution of sodium (aminomethyl)cyclopentadienide (0.818g, 0.0034 mol, 1.05 eq.) in 25 mL of THF was added to a solution of CpTiCl3 (0.718 g, 0.0032 mol, 1.0 eq.) in 20 mL of toluene, by cannulation at -30°C. The resulting solution was left to warm up to room temperature and then stirred overnight. The crude red solution was first evaporated. The deep red oil obtained was dissolved in 20 mL of CH2Cl2, filtered through diatomaceous earth, which was then washed by 2 x 5 mL of CH2Cl2. The deep red extracts obtained were concentrated by evaporation of 3 /4 of the solvent and the compound was precipitate by addition of pentane with vigorous stirring. The orange-red precipitate was filtered off, and dried to yield the desired compound.

Synthesis of 6 (orange-red powder, 0.680 g, yield = 54 %). Amount of substrates/reactants: 1 (0.818 g, 0.0034 mol, 1.05 eq.); CpTiCl3 (718 mg, 0.0032 mol, 1.0 eq.). HMRS (Positive mode ESI, dichloromethane): m/z calcd. for C19H21NClTi [M-Cl] + 346.08388; found 346.08446 (Rel. ab.: 90%, 2.3 ppm). Elemental Analysis: % calcd for C19H19NCl2Ti: C,59.72;H,5.54;N,3.67;Found: C,58.93;H,5.50;N,3.58. 1 H NMR (600.23 MHz,300 K,: δ 1 H: 7.29 (m, 2H, o-Ph), 7.18 (m, 2H, m-Ph), 7.10 (m, 1H, p-Ph) 6.26 (m, 2H, H2 of Cp'), 5.98 (s, 5H, Cp), 5.68 (m, 2H, H3 of Cp'), 3.74 (s, 2H, NCH2C5H4), 3.33 (s, 2H, NCH2Ph), 2.02 (s, 3H, NCH3). 13 C{ 1 H} NMR (125.77 MHz, 300 K, benzene-d6): δ 13 C: 139.6 (i-Ph), 133.2 (i-Cp'), 129.3 (o-Ph), 128.6 (m-Ph), 127.4 (p-Ph), 124.6 (C2 of Cp'), 119.5 (Cp), 115.1 (C3 of Cp'), 62.1 (NCH2Ph), 57.7 (NCH2C5H4), 42.2 (NCH3).

Synthesis of 7 (orange-red powder, 2.74 g, yield = 69 %). Amount of substrates/reactants: 2 (2.25 g, 0.0120 mol, 1.05 eq.); CpTiCl3 (2.51 g, 0.0114 mol, 1.0 eq.). HMRS (Positive mode ESI, dichloromethane): m/z calcd. for C15H19NClTi [M-Cl] + 296.06717; found 296.06819 (Rel. ab.: 50%, -2.8 ppm). Elemental Analysis: % calcd for C15H19NCl2Ti: C, 54. 25;H,5.77;N,4.22;Found: C,54.66;H,5.51;N,4.26. 1 H NMR (600 MHz, 300 K, dichloromethane-d2): δ 1 H: 6.57 (s, 5H, Cp), 6.52 (m, 2H, H2 of Cp'), 6.46 (m, 2H, H3 of Cp'), 3.67 (s, 2H, NCH2C5H4), 2.55 (broad, 4H, NCH2CH2), 1.77 (broad, 4H, NCH2CH2). 13 C{ 1 H} NMR (150.94 MHz, 300 K, dichloromethane-d2): δ 13 C: 133.9 (i-Cp'), 124.7 (C2 of Cp'), 120.6 (Cp), 116.8 (C3 of Cp'), 55.8 (NCH2C5H4), 54.7 (NCH2CH2), 24.1 (NCH2CH2).

Synthesis of 8 (orange-red powder, 1.61 g, yield = 80 %). Amount of substrates/reactants: 3 (1. 19 g, 0.0055 mol, 1.05 eq.); CpTiCl3 (1.14 g, 0.0052 mol, 1.0 eq.). HMRS (Positive mode ESI, dichloromethane): m/z calcd. for C17H26NClTi [M+H] + 362.09189; found 362.09285 (Rel. ab.: 100%, 3.4 ppm). Elemental Analysis: % calcd for C17H25Cl2NTi: C,56.38;H,6.96;N,3.87;Found: C,55.99;H,6.96;N,3.88. 1 H NMR (600.23 MHz,300 K,: δ 1 H: 6.54 (s, 5H, Cp), 6.52 (m, 2H, H2 of Cp'), 6.44 (m, 2H, H3 of Cp'), 3.71 (s, 2H, NCH2Cp), 2.97 (sept, 3 JHH = 6.6 Hz, 2H, NCH), 1.01 (d,3 JHH = 6.6 Hz,12H,NCHCH3). 13 C{ 1 H} NMR (150.94 MHz, 300 K, dichloromethane-d2): δ 13 C: 140.4 (i-Cp'), 124.1 (C2 of Cp'), 120.3 (Cp), 117.2 (C3 of Cp'), 49.2 (NCH), 45.6 (NCH2C5H4), 21.3 (NCHCH3).

