Sarah

Sarah Hadi

and lovely spirit and the confidence he gave me. My thanks to my friend and best "intern" Kajetan who inspired me a lot with his motivation, for the joyful time we spend together in preparing cryptophanes and of course for the chemistry songs he used to write. "Thank you" for my best friends Batoul, Riva,

Table of Content

Ce travail nécessite toutefois de développer en amont de nouveaux cryptophanes présentant toutes les caractéristiques requises pour une utilisation optimale en milieu physiologique. La mise en oeuvre de composés présentant une bonne solubilité est par exemple cruciale pour éviter les phénomènes d'agrégation pouvant se produire dans les milieux aqueux.

Le greffage sélectif d'une tête de reconnaissance présentant une activité biologique prédéfinie constitue également un verrou important qu'il faut prendre en compte. Au cours de ces 20 dernières années des résultats importants ont été rapportés dans la littérature et ont permis de démontrer l'intérêt de ces systèmes supramoléculaires pour l'imagerie RMN. Cependant, les systèmes développés jusqu'ici n'ont pas permis de résoudre de manière satisfaisante les deux points soulevés ci-dessus.

General Introduction

Cryptophanes are container-like molecular structures that have the ability to encapsulate various substrates in their lipophilic cavity. This structure, which is elegantly tailored, has offered different lessons in supramolecular chemistry especially in host-guest interactions. The high affinity of cryptophanes to xenon has been smartly exploited to design 129 Xe-NMR/MRI sensors.

Unlike the usual proton-based MRI tracers, 129 Xe-cryptophane combination allows accessing smart MRI biosensors that are characterized by their fast response and high sensitivity. Since 2001, a large number of hyperpolarized 129 Xe-cryptophane biosensors has been reported for the detection of various physical and biological targets like pH, temperature, enzymatic activity and ionic analytes. The synthesis of these molecular structures remains challenging and this thesis is dedicated to develop a new strategy aimed at facilitating the elaboration of these cryptophanes based biosensors.

The first part of this manuscript describes the synthesis of cryptophane platforms for biosensing applications. In the introductory chapter of this part, we gave a general view on cryptophane derivatives and their main features. Additionally, we delivered a non-exhaustive description of the new imaging and hyperpolarization techniques and the biosensors reported in literature. In a second chapter, we focused our attention on the synthesis and development of new cryptophane- [223] platforms, characterized by their improved water solubility and easy functionalization via the two available reactive sites. The sensors elaborated from these new cages were studied by hyperpolarized 129 Xe NMR spectroscopy and ITC experiments and the results are reported in the third chapter.

A second part of this manuscript is dedicated to the synthesis and the characterization of cryptophane- [223] derivatives subjected to self-encapsulation. An introductory chapter of this part describes the synthesis of enantiopure cryptophanes and their characterization by different

Introduction

The knowledge in biological systems' chemistry is inevitably inspiring. 1 Inspirited by natural processes, supramolecular chemistry or "the chemistry beyond the molecule" has emerged, as the study of the structure and function of intermolecular assemblies among molecules driven by weak non-covalent interactions. [START_REF] Lehn | Cryptates: Inclusion Complexes of Macropolycyclic Receptor Molecules[END_REF] Supramolecular chemistry has served, to a great extent in a better understanding of biological processes, which are vastly governed by non-covalent bonds like hydrogen bonding, Van der Waals interactions, solvophobic effect and coulombic interactions. [START_REF] Lehn | Towards Complex Matter: Supramolecular Chemistry and Self-Organization[END_REF] From protein folding, enzyme-substrate assembly, genetic information storage, transcription, to the most complicated cells interaction, highly selective and delicate cascade of events takes place, starting from the selective recognition of a receptor or a "host" to a substrate or a "guest". [START_REF] Kyba | Host-Guest Complexation. 1. Concept and Illustration[END_REF][START_REF] Fischer | Einfluss der Configuration auf die Wirkung der Enzyme[END_REF][START_REF] Schneider | Limitations and Extensions of the Lock-and-Key Principle: Differences between Gas State, Solution and Solid State Structures[END_REF] This recognition is based on complementarity, the strength and kind of interactions and the extent of preorganization of the non-complexed species before the binding event. [START_REF] Cram | Molecular Container Compounds[END_REF] Exploiting this knowledge, Pedersen in 1967 [START_REF] Pedersen | Cyclic Polyethers and Their Complexes with Metal Salts[END_REF] , Lehn and Sauvage in 1969 [START_REF] Dietrich | Diaza-Polyoxa-Macrocycles et Macrobicycles[END_REF] and Cram in 1973 [START_REF] Kyba | Chiral Recognition in Molecular Complexing[END_REF] have developed new families of structurally ion-selective complexes, establishing by this the foundations of the field. Greater complexity has been added to this field by introducing photo or electro-responsive motifs, making dynamic molecular assemblies, Figure 1. [START_REF] Shi | Selective Extraction of C70 by a Tetragonal Prismatic Porphyrin Cage[END_REF] Figure 1: earliest receptor crown ether by Pedersen, Cram's spherand, Lehn's macrobicylic cryptands and Stoddart's modern containers.