Synthesis of 9 (red powder, 0.923 g, yield = 46 %). Amount of substrates/reactants: 3 (1.00 g, 0.0046 mol, 1.05 eq.) Cp * TiCl3 (1.27 g, 0.0044 mol, 1.0 eq.). HMRS (Positive mode ESI, dichloromethane): m/z calcd. for C22H36NCl2Ti [M+H] + 432.17019; found 432.16878 (Rel. ab. 100%, -2.5 ppm). Elemental Analysis: % calcd for C22H35NCl2Ti: C, 61.12; H, 8.16; N, 3.24; Found: C, 61.13; H, 8.26; N, 3.40. 1 H NMR (600.23 MHz, 300 K, dichloromethane-d2): δ 1 H: 6.19 (m, H2 of Cp'), 5.99 (m, 2H, H3 of Cp'), 3.69 (s, 2H, NCH2C5H4), 2.97 (sept, 3 JHH = 6.7 Hz, 2H, NCH), 2.01 (s, 15H, CH3 of Cp*), 1.00 (d, 3 JHH = 6.7 Hz, 12H, NCHCH3). 13 C{ 1 H} NMR (150.94 MHz, 300 K, dichloromethane-d2): δ 13 C: 139.5 (i-Cp'), 130.0 (i-Cp*), 124.4 (C2 of Cp'), 115.9 (C3 of Cp'), 49.1 (NCH), 45.4 (NCH2C5H4), 21.3 (NCHCH3), 13.8 (CH3 of Cp*).

Synthesis of 10 (orange-red powder, 4.0 g, yield = 80 %). Amount of substrates/reactants: 4 (3.76 g, 0.0119 mol, 1.05 eq.); CpTiCl3 (2.50 g, 0.0114 mol, 1.0 eq.). HMRS (Positive mode ESI, dichloromethane): m/z calcd. for C25H26NCl2Ti [M+H] + 458.09196; found 458.09393 (Rel. ab.: 100%, 5.0 ppm). Elemental Analysis: % calcd: C, 65.53; H, 5.50; N, 3.06; Found: C, 66.52; H, 5.57; N, 3.20. 1 H NMR (600.23 MHz, 300 K, dichloromethane-d2): δ 1 H: 7.38 (m, 4H, o-Ph), 7.33 (m, 4H, m-Ph), 7.25 (m, 2H, p-Ph), 6.52 (m, 2H, H2 of Cp'), 6.43 (m, 2H, H3 of Cp'), 6.38 (s, 5H, Cp), 3.60 (s, 2H, NCH2C5H4), 3.57 (s, 4H, NCH2Ph). 13 C{ 1 H} NMR (150.94 MHz, 300 K, dichloromethane-d2): δ 13 C: 139.9 (i-Ph), 133.5 (i-Cp'), 129.5 (o-Ph), 128.8 (m-Ph), 127.6 (p-Ph), 124.5 (C2 of Cp'), 120.5 (Cp), 117.6 (C3 of Cp'), 58.8 (NCH2Ph), 53.6 (NCH2C5H4).

Synthesis of 11 (orange powder, 0.870 g, yield = 68 %). Amount of substrates/reactants: 5 (0.693 g, 0.0032 mol, 1.05 eq.); CpTiCl3 (0.800 g, 0.0031 mol, 1.0 eq.). HMRS (Positive mode ESI, dichloromethane): m/z calcd. for C20H30NCl2Ti [M+H] + 402.12322; found 402.12089 (Rel. ab.: 90%, -5.1 ppm). Elemental Analysis: % calcd for C20H29NCl2Ti: C, 59.72; H, 7.27; N, 3.48; Found: C, 59.88; H, 7.71; N, 3.44. 1 H NMR (600.23 MHz, 300 K, benzene-d6): δ 1 H: 6.23 (broad, 2H, H2 of Cp'), 6.01 (s, 5H, Cp), 5.70 (broad, 2H, H3 of Cp'), 4.02 (s, 2H, NCH2C5H4), 1.43 (m, 2H, NC(CH3)2CH2CH2), 1.37 (m, 4H, NC(CH3)2CH2), 1.01 (s, 12H, NC(CH3)2). 13 C{ 1 H} NMR (150.94 MHz, 300 K, benzene-d6): δ 13 C: 146.2 (i-Cp'), 121.9 (C2 of Cp'), 119.1 (Cp), 115.6 (C3 of Cp'), 55.6 (NC(CH3)2), 45.6 (NCH2C5H4), 41.9 (NC(CH3)2CH2), 28.2 (broad, NC(CH3)2), 18.2 (NC(CH3)2CH2CH2).

Synthesis of 12.

A solution of 5 (0.586 g, 0.0027 mol, 1.0 eq.) in 35 mL of THF was added to a solution of CpZrCl3 (0.700 g, 0.0027 mol, 1.0 eq.) in 35 mL of THF cooled down to -30°C. The resulting solution was left to warm up to room temperature and then stirred overnight. The crude solution was evaporated and the residue was treated with 30 mL of dichloromethane. After filtration through diatomaceous earth, the filtrate was evaporated and the residue triturated with 15 mL of pentane in order to form a white powder. The supernatant was removed and the precipitate washed twice with 5 mL of pentane. The desired compound was obtained after removal of all volatiles as a white powder. (0.640 g, 54%). [START_REF]Satisfactory elemental analyses were not obtained due to the presence of residual LiCl[END_REF] HMRS (Positive mode ESI, dichloromethane): m/z calcd. for C20H30NCl2Zr [M+H] + 444.07969; found 444.07893 (Rel. ab.: 10%, -1.701 ppm), C15H26N [M-CpZrCl2+H] + 220.20652 found 220.20539 (Rel. ab.: 100%, -5.1 ppm). 1 H NMR (600.23 MHz, 300 K, tetrahydrofuran-d8): δ 1 H: 6.45 (s, 5H, Cp), 6.38 (m, 2H, H3 of Cp'), 6.31 (m, 2H, H2 of Cp'), 3.82 (s, 2H, NCH2C5H4), 1.55 (m, 2H, NC(CH3)2CH2CH2), 1.42 (m, 4H, NC(CH3)2CH2), 1.03 (s, 12H, NC(CH3)2). 13 C{ 1 H} NMR (150.94 MHz, 300 K, tetrahydrofuran-d8): δ 13 C: 142.6 (i-Cp'), 117.6 (C2 of Cp'), 116.6 (Cp), 113.9 (C3 of Cp'), 56.2 (NC(CH3)2), 45.4 (NCH2C5H4), 42.7 (NC(CH3)2CH2), 28.6 (NC(CH3)2), 18.9 (NC(CH3)2CH2CH2).

General procedure for the synthesis of 13-17. To a solution of (aminomethyl)titanocene dichloride (0.0003 mol, 1 eq., 5 mL toluene) was added dropwise a freshly-prepared HCl solution (5 mL of toluene). A red precipitate was directly formed. The reaction mixture was left to stir for 15 min. The precipitate was then filtered off and washed with 3 x 5 mL of pentane under aerobic conditions, yielding the desired product.