Among the vast majority of supramolecular designs, molecular containers possessing hollow, concave hydrophobic surfaces have gained special attention. Mimicking enzyme's hydrophobic pocket, these containers can selectively recognize smaller convex molecules based on the size, shape and nature of the inner surface. 12,[START_REF] Moran | Synthetic Molecular Vessels[END_REF] The residency time of such complexes is strongly dependent on the host's structure. For example, molecules with rigid enclosed spheres can permanently encapsulate guests and these are referred to as carcerands. 14,[START_REF] Bryant | Host-Guest Complexation. 55. Guest Capture during Shell Closure[END_REF] Other structures possess dynamic portals [START_REF] Liu | Gated Container Molecules[END_REF] allowing the exchange of the contained molecule with the bulk such as cavitands, deep cavitands, [START_REF] Rudkevich | Nanoscale Molecular Containers[END_REF] cryptophanes, [START_REF] Gabard | Synthesis of a (D3)-Bis(cyclotriveratrylenyl) Macrocage by Stereospecific Replication of a (C3)-Subunit[END_REF] hemicarcerands, [START_REF] Sherman | Syntheses and Properties of Soluble Carceplexes[END_REF] cyclodextrins, [START_REF] Szejtli | Introduction and General Overview of Cyclodextrin Chemistry[END_REF] hemicryptophanes, [START_REF] Zhang | Emergence of Hemicryptophanes: From Synthesis to Applications for Recognition, Molecular Machines, and Supramolecular Catalysis[END_REF] and calixarenes, [START_REF] Szejtli | Introduction and General Overview of Cyclodextrin Chemistry[END_REF] Figure 2. Once the complex formed, a change in the guest behavior is expected, due to the change in the surrounding environment. These include physical, mainly magnetic properties, which are well characterized by NMR spectroscopy, conformational and catalytic properties. 12 This effect is especially important because it paves the way for many applications such as catalysis, stabilization of highly reactive intermediates, sensors, nano-scale molecular reactors and separation agents. 23, 24 ,21 Figure 2: examples of cavity-containing macrocycles like cyclodextrins A, calixarenes B, cavitand C and hemicryptophane D.

The elegantly elaborated cryptophanes constitute a family of the earliest molecular containers with a hydrophobic cavity, Figure 3. Reported by André Collet in 1981, cryptophanes are initially

A B D C
made up of two C3-symmetric cyclotribenzylene CTB units connected by three modifiable bridges. The first cryptophane synthesized was the cryptophane-A, which is still very useful nowadays to prepare new cryptophane derivatives. This cryptophane was initially constructed to bind lipophilic molecules like halogenomethanes and isobutanes constituting by this, the first family of molecular containers to isolate lipophilic guests from the bulk. [START_REF] Gabard | Synthesis of a (D3)-Bis(cyclotriveratrylenyl) Macrocage by Stereospecific Replication of a (C3)-Subunit[END_REF] Remarkably, enantioenriched cryptophanes were the first molecular hosts to isolate and discriminate between the two enantiomers of CHFClBr, one of the smallest chiral molecules. For a long period of time, cryptophanes have been mainly framed in fundamental research, where the study of their complexes has served to understand molecular recognition. Since 2001, these molecules have gained a lot of attention in xenon based sensing applications. The remarkable structural features of cryptophanes, from chirality, tunable cavity size and solubility have been heavily exploited to finely tune properties to applications. In this chapter a detailed description of cryptophanes' structure, symmetry, conformations, synthesis, complexes and applications will be discussed.

Figure 3: structure of cryptophane- [222] (OMe)6 or the so-called cryptophane-A, under the IUPAC name: 3,22-(Epoxyethanoxy) -6,9:25,28-dietheno-7,36:17,26-dimethano 14,18:33,37 dimetheno18H,37 Hdibenzo[j,na1].

Cryptophane-A

Cryptophanes: General Features

Structure and Nomenclature:

Cryptophanes are constructed from two C3-symmetric CTB units, connected by three bridges whose length and nature can be varied. Numerous cryptophane derivatives have been prepared so far and the readers can refer to different reviews made by Collet, Brotin, Dutasta and Holman to appreciate the large diversity of cryptophane derivatives, which have been prepared to date. [START_REF] Collet | Cyclotriveratrylenes and Cryptophanes[END_REF][START_REF] Brotin | Cryptophanes and Their Complexes -Present and Future[END_REF]27 To talk about the CTB units, they have been synthesized since 1915 but correctly described in 1965. [START_REF] Lindsey | The Structure of Cyclotriveratrylene (10,15-Dyhydro-2,3,7,8,12,13-Hexamethpxy-5H-Tribenzo[a,d,g]Cyclononene) and Related Compounds[END_REF] These units are synthesized from three consecutive Friedel-Crafts alkylations of electron rich 3-4-disubstituted benzyl alcohols, which adopt a crown-shaped conformation. Depending on its substituents, CTB units can have an achiral C3v-symmetry if the substituents on the CTB rims, Rx and Ry are identical or a chiral C3 or C1-symmetry if they are different, Figure 4. [START_REF] Canceill | Analytical Optical Resolution of Bromochlorofluoromethane by Enantioselective Inclusion into a Tailor-Made "Cryptophane" and Determination of Its Maximum Rotation[END_REF] The two enantiomers of the chiral CTB units possess helical chirality, hence they can adopt an M or P helicity, which can interconvert at high temperature as will be seen later. CTB, by themselves, have been utilized for many applications, including binding of neutral molecules like fullerene, cations and anions and form liquid crystals. [START_REF] Steed | Ball and Socket Nanostructures: New Supramolecular Chemistry Based on Cyclotriveratrylene[END_REF][START_REF] Jurisson | Inclusion of Neutral and Anionic Guests within the Cavity of π-Metalated Cyclotriveratrylenes[END_REF][START_REF] Canceill | Macropolycyclic Receptor Cages Based on Binding and Shaping Sub-units. Synthesis and Properties of Macrocycle Cyclotriveratrylene Combinations. Preliminary Communication[END_REF] To talk about the cryptophanes' bridges, they can vary in nature and length from alkyl, alkenyl, aryl or alkynyl chains. [START_REF] Gabard | New D3 and C3h Cryptophanes with Ethelynic Bridges Structural Assignments, Absolute Confugrations and Circular Dichriosm of D3 Isomers[END_REF]34,[START_REF] Akabori | The Complexing Abilities of Diethyleneoxy-and Xylene-Bridged Cryptophanes with Alkanes[END_REF] The CTB and the bridges, combined, can determine the properties of the resulting cage like cavity size, solubility, chirality and their affinities to the guests.

3v if R X = R y C 3 if R X ≠ R y M P R Y > R x
The IUPAC nomenclature of cryptophanes is not adopted due to its complexity and length, Figure 3. Since the first reported cage, simpler names were given by A. Collet to different cages like A, B, C, E, etc... For example, cryptophane-A was the first cage prepared by Collet and coworkers. This cage possesses three ethylenedioxy linkers and six methoxy groups on the two CTB units. Cryptophane-B, however is the syn-isomer of cryptophane-A, which is recently reported by Brotin et al. [START_REF] Mulatier | Synthesis of Cryptophane-B: Crystal Structure and Study of Its Complex with Xenon[END_REF] These simplified names reflect neither the structure nor the time at which the cages were synthesized. Henceforth, a more generalized naming system is adopted.

This system is based on indicating the length of the linkers and the nature of substituents on the CTB caps whenever possible. For example cryptophane-A becomes [222] (OMe)6. For the moment no concrete naming system is adopted and each research group tends to name cryptophanes on their own way.

Cryptophane Symmetry:

Molecular symmetry of cryptophanes is one of the key features to be studied to better understand their mode of complexation and interactions with the guest. Inherited from the chirality of CTB caps and the arrangement of the substituents around the linkers, cryptophanes exist in two different diastereomers, the syn and the anti. This modification can have a huge impact on the binding properties of these derivatives, Figure 5.

Syn Anti

Compared to syn-isomer, anti-cryptophane is more flexible and has better binding properties. The anti-diastereomer forms when two CTB with the same absolute configuration are connected (MM or PP). This derivative is always chiral whatever the nature of the substituents or the linkers are, like cryptophane-A with D3 symmetry. The syn isomer is formed when the two CTB connected have opposite handedness (MP). This isomer is achiral, provided that the substituents and the linkers are identical (R1=R2 and X=Z), like cryptophane-B with C3h symmetry, else it will be chiral like cryptophane-D (syn-Cryptophane-[222] (OMe)3) with C3 symmetry. Table 1 provides the details of the point group and the chirality of the two diastereomers depending on the nature of the substituents and the linkers. [START_REF] Brotin | Cryptophanes and Their Complexes -Present and Future[END_REF] Table 1: summary of cryptophanes' symmetry and chirality.

Configuration Substituents and linkers

Symmetry Chirality

Syn

R1=R2, X=Z [START_REF] Mulatier | Synthesis of Cryptophane-B: Crystal Structure and Study of Its Complex with Xenon[END_REF][START_REF] Canceill | Two Step Synthesis of D3 and C3h Cryptophanes[END_REF] R1=R2, X≠Z R1≠R2, X=Z R1≠R2, X=Z [START_REF] Collet | Absolute Configuration of Bromochlorofluoromethane from Experimental and Ab Initio Theoretical Vibrational Raman Optical Activity[END_REF][START_REF] Berthault | Accurate PH Sensing via Hyperpolarized Xe NMR[END_REF] R1≠R2, X≠Z 41 The CTB bowls of cryptophanes can adopt two different conformations, a stable "crown" as proved by X-ray structure and a less stable "saddle" shaped conformation (disfavored by 13-16 kJ/mol). The formation of the "crown" conformer is usually evidenced by 1 H NMR spectroscopy, which shows distinct signals for the two diastereotopic protons of the methylene bridge (axial and equatorial protons). The latter pseudo-rotating "saddle" conformer is an intermediate in the interconversion of the two "crown" conformational isomers (M and P) obtained at higher temperature (at 100 °C, the interconversion takes place in few minutes), Figure 6. It is specifically formed by an inward flipping of the CTB methylene bridge. The lack of stability of the latter compared to "crown" conformation is due to the repulsion of the two hydrogens on the methylene bridge. [START_REF] Malthete | Inversion of the Cyclotribenzylene Cone in a Columnar Mesophase: A Potential Way to Ferroelectric Materials[END_REF][START_REF] Dale | Multistep Conformational Interconversion Mechanisms[END_REF] The two conformations show distinct NMR signals and they can be easily identified. Similar to CTB, tribenzotriquinacenes (TBTQ) are a family of three mutually fused indanes, developed by D. Kuck and coworkers. [START_REF] Markopoulos | Tribenzotriquinacene: A Versatile Synthesis and C3-Chiral Platforms[END_REF][START_REF] Niu | Synthesis and Optical Resolution of Inherently Chiral Difunctionalized Tribenzotriquinacenes[END_REF] These molecules are similar to CTB in symmetry and shape but differ in conformational rigidity. TBTQ are usually more rigid than CTB thanks to the bridge fusing the three methylene groups. With their original structure and rigidity, these molecules are usually hard to obtain in C3-symmetry. Racemic mixture of TBTQ trialdehydes has

Crown (C) Saddle (S) Inverted Crown (Ci)

been also used to construct cryptophanes by dynamic covalent assembly with enantiopure (1S,2S)-diamino-cyclohexane. This strategy led to the formation of three diastereomeric mixtures of multiple Schiff's bases and their corresponding hydrolysis gave rise to the enantiopure M and P-TBTQ, Figure 7. [START_REF] Wang | C3-Symmetrical Tribenzotriquinacene Derivatives: Optical Resolution through Cryptophane Synthesis and Supramolecular Self-Assembly into Nanotubes[END_REF] Figure 7: structure of a TBTQ derivative and the cryptophane derived by dynamic covalent assembly.

Cryptophanes derived from CTB, however show some flexibility and undergo a similar type of conformational conversion to that of the CTB caps. These forms are the following, out-out (CC), in-out (CCi), saddle-out (CS) and in-in (CiCi) conformers, Figure 8. It is noteworthy that the presence of several conformers can complicate the characterization of the cryptophane derivatives.