Synthesis of 13 (red powder, 0.074 g, yield: 67 %). Amount of substrates/reactants: 6 (0.100 g, 0.0003 mol, .01 eq., 5 mL toluene); 5 mL of a HCl-saturated toluene solution. HMRS (Positive mode ESI, dichloromethane): m/z calcd. for C19H22NCl2Ti [M] + 382.06060; found 382.05935 (Rel. ab.: 10%, -2.6 ppm). Elemental Analysis: % calcd for C19H22Cl3NTi: C, 54.51; H, 5.30; N, 3.35; Found: C, 54.50; H, 5.44; N, 3.69. 1 H NMR (600.23 MHz, 300 K, chloroform-d1): δ 1 H: 12.83 (s, 1H, NH), 7.60 (m, 2H, o-Ph), 7.46 (m, 3H, m-Ph overlapping with p-Ph), 7.07 (q, 3 JHH = 4 JHH = 2.6 Hz, 1H, H2 of Cp'), 6.97 (q, 3 JHH = 4 JHH = 2.6 Hz, 1H, H5 of Cp'), 6.69 (s, 5H, Cp), 6.60 (q, 3 JHH = 4 JHH = 2.6 Hz, 1H, H3 of Cp'), 6.57 (q, 3 JHH = 4 JHH = 2.6 Hz, 1H, H4 of Cp'), 4.38 (dd, 2 JHH = 13.2 Hz 3 JHH = 3.6 Hz, 1H, NCH2Ph), 4.37 (dd, 2 JHH = 13.7 Hz 3 JHH = 4.6 Hz, 1H, NCH2C5H4), 4.13 (dd, 2 JHH = 13.7 Hz, 3 JHH = 6.0 Hz, 1H, NCH2C5H4), 4.04 (dd, 2 JHH = 13.2 Hz, 3 JHH = 6.6 Hz, 1H, NCH2Ph), 2.60 (d, 3 JHH = 2.6 Hz, 3H, NCH3). 13 C{ 1 H} NMR (150.94 MHz, 300 K, chloroform-d1): δ 13 C: 131.5 (o-Ph), 130.5 (m-Ph), 129.7 (p-Ph), 128.2 (i-Ph), 125.9 (C5 of Cp'), 124.8 (C2 of Cp'), 122.0 (i-Cp'), 121.3 (Cp), 117.0 (C4 of Cp'), 116.4 (C3 of Cp'), 60.0 (NCH2Ph), 55.4 (NCH2Cp), 39.2 (NCH3).

Synthesis of 14 (red powder, 1.60 g, yield: 52 %). Amount of substrates/reactants: 7 (3.08 g, 0.0093 mol, 1.0 eq., 20 mL dichloromethane); 20 mL of a HCl-saturated toluene solution. HMRS (Positive mode ESI, dichloromethane): m/z calcd. for C15H20NCl2Ti [M-Cl+H] + 332.04492; found 332.04335 (Rel. ab.: 100%, -4.0 ppm). Elemental Analysis: % calcd for C15H20Cl3NTi: C,48.88;H,5.44;N,3.69;Found: C,49.14;H,5.82;N,3.89. 1 H NMR (600 MHz,300 K,: δ 1 H: 12.77 (broad, 1H, NH), 6.92 (m, 2H, H2 of Cp'), 6.67 (s,5H,Cp),6.56 (m,2H,H3 of Cp'),4.27 (d,3 JHH = 6.0 Hz, 2H, NCH2C5H4), 3.68 (m, 2H, NCH2CH2), 2.89 (m, 2H, NCH2CH2), 2.25 (m, 2H, NCH2CH2), 2.10 (m, 2H, NCH2CH2). 13 C{ 1 H} NMR (150.94 MHz, 300 K, chloroform-d1): δ 13 C: 125.0 (C2 of Cp'), 123.1 i-Cp'), 121.1 (Cp), 116.2 (C3 of Cp'), 54.3 (NCH2C5H4), 53.8 (NCH2CH2), 23.5 (NCH2CH2). 15 (red powder, 0.490 g, yield: 66 %). Amount of substrates/reactants: 8 (0.725 g, 0.0018 mol, 1 eq., 5 mL toluene); 5 mL of a HCl-saturated toluene solution. HMRS (Positive mode ESI, dichloromethane-methanol): m/z calcd. for C19H21NClTi [M-Cl] + 362.09189; found 362.09282 (Rel. ab.: 85%, 3.3 ppm). Elemental Analysis: % calcd for C25H26Cl3NTi: C, 51. [START_REF] Armarego | Purification of laboratory chemicals 6th ed[END_REF]H,6.57;N,3.51;Found: C,50.95;H,7.64;N,3.52. 1 H NMR (600.23 MHz,300 K,: δ 1 H: 11.62 (broad, 1H, NH), 7.00 (m, 2H, H2 of Cp'), 6.68 (s,5H,Cp),6.57 (m,2H,H3 of Cp'),4.35 (d,3 JHNH = 5.2 Hz, 2H, NCH2C5H4), 3.71 (dsept, 3 JHH = 6.7 Hz, 3 JHNH = 3.4 Hz, 2H, NCH), 1.56 (d,3 JHH = 6.7 Hz, 6H, NCHCH3), 1.51 (d,3 JHH = 6.7 Hz, 6H, NCHCH3). 13 C{ 1 H} NMR (150.94 MHz, 300 K, chloroform-d1): δ 13 C: 127.0 (C2 of Cp'), 122.8 (i-Cp'), 120.9 (Cp), 115.0 (C3 of Cp'), 55.2 (NCH), 46.6 (NCH2C5H4), 19.3 (NCHCH3), 18.1 (NCHCH3).

Synthesis of

Synthesis of 16 (red powder, 0.100 g, yield: 60 %). Amount of substrates/reactants: 10 (0.150 g, 0.0005 mol, 1.0 eq., 5 mL toluene); 5 mL of a HCl-saturated toluene solution. HMRS (Positive mode ESI, dichloromethane-methanol): m/z calcd. for C25H26NCl2Ti [M-Cl] + 458.09196; found 458.09056 (Rel. ab.: 15%, -2.3 ppm). Elemental Analysis: % calcd for C25H26Cl3NTi: C,60.70;H,5.30;N,2.83;Found: C,60.54;H,5.13;N,2.92. 1 H NMR (600.23 MHz,300 K,: δ 1 H: 12.90 (broad, 1H, NH), 7.61 (m, 4H, o-Ph), 7.45 (m, 6H, m-Ph and p-Ph), 6.96 (m, 2H, H2 of Cp'), 6.61 (s, 5H, Cp), 6.47 (m, 2H, H3 of Cp'), 4.25 (dd, 2 JHH = 13.3 Hz, 3 JHNH = 4.5 Hz, 2H, NCH2Ph), 4.16 (d, 3 JHNH = 4.2 Hz, 2H, NCH2C5H4), 4.15 (dd, 2 JHH = 13.3 Hz, 3 JHNH = 4.5 Hz, 2H, NCH2Ph). 13 C{ 1 H} NMR (150.94 MHz, 300 K, chloroform-d1): δ 13 C: 131.7 (o-Ph), 130.4 (p-Ph), 129.7 (m-Ph), 128.4 (i-Ph), 124.9 (C2 of Cp'), 122.5 (i-Cp'), 121.2 (Cp), 117.3 (C3 of Cp'), 57.5 (NCH2Ph), 52.5 (NCH2C5H4). (red powder, 1.86 g, yield: 73 %). Amount of substrates/reactants: 11 (2.55 g, 0.006 mol, 1.0 eq., 5 mL toluene); 5 mL of a HCl-saturated toluene solution. (d, 3 JHH = 3.5, 2H, NCH2C5H4), 2.77 (td 2 JHH = 3 JHH = 13.7 Hz, 3 JHH = 3.8 Hz, 2H, NC(CH3)2CH2), 1.79 (s, 6H, NC(CH3)2), 1.78 (1 overlapping m, 1H, NC(CH3)2CH2CH2), 1.73 (m, 1H, NC(CH3)2CH2CH2), 1.56 (dt, 2 JHH = 13.7 Hz, 3 JHH = 3.0 Hz, 2H, NC(CH3)2CH2), 1.46 (s, 6H, NC(CH3)2). 13 C{ 1 H} NMR (150.94 MHz, 300 K, chloroform-d1): δ 13 C: 129.9 (C2 of Cp'), 122.9 (i-Cp'), 120.6 (Cp), 112.6 (C3 of Cp'), 66.9 (NC(CH3)2), 46.1 (NCH2C5H4), 36.8 (NC(CH3)2CH2), 30.2 (NC(CH3)2), 22.1 (NC(CH3)2), 16.2 (NC(CH3)2CH2CH2). Synthesis of 18. TiCl4(THF)2 (0.268 g, 0.001 mol, 1 eq.) dissolved in 5 mL of Et2O was added dropwise at 0°C by cannulation on 4 (0.500 g, 0.002 mol, 2.0 eq.) dissolved in 10 mL of Et2O The resulting solution was left to warm up to room temperature and then stirred 30 min. The crude red/brown solution was filtered through diatomaceous earth which was then washed 2 x 5 mL of Et2O and residual starting material. The brown solution was then evaporated to yield a dark brown oil. The compound was purified by crystallization thanks to a biphasic (Dichloromethane: Pentane 1: 3) in the Glove box. After removing the mother liquor, red crystals were obtained after washing with pentane and drying (0.080 g, yield = 15 %). [30] HMRS (Positive mode ESI, dichloromethane): m/z calcd. for C40H41N2Cl2Ti [M+H] + 667.21258; found 667.21217 (Rel. ab.: 35%, 0.137 ppm), C40H40N2ClTi [M-Cl] + 631.23586; found 631.23645 (Rel. ab.: 5%, 1.7 ppm). 1 H NMR (600.23 MHz, 300 K, dichloromethane-d2): δ 1 H: 7.36 (m, 8H, o-Ph), 7.32 (m, 8H, m-Ph), 7.24 (m, 4H, p-Ph), 6.35 (m, 2H, H2 of Cp'), 6.25 (m, 2H, H3 of Cp'), 3.53 (broad, 8H, NCH2Ph), 3.52 (broad, 4H, NCH2C5H4). 13 C{ 1 H} NMR (150.94 MHz, 300 K, dichloromethane-d2): δ 13 C: 139.9 (i-Ph), 132.2 (i-Cp'), 129.4 (o-Ph), 128.8 (m-Ph), 127.6 (p-Ph), 124.5 (C2 of Cp'), 117.1 (C3 of Cp'), 58.7 (NCH2Ph), 53.6 (NCH2C5H4).