Racemic mixture TBTQ CC CS C i C i CC i

The (CS) conformer or the so-called imploded form possesses a unique 1 H NMR signature compared to the (CC) conformer. [START_REF] Mough | Isolation and Structure of an "Imploded[END_REF][START_REF] Haberhauer | Strongly Underestimated Dispersion Energy in Cryptophanes and Their Complexes[END_REF][START_REF] Thorp-Greenwood | Fully Collapsed Imploded Cryptophanes in Solution and in the Solid State[END_REF] This form was initially observed in cryptophanes with long flexible linkers or in the presence of a solvent that does not fill the cavity. [START_REF] Huber | Water Soluble Cryptophanes Showing Unprecedented Affinity for Xenon: Candidates as NMR-Based Biosensors[END_REF] Surprisingly, smaller cryptophane cages (i.e. cryptophane-[222] derivatives) are also capable to implode like cryptophane- [222] (OCH2CO2H)6. The imploded form can readily be formed upon heating or storing the compounds for a long time in the solid state. For example, the imploded form of mxylyl-bridged cryptophane was obtained at high temperature (210 °C). Purification of this form offered a clean detailed 1 H NMR and single crystals were also analyzed assuring the (CS) conformation, Figure 9. [START_REF] Mough | Isolation and Structure of an "Imploded[END_REF] Recently, we have discovered a new type of conformational isomer with cryptophane- [223] bearing a substituent on the central propylenedioxy linker. This conformation corresponds to the position of the central substituent that can be inside or outside the cavity, which is referred to as "self-encapsulation". This new conformational change represents an easy way for cryptophanes to fill their cavity with low energetic cost. The process is solvent dependent and will be thoroughly discussed in the second part of this thesis.

Cryptophane Complexes:

As mentioned previously, cryptophanes possess a lipophilic cavity suitable to accommodate different guest molecules. Being a container molecule, cryptophanes' binding process is governed by constrictive binding property for the in-out exchange of the guest, Figure 10. Cryptophanes binding portals are the linkers and its cavity is defined by the three dimensional arranged six aromatic rings. Cryptophanes' constrictive binding is based on "sliding door" mechanism, that is the optimization of the cryptophanes' portals by squeezing or expansion to accommodate the corresponding guest. [START_REF] Liu | Gated Container Molecules[END_REF] This mechanism involves conformational changes of its flexible linkers. In cryptophanes, the stability of the guest@cryptophane complex is governed by several factors, mainly the volume compatibility (very large or very small guests do not fit properly based on ratio of coefficient packing V guest/ V cryptophane = 0.55) [START_REF] Mecozzi | The 55% Solution: A Formula for Molecular Recognition in the Liquid State[END_REF] , size and shape of the guest (globularly shaped guests) and electronic effect (highly polarizable atoms like xenon) and of course the symmetry of the cage itself, which greatly affects the binding properties. 27 Figure 10: constrictive binding of guests by sliding the portals (linkers), followed by the adjustment of the cavity size.

Complexation studies are investigated in both organic and aqueous media depending on the cage solubility and the guest of interest. 1 H NMR spectroscopy serves as a leading tool to study such interactions, showing a high shielding of the encapsulated guest by the effect of the cage's ring

Flexible portals Adjustment of the cage upon complexation

Guest Guest Guest current. Isothermal calorimetric titration (ITC) appears as another useful method to measure accurately the association constant in water or in organic solvents. In addition, this method gives access to thermodynamic parameters of complexation (ΔH°, ΔS° and ΔG°). Thus ITC is a very important analytical tool to understand how the binding process between a cryptophane and a guest molecule takes place. Brotin and co-workers have used intensively this method to determine the association constant between water-soluble cryptophanes and cationic species.

Below is a representative ITC plot for the titration of cryptophane-[222] (OH)5 dissolved in LiOH (0.1M) and Tl(OAc) performed at 298K. This titration reveals the strong association constant (Ka = 2.39 10 9 M -1 ) of Tl + by this cryptophane derivative, Figure 11. 52 In the coming sections, we will describe the different types of guests encapsulated by cryptophanes in solution.

Encapsulation of Neutral Species:

Cryptophanes are well known for their interesting ability to encapsulate neutral molecules like CHCl3, CH2Cl2, isobutane, CHFClBr, CHFClI, epoxides, and gases like methane and xenon. [START_REF] Canceill | Structure and Properties of the Cryptophane-E/CHCl3, Complex, a Stable van Der Waals Molecule[END_REF][START_REF] Little | Synthesis and Methane-Binding Properties of Disulfide-Linked Cryptophane-0.0.0[END_REF][START_REF] Fogarty | A Cryptophane Core Optimized for Xenon Encapsulation[END_REF][START_REF] Canceill | Analytical Optical Resolution of Bromochlorofluoromethane by Enantioselective Inclusion into a Tailor-Made "Cryptophane" and Determination of Its Maximum Rotation[END_REF][START_REF] Garel | Complexation of Methane and Chlorofluoro-Carbons by Cryptophane-A in Organic Solution[END_REF][START_REF] Soulard | Chlorofluoroiodomethane as a Potential Candidate for Parity Violation Measurements[END_REF] These complexation studies are done in solution. 1,1,2,2-tetrachloroethane appears as the solvent of choice because it is too big to enter the cavity of the smallest cryptophanes (cryptophane- [111], cryptophane-A, cryptophane-C). Thus it does not act as a competitor with the guest studied. Other non-competing solvents might also be employed like bis(trichlromethyl)ketone that was used by Crams and coworkers. [START_REF] Cram | Two Chiral [1.1.1Orthocyclophane Units Bridged by Three Biacetylene Units as a Host Which Binds Medium-Sized Organic Guests[END_REF] Recent studies show that 1,1,2,2-tetrachloroethane can partially enter the cavity of cryptophane-E and larger cryptophanes. [START_REF] Haberhauer | Strongly Underestimated Dispersion Energy in Cryptophanes and Their Complexes[END_REF] What is quite interesting in these complexes is the ability of cryptophanes to scavenge lipophilic guests in organic solvents, without having a hydrophobic effect. Another remarkable feature is that cryptophanes differentiate guests based on little volume changes. For example, cryptophane-E has a higher affinity to CHCl3 compared to CHCl2Br. 59