Synthesis of 17

Synthesis of 19.

A solution of 4 (1.20 g, 0.004 mol, 2.1 eq.) in 15 mL of THF was added to a freshly prepared solution of ZrCl4(THF)2 (0.425 mg of ZrCl4 in 7 mL of THF, 0.002 mol, 1.0 eq.), by cannulation at -10°C. The resulting solution was left to warm up to room temperature and then stirred overnight. The crude brown solution was first evaporated. The brown oil obtained was dissolved in 25 mL of toluene, filtered through diatomaceous earth which was then washed by 4 x 10 mL of toluene and residual starting material. The yellow solution was then evaporated to form a brown sticky oil which was stirred overnight in a mixture of Dichloromethane: pentane (1: 3) to yield a white precipitate. The precipitate was filtered off, washed with pentane (2 x 10 mL) and dried to yield the desired compound. (0.400 g, yield = 31 %). [START_REF]Satisfactory elemental analyses were not obtained due to the presence of residual LiCl[END_REF] HMRS (Positive mode ESI, dichloromethane): m/z calcd. for C40H41N2Cl2Zr [M+H] + 709.16884; found 709.16653 (Rel. ab.: 15%, -3.3 ppm). 1 H NMR (600.23 MHz, 300 K, benzene-d6): δ 1 H: 7.32 (m, 8H, o-Ph), 7.20 (m, 8H, m-Ph), 7.10 (m, 4H, p-Ph), 6.06 (m, 4H, H2 of Cp'), 5.72 (m, 4H, H3 of Cp'), 3.62 (s, 4H, NCH2C5H4), 3.38 (s, 8H, NCH2Ph).

13 C{ 1 H} NMR (150.94 MHz, 300 K, benzene-d6): δ 13 C: 139.6 (i-Ph), 129.2 (o-Ph), 128.7 (i-Cp'), 128.6 (m-Ph), 127.4 (p-Ph), 118.4 (C2 of Cp'), 113.2 (C3 of Cp'), 58.1 (NCH2Ph), 52.7 (NCH2C5H4).

Synthesis of 20.

A solution of 3 (3.0 g, 0.014 mol, 2.0 eq.) in 70 mL of THF was added to a freshly prepared solution of ZrCl4(THF)2 (1.77 g of ZrCl4 in 30 mL of THF, 0.008 mol, 1.1 eq.), by cannulation at -10°C. The resulting solution was left to warm up to room temperature and then stirred overnight. The crude brown solution was filtered through diatomaceous earth to remove KCl salts. Solvent was removed by vacuum pumping and the white precipitate obtained was wash with pentane (2 x 20 mL) to afford the desired compound in good yield (2.75 g, yield = 76 %). [START_REF]Satisfactory elemental analyses were not obtained due to the presence of residual LiCl[END_REF] HMRS (Positive mode ESI, dichloromethane): not detected. 1 H NMR (600.23 MHz, 300 K, dichloromethane-d2): δ 1 H: 6.35 (m, 4H, H2 of Cp'), 6.33 (m, 4H, H3 of Cp'), 3.66 (s, 4H, NCH2C5H4), 2.97 (sept, 3 JHH = 6.5 Hz, 4H, NCH), 1.01 (d, 3 JHH = 6.5 Hz, 24H, NCHCH3). 13 C{ 1 H} NMR (150.94 MHz, 300 K, dichloromethane-d2): δ 13 C: 136.0 (i-Cp'), 118.4 (C2 of Cp'), 113.0 (C3 of Cp'), 48.8 (NCH), 44.8 (NCH2C5H4), 21.2 (NCHCH3).

Synthesis of 22.

A solution of 5 (1.0 g, 0.004 mol, 1.0 eq.) in 30 mL of THF was cooled down at -70°C. Trimethylsilyl chloride (0.55 mL, 4.3mmol, 1.1 eq.) was added dropwise to the solution and the resulting solution was warmed up to room temperature and stirred for 3h. All volatiles were removed by evaporation to yield a yellow sticky solid. Dissolution of the solid in 30 mL of pentane and filtration through diatomaceous earth were used to remove KCl salts. Pentane was evaporated to yield the corresponding silylated amino cyclopentadiene (0.970 g, 0.003 mol, 85 %) as a yellow solid. It was dissolved in 40 mL of toluene, and added at -30°C to a toluenic solution of TiCl4 (0.36 mL, 0.003 mol, 1.0 eq.). The resulting brown solution was left to warm up to room temperature and stirred overnight. A dark brown solution with a black precipitate was obtained. The heterogeneous solution was dried in vacuo. The brownish residue was triturated in pentane (30 mL) and dried in vacuo. The sticky residue was triturated in cold pentane (10 mL) and filtered to yield a dark brown precipitate (1.08 g, 87% step yield, 74% global yield). HMRS (Positive mode ESI, dichloromethane / MeOH): m/z calcd. for C16H28NCl2TiO [M-Cl+OCH +H] + 368.10244; found 368.1074 (Rel. ab.: 100%, 2.3 ppm). Elemental Analysis: % calcd for C15H24NCl3Ti: C,48.35;H,6.49;N,3.76;Found: C,48.67;H,6.69;N,3.63. 1 H NMR (600.23 MHz,300 K,: δ 1 H: 6.40 (m, 2H, H2 of Cp'), 6.15 (m, 2H, H2 of Cp'), 3.75 (s, 2H, NCH2C5H4), 1.36 (m, 2H, NC(CH3)2CH2CH2), 1.26 (m, 4H, NC(CH3)2CH2), 0.82 (s, 12H, NC(CH3)2). 13 C{ 1 H} NMR (150.94 MHz, 300 K, benzene-d6): δ 13 C: 151.0 (i-Cp'), 123.9 (C2 of Cp'), 123.5 (C2 of Cp'), 55.3 (NC(CH3)2), 45.5 (NCH2C5H4), 41.2 (NC(CH3)2CH2), 27.9 (broad, NC(CH3)2), 17.8 (NC(CH3)2CH2CH2).

Scheme 2 .

 2 Scheme 2. Reagents and conditions: i) CpNa, Et2O, r.t., 4 h (for benzyl derivatives, reflux, 12 h); ii) H2O; iii) KHMDS, toluene / Et2O, 0 °C (10') to r.t. (2 h); iv) C5H5MCl3 (M = Ti, Zr), or C5Me5TiCl3, toluene / THF, -30 °C (30') to r.t.,12 h.

Figure 1 .

 1 Figure 1. ORTEP views of complexes 9-12 (hydrogen atoms as well as the second independent molecules for 9 and 11 are omitted for clarity) and definitions of the torsion angles ω and ϕ (Clm is the centroid of Cl1/Cl2). Ct1 and Ct2 are centroids of Cp' and Cp (Cp*) rings, respectively.

Figure 2 .

 2 Figure2. ORTEP view of 14,15 and 17 (hydrogen atoms removed for clarity except NH as well as counter anion and solvent). Selected bond lengths (Å) and angles(°): 14: Ct1-Ti = 2.062(3), Ct2-Ti = 2.057(6), Cl1-Ti = 2.3477(16), Cl2-Ti = 2.3645(16), Cl1-Ti-Cl2 = 92.43(6), Ct1-Ti-Ct2 = 131.9(2), ω = -0.99(18), ϕ = 176.1(4). 15: Ct1-Ti = 2.0617(6), Ct2-Ti = 2.0548(6), Cl1-Ti = 2.3461(4), Cl2-Ti = 2.3638(4), Cl1-Ti-Cl2 = 92.920(13), Ct1-Ti-Ct2 = 130.97(3), ω = 8.55(3), ϕ = -148.28(7). 17a (bisecting conformation): Ct1-Ti = 2.191(3), Ct2-Ti = 2.051(4), Cl1-Ti = 2.335(3), Cl2-Ti = 2.363(5), Cl1-Ti-Cl2 = 93.17(15), Ct1-Ti-Ct2 = 129.45(15), ω = 16.43(15), ϕ = -154.41(14). 17b (non-bisecting conformation): Ct1-Ti = 1.955(3), Ct2-Ti = 2.056(3), Cl1-Ti = 2.366(5), Cl2-Ti = 2.362(3), Cl1-Ti-Cl2 = 95.54(13), Ct1-Ti-Ct2 = 127.00(14), ω = 86.6(2), ϕ = -147.51(14).