Enantioselective Binding of Guests:

Enantioselective binding of chiral guests is also possible with cryptophanes. Unfortunately, the number of examples reported in the literature is rare because enantiopure cryptophanes are hard to obtain and the number of small chiral guests that can enter their cavity is limited and difficult to obtain in their enantiopure form. This enantioselective binding was first demonstrated by distinguishing the two enantiomers of CHFClBr by 1 H NMR spectroscopy using cryptophane-C ([222] (OMe)6). The difference in binding constants between the two diastereomeric complexes affects the orientation adopted by the guest inside the cavity and its magnetic properties, which will be readily detected by 1 H NMR spectroscopy. Enantioselective complexation of R-methyl oxirane was also realized in water by enantiopure PP-cryptophane- [222] (OH)5 with a free energy difference 1.7 kJ/mol compared to the less stable S-methyl oxirane@PP-cryptophane, Figure 12. Recently, the complexation of additional oxiranes by the same cryptophane derivative was also tested and studied by 1 H NMR and electronic circular dichroism spectroscopy, ECD.

Most interestingly, 2,2′-bioxirane, which has two chiral centers shows three diastereomers in the presence of enantiopure cryptophane. These diastereoisomers are readily distinguished by 1 H NMR spectroscopy. [START_REF] Bouchet | Enantioselective Complexation of Chiral Propylene Oxide by an Enantiopure Water-Soluble Cryptophane[END_REF][START_REF] De Rycke | Enantioselective Complexation of Chiral Oxirane Derivatives by an Enantiopure Cryptophane in Water[END_REF] Figure 12: enantioselective encapsulation of (R)-epichlohydrin cryptophane by MM-cryptophane- [222] (OH)5.

Encapsulation of Cationic Species:

Cryptophane cages are also suitable for cation complexation. The encapsulation of organic (ammonium) cations has been thoroughly studied in the past. However, complexation of metallic cation is more recent. Remarkably, the binding of alkali cations (rubidium, cesium) occurs in water, a strong competing solvent, and under basic conditions in order to solubilize the cryptophane derivative. As revealed by titration experiments, this work has shown that thallium (Tl + ) (caution: very toxic) is very well recognized under these conditions. More recently, some measurements have also been obtained at pH close to 7, with new cryptophane derivatives. In cation complexation, the presence of phenol groups seems mandatory to observe an efficient binding and the replacement of these groups by carboxylic acid moieties results in a lack of complexation. For instance, cryptophane hexa-carboxylate does not bind cationic species such as cesium or thallium cations under the same conditions. [START_REF] Garel | Remarkable Effect of Receptor Size in the Binding of Acetylcholine and Related Ammonium Ions to Water-Soluble Cryptophanes[END_REF][START_REF] Garcia | Kinetic and Thermodynamic Consequences of the Substitution of SMe for OMe Substituents of Cryptophane Hosts on the Binding of Neutral and Cationic Guests[END_REF][START_REF] Chapellet | Experimental and Theoretical Study of the Complexation of Cesium and Thallium Cations by a Water-Soluble Cryptophane[END_REF][START_REF] Brotin | High Affinity of Water-Soluble Cryptophanes for Cesium Cations[END_REF][START_REF] Brotin | Water-Soluble Molecular Capsule for the Complexation of Cesium and Thallium Cations[END_REF][START_REF] Brotin | Influence of the Cavity Size of Water-Soluble Cryptophanes on Their Binding Properties for Cesium and Thallium Cations[END_REF] 1.4.4. Encapsulation of Anionic species:

The

Synthesis of Cryptophanes

The construction of cryptophane cages has been achieved in three different ways, the simplest direct method, the template method and the coupling method, all starting from the vanillic alcohol building block. These methods mainly differ in the way of creating and/or connecting the two CTB units. 27 Each method has its own advantages and inconveniences and we will give one or two examples per each.

Direct Method or Two-Step Cryptophane Synthesis:

The direct method consists of the trimerization of properly bridged and substituted vanillic alcohol dimers. With six consecutive Friedel-Crafts alkylations under acidic conditions (usually formic acid works very well), the direct method provides cryptophanes in low yields and it gives a lot of side-product (mainly polymerized byproducts). The reaction is controlled by the diffusion, so it is not possible to use diluted conditions to decrease the formation of the byproducts. This strategy allows the access of exclusively D3 and C3h symmetrical cages, and it is more suitable for the construction of intermediate size cages (bridge size < 6) as the increase in the bridge length drops the yield to the favor of the intramolecular condensation. [START_REF] Canceill | Two Step Synthesis of D3 and C3h Cryptophanes[END_REF][START_REF] Akabori | Syntheses of Diethyleneoxy Bridged Cryptophanes and Their Complexing Abilities with Alkali Metal and Alkylammonium Cations[END_REF] The approach has been used by chemists to obtain cryptophanes rapidly for complexation studies but it is less used to obtain sophisticated functionalized cages. An example is given with the synthesis of cryptophane-E, which has been obtained with a relatively high yield of 20%, Figure 14. In the case of cryptophane-A, the same approach gives rise to this derivative with an extremely low yield (1-2%).

Figure 14: synthesis of cryptophane-E starting from vanillic alcohol by the direct method where it is obtained in 20% yield.

Cryptophane-E

Template Method:

The template method is the earliest and by far the most employed approach to prepare cryptophane derivatives, because it allows the access to a wide range of cages of different symmetry and functionalization. [START_REF] Gabard | Synthesis of a (D3)-Bis(cyclotriveratrylenyl) Macrocage by Stereospecific Replication of a (C3)-Subunit[END_REF] It is also the most appropriate method to obtain fair quantities of cryptophanes. In this method a CTB unit is first constructed, then elaborated by the attachment of three bridges with peripheral benzyl alcohols to form the so-called "template". The cyclization of the second CTB is usually carried under diluted acidic conditions usually 10 -3 M in pure formic acid or in equal mixture of formic acid/CHCl3. Other conditions have been used to conduct the second intramolecular cyclization like perchloric acid/methanol mixture, and scandium triflate/ acetonitrile mixture. The latter provides a milder condition and makes purification easier. [START_REF] Taratula | Shorter Synthesis of Trifunctionalized Cryptophane -A Derivatives[END_REF] The main advantage of this method compared to the direct method is that it is not diffusion controlled, this indicates that more diluted conditions can be used, thus decreasing the polymeric sideproducts. With this strategy, accessible synthesis of C1-symmetric cages with different bridges and substituents is achieved. A wide range of syn and anti-configurations are constructed. The diastereoselectivity of the cyclization is dependent on many conditions like substituents and linker's length, which are usually hard to control and predict. For example, it was believed that cryptophanes with an odd number of carbons on the linker tend to give syn-configurations, whereas, even ones give anti-configurations. Opposite to this claim, nona-and dodecamethoxy-

[222] cryptophanes, give rise to anti and syn configurations, although they have even number of carbons on the linkers. [START_REF] Brotin | Chiroptical Properties of Nona-and Dodecamethoxy Cryptophanes[END_REF] As an illustration of the higher yields obtained with template method compared to the direct method, we can talk about cryptophane-A, which is obtained in a higher yields (80%) compared to 1-2% yield, Figure 15.

Figure 15: synthesis of cryptophane-A by the template method with 80% yield.

Coupling Method:

As its name implies, the method relies on the coupling of two properly functionalized CTB units. Unlike the first two methods, in the coupling approach no need to use acidic conditions.

Coupling method was first utilized by Cram and coworkers in 1991 to couple a bis-alkynebridged CTB by cupric oxidative coupling. [START_REF] Cram | Two Chiral [1.1.1Orthocyclophane Units Bridged by Three Biacetylene Units as a Host Which Binds Medium-Sized Organic Guests[END_REF] This method was later employed to synthesize other cryptophanes like the cryptophane-[111] by Brotin, Dutasta and co-workers, Figure 16, [START_REF] Fogarty | A Cryptophane Core Optimized for Xenon Encapsulation[END_REF] and cryptophane-[000] by Hardie and co-workers. [START_REF] Little | Synthesis and Methane-Binding Properties of Disulfide-Linked Cryptophane-0.0.0[END_REF] Asymmetric cryptophanes- [221] and [112] by Rousseau and co-workers [START_REF] Kotera | Design and Synthesis of New Cryptophanes with Intermediate Cavity Sizes[END_REF] and aryl-bridged cryptophane by Holman and co-workers [START_REF] Holman | Anion Binding, Aryl-Extended Cyclotriguaiacylenes and an Aryl-Bridged Cryptophane That Provides Snapshots of a Molecular Gating Mechanism[END_REF] have also been prepared in low yields by using a similar approach.

Figure 16: synthesis of cryptophane-[111] by coupling method.

Cryptophane-A 80 %

Functionalization and Solubilization of Cryptophanes:

As many other symmetrical lipophilic supermolecules, the main synthetic challenge relies on the selective functionalization and solubilization of the core. These two different aspects are very important for the design of xenon-biosensor, as it will be seen later. Added functionalities can eventually be used to introduce a sensing unit, water solubilizing groups, a chiral moiety to optically resolve cryptophanes enantiomers or an anchoring arm to attach them to nanoparticles.

These functionalities can be introduced either before or after the cage cyclization. Herein, a detailed description of the functionalization strategies is addressed.

Readily formed cryptophanes can be modified by substituting the existing functionalities, mostly methoxy groups, with other ones via alkylation, acetylation or dehydroxylation. Since 1987, André Collet reported an efficient selective procedure to remove the six methoxy groups on cryptophane-A core, using lithium diphenylphosphide producing a water soluble cage with six phenols. Since then, Brotin and co-workers have reported an improved approach. The six phenol moieties are then replaced by other functionalities like acetates or methyl esters, Figure 17. The latter are then hydrolyzed to give six carboxylic acid groups providing a water soluble cage up to pH = 5. [START_REF] Canceill | Water-Soluble Cryptophane Binding Lipophilic Guests in Aqueous Solution[END_REF]34 Other cages of different linker size were also functionalized by this way offering water soluble cryptophanes. [START_REF] Brotin | Influence of the Cavity Size of Water-Soluble Cryptophanes on Their Binding Properties for Cesium and Thallium Cations[END_REF] Figure 17: functionalization of cryptophanes after a demethylation step either by alkylation or acylation reactions.

Water soluble hexa-phenol Water soluble hexa-acid Hexa-acetate derivative Hexa-methoxy derivative

The template method is the method of choice to synthesize asymmetric cages with different linkers' length and substituents. This method is widely employed to construct different cryptophanes families like mono, tri and dually functionalized cages. In this strategy, a suitably decorated linker or linkers are grafted on the CTB followed by the formation of the second CTB cap.

To talk about the mono functionalized cryptophanes, Brotin and co-workers were able to successfully synthesize mono-allyl protected cryptophane-A derivative, Figure 18. Exploiting the template method, a single ethylenedioxy arm possessing an allyl protected benzyl alcohol was introduced to a doubly alkylated CTB, cyclization under acidic conditions yielded the corresponding product, which was further utilized to construct a wide range of symmetric, asymmetric and dimeric cages. [START_REF] Darzac | Synthesis and Application of Cryptophanol Hosts: 129 Xe NMR Spectroscopy of a Deuterium-Labeled (Xe)2@bis-Cryptophane Complex[END_REF][START_REF] Darzac | New Versatile Compounds for the Synthesis of Functionalized Cryptophanes and Polycryptophanes[END_REF][START_REF] Mari | 129 Xe NMR-Based Sensors: Biological Applications and Recent Methods[END_REF][START_REF] Cavagnat | Synthesis and Chiroptical Properties of Cryptophanes Having C1-Symmetry[END_REF] Figure 18: synthesis of mono-allyl protected cryptophane-A from its corresponding template.

Using the same strategy, Dmochowski and co-workers reported the synthesis of monopropargylic cryptophane-A derivative, which was then functionalized by copper catalyzed cycloaddition. [START_REF] Wei | Designing 129 Xe NMR Biosensors for Matrix Metalloproteinase Detection[END_REF] By the same group, successful allyl, benzyl or propargyl tri-functionalized- [222] cages were described using scandium triflate or perchloric acid/methanol for the cyclization step. [START_REF] Taratula | Shorter Synthesis of Trifunctionalized Cryptophane -A Derivatives[END_REF][START_REF] Hill | Thermodynamics of Xenon Binding to Cryptophane in Water and Human Plasma[END_REF] Equally, Rousseau and co-workers described a water soluble tri-functionalizable cage with three polyethylene glycol groups (PEG) and three propargyl groups on the second CTB, which proved their reactivity for Huisgen cycloaddition. [START_REF] Delacour | Clickable" Hydrosoluble PEGylated Cryptophane as a Universal Platform for 129 Xe Magnetic Resonance Imaging Biosensors[END_REF] Schröder and co-workers also reported a dually dendronized cage with enhanced water solubility, Figure 19. [START_REF] Tyagi | Dendronized Cryptophanes as Water-Soluble Xenon Hosts for 129 Xe Magnetic Resonance Imaging[END_REF][START_REF] Jayapaul | Complete Generation of a 129 Xe Biosensor on the Solid Support by Systematic Backbone Assembly[END_REF] In the latter case, it is worth mentioning that the corresponding di-acidic derivative has never been described.

According to our knowledge in cryptophane synthesis, we believe that the two acidic moieties are grafted on the same CTB units, and that the structure of the dendrimer substituted cage is not correct. Introducing water-solubilizing substituent is even more complicated with other cryptophanes such as cryptophane- [111] skeleton since this compound is devoid of substituents. Indeed, in absence of any existing functionality on cryptophane-[111] core, [START_REF] Fogarty | A Cryptophane Core Optimized for Xenon Encapsulation[END_REF] new methods have been developed to render it water soluble and functionalizable. Cryptophane-[111] core was first solubilized in water by the metalation of the arene rings with six [Cp*Ru] + moieties. [START_REF] Fairchild | A Water-Soluble Xe@cryptophane-111 Complex Exhibits Very High Thermodynamic Stability and a Peculiar 129 Xe NMR Chemical Shift[END_REF] Introduction of a single substituent was also possible by introducing iodine or bromine followed by palladium catalyzed coupling reactions. [START_REF] Traoré | Scalable Synthesis of Cryptophane-1.1.1 and Its Functionalization[END_REF][START_REF] Dubost | Synthesis of a Functionalizable Water-Soluble Cryptophane-111[END_REF][START_REF] Berthault | The First Metal-Free Water-Soluble Cryptophane-111[END_REF] Metal-free water soluble cryptophane-[111] was also synthesized by Rousseau and co-workers, by an amide coupling of the mono-carboxylic acid derivative with a single tri-sulfonated linker, Figure 21.

Dually functionalized cage Water soluble hexa-phenol

Water soluble hexa-carboxylic acid

Xenon-cryptophane Properties and Applications

Xenon is an inert noble gas discovered by Ramsay and Morris Travers in 1898 and obtained by fractional distillation of the liquid air. [START_REF] Ramsay | Argon and Its Companions[END_REF] The biocompatibility of the gas has been demonstrated in 1951, by Stewart C. Cullen who showed its anesthetic effect. [START_REF] Cullen | The Anesthetic Properties of Xenon in Animals and Human Beings, with Additional Observations on Krypton[END_REF] Xenon is a doping agent that enhances athletic performance and it has been forbidden since 2015 by the World Anti-Doping Agency (WADA). [START_REF] Frampas | Xenon: From Medical Applications to Doping Uses[END_REF][START_REF] Thevis | Urine Analysis Concerning Xenon for Doping Control Purposes[END_REF][START_REF] Harris | The Uses of Helium and Xenon in Current Clinical Practice[END_REF] Among the eight naturally occurring isotopes, only 129 Xe and 131 Xe are stable with natural abundance of 26.5% and 21.1% and nuclear spins of 1/2 and 3/2, respectively, making them NMR active. 129 Xe is the most used isotope thanks to its higher abundance, simple NMR signature and the possibility to be hyperpolarized, which will be seen later. Bearing 54 electrons and a volume of 42 Å 3 , xenon electronic cloud is highly polarizable, and it interacts strongly with the surrounding environment via London dispersion forces and hydrophobic interactions. This high polarizability is reflected by the large chemical shift range (about 300 ppm in water for non-covalently bound xenon) indicating also the large sensitivity of the gas to its surrounding environment. 93 Apart from sensitivity, high polarizability renders the gas lipophilic and this may explain its anesthetic behavior. [START_REF] Cullen | The Anesthetic Properties of Xenon in Animals and Human Beings, with Additional Observations on Krypton[END_REF] Ostwald solubility or the ratio of the volume of gas absorbed to the volume of the absorbing liquid at the same pressure and temperature is given in table 2, which shows a moderate and high solubility of the gas in aqueous and organic media, respectively. For example the solubility of the gas in fat tissues (1.3) is almost 10 times higher than that in blood (0.14). In addition to the high sensitivity, wide range solubility and chemical inertness, long relaxation time is another important property to be added to the list. Unlike proton relaxation mechanisms, xenon relaxation times are affected by intrinsic and extrinsic parameters (transient and persistent

Xe-Xe van der Waals dimer relaxations as the intrinsic factors (spin-rotation); atomic diffusion through gradients of the magnetic field and interaction with the cell wall relaxations as the extrinsic factors (dipole relaxation)). These relaxation processes contribute differently if xenon is in the solid, liquid or gas phase. For liquid state, relaxations are mostly decreased by wall and solvent interactions and the presence of oxygen or any paramagnetic species drastically decrease the relaxation times T1 and T2 of xenon. [START_REF] Anger | Gas-Phase Spin Relaxation of Xe-129[END_REF] The simplest way to introduce xenon into the body is by inhalation. [START_REF] Ruppert | Exploring Lung Function with Hyperpolarized 129Xe Nuclear Magnetic Resonance[END_REF] Thanks to this, real-time high-resolution lung imaging was achieved, which is usually hard to attain by the conventional MRI, due to low proton density. This is currently a hot research topic that may offer new imaging utilities for medical applications. [START_REF] Ruppert | Using Hyperpolarized Xenon-129 MRI to Quantify Early-Stage Lung Disease in Smokers[END_REF][START_REF] Torres | Structure-Function Imaging of Lung Disease Using Ultrashort Echo Time MRI[END_REF] Some 129 Xe MRI brain experiments have been also realized in rodents. Intravenous and arterial introduction of the gas allows a specified organs imaging. [START_REF] Goodson | In Vivo NMR and MRI Using Injection Delivery of Laser-Polarized Xenon[END_REF]100 All these experiments are promising and suggest that xenon can be used as an exogenous tracer for biomedical studies.

As mentioned previously, the characteristic volume of xenon (42 Å 3 ) allows its encapsulation in different hosts of matching cavity volume. In 1998, Bartik et al. reported the first 129 Xenon@Cryptophane-A inclusion complex in 1,1,2,2-tetrachloroethane-d2. The binding constant is impressively high, of about 4000 M -1 even higher than that of chloroform and dichloromethane in 1,1,2,2-tetrachloroethane-d2. The 129 Xe@Cryptophane complex is stabilized by London dispersion forces between the polarizable atom and electron rich aromatic rings. Over

Different Hosts for 129 Xe Inclusion:

α-cyclodextrins are cyclic oligosaccharides that possess a relatively apolar internal cavity of volume in the order of 140 Å 3 . This large cavity size makes the 129 Xe association constant very weak (Ka = 22.9 M -1 ) in water and the in-out exchange is relatively fast. The formation of xenon@cyclodextrin complex in that case was proved by proton-xenon heteronuclear NOE experiments. 101 Recent study investigated a cyclodextrin based pseudo-rotaxane, where the large cavity was threaded with a disubstituted alkyl chain, to decrease its volume, hence slowed the in-out exchange rates. 102 Thiacalix [4]arene also showed weak binding constants (Ka = 13.6 M -1 ) to xenon, with a concentration dependent chemical shift variations. 103 Ternary complexes of pillar [5] 22. 105,106,107,108 The first in-vivo imaging for the detection 129 Xe@CB [6] in water was also performed, where it was possible to localize the cage in brain, heart, kidneys, liver and aorta. 109

Figure 22: 129 Xenon NMR spectra of xenon in the presence of CB* [6] in pure water (above) and aqueous Na2SO4 solution at 298 K.

129 Xenon@Cryptophane Complexes:

Compared to the previously discussed molecules, cryptophane appears as the host of choice for xenon encapsulation due to many different reasons. 110 Along with the high binding constants, slow exchange at the NMR time-scale and relatively optimized in-out kinetics, the possibility to functionalize cryptophanes is a significant property to be added on the list. With this property, successful grafting of water solubilizing or sensing units is made possible. In addition, the latter allows a perfect tuning of xenon complexation properties like chemical shifts, binding constant, and kinetics. For the moment, cryptophanes are the most investigated hosts in this domain, where a notable number of cages have been developed with various cavity size, substituents and applications. Each of these cages has different binding affinity and chemical shift depending on their structures and this will be detailed in the next paragraphs.

129 Xenon@Cryptophane Binding Constants:

One factor affecting xenon binding affinity is the cavity volume. In organic media, usually 1,1,2,2-tetrachloethane, smaller cryptophanes like cryptophane-[111] and [222] (OMe)6 (A), with a cavity volumes of about 81 and 95 Å 3 respectively, tend to have high binding constants, of 10000 M -1 and 3900 M -1 , respectively. [START_REF] Fogarty | A Cryptophane Core Optimized for Xenon Encapsulation[END_REF]110 Larger cages showed a lower binding constant as expected, like cryptophanes [223] (OMe)6, [233] (OMe) 6 and [333] (OMe)6 (E), with binding constants of about 2800, 800 and 10 M -1 respectively. 111 This trend confines with the packing coefficient of the guest to host volumes described earlier that must be 0.55. For example in cryptophane-[111] and cryptophane-A, these coefficients are equal to 0.49 and 0.6, respectively, which are more or less optimal for these complexations. In aqueous media, however, higher binding constants are usually observed due to the hydrophobic effect of the lipophilic cavity. The interaction in aqueous media is entropically favored, where water molecules filling possibly the hydrophobic cavity tend to leave to be replaced by the lipophilic gas. For example, cryptophane-

[111] (RuCp*)6Cl6 exhibits a binding constant of about 29000 M -1 . [START_REF] Fairchild