Figure 3 .

 3 Figure 3. ORTEP views of complexes 18 and 19 (hydrogen atoms removed for clarity). Selected bond lengths (Å) and angles(°): 18: Ti-Cl = 2.3586(4), Ti-Ct = 2.0633(6), Cl-Ti-Cl = 94.37(2), Ct-Ti-Ct = 133.01(3), ω = 0.93(4), ϕ = 170.4(1). 19: Zr-Cl = 2.4468(4), Zr-Ct = 2.2058(8), C1-C6 = 1.504 (2), C6-N = 1.475 (2), Cl-Zr-Cl = 96.42(2), Ct-Zr-Ct = 130.08(3), ω = -9.91(5), ϕ = -160.34(11).

  Scheme 5. Synthesis of the half-sandwich titanium complex 22.

Figure 4 .

 4 Figure 4. ORTEP view of 22 (hydrogen atoms removed for clarity). Selected bond lengths (Å) and angles(°): Ct-Ti = 2.0162(10), Cl1-Ti = 2.2360(6), Cl2-Ti = 2.2308(6), Cl3-Ti =2.2425(6) , C1-C6 = 1.519(3), C6-N = 1.461(2), Ct-Ti-Cl1 = 116.78(3), Ct-Ti-Cl2 = 115.36(3), Ct-Ti-Cl3 = 113.27(3), Cl1-Ti-Cl2 = 100.32(3), Cl1-Ti-Cl3 = 102.32(3), Cl2-Ti-Cl3 = 106.94(3), ϕ = 107.03(11).

  d'Analyses Chimiques et de Synthèse Moléculaire de l'Université de Bourgogne". The identity and purity (> 95%) of the complexes were unambiguously established using elemental analysis, high-resolution mass spectrometry (Electrospray Ionization), NMR and IR spectroscopy. Elemental analyses were obtained on a Flash EA 1112 CHNS-O Thermo Electron Flash instrument. Exact masses of the isolated compounds were obtained on a Bruker micrOTOF-Q ESI-MS. 1 H (300.13, 500.03, or 600.23 MHz), 11 B (160.42 MHz), 13 C (125.77 or 150.94 MHz), 15 N (43.3 MHz) and 19 F (470.45 MHz) NMR spectra were recorded on Bruker 300 Avance III, 500 Avance III, or 600 Avance II spectrometers. Chemical shifts are quoted in parts per million (δ) relative to TMS ( 1 H and 13 C), or CH3NO2 (for 15 N). IR spectra were recorded on a FT BRUKER Vertex 70v spectrophotometer (Globar MIR and Hg FIR sources, Ge/KBr (MIR) and silicium (FIR) beamsplitters and Ge/KBr (MIR) and silicium (FIR) detectors).

  For C15H24N [M-K] -218.19033; found 218.19111 (Rel. ab.: 100%, -1.4 ppm). 1 H NMR (600.23 MHz, 300 K, tetrahydrofuran-d8): δ 1 H: 5.46 (m, 2H, H2 of Cp'), 5.38 (m, 2H, H3 of Cp'), 3.55 (s, 2H, NCH2C5H4), 1.57 (m, 2H, NC(CH3)2CH2CH2), 1.43 (m, 4H, NC(CH3)2CH2), 1.12 (s, 12H, NC(CH3)2). 13 C{ 1 H} NMR (150.94 MHz, 300 K, tetrahydrofuran-d8): δ 13 C: 123.5 (i-Cp'), 105.6 (C2 of Cp'), 104.1 (C3 of Cp'), 55.6 (NC(CH3)2), 45.0 (NCH2C5H4), 42.9 (NC(CH3)2CH2), 29.0 (NC(CH3)2), 19.2 (NC(CH3)2CH2CH2).

Acknowledgements

This work is supported by the CNRS, Université de Bourgogne, Conseil Régional de Bourgogne through the plan d'actions régional pour l'innovation (PARI), the fonds européen de développement regional (FEDER) programs, the Agence Nationale de la Recherche and Deutsche Forschungsgemeinschaft (MENOLEP project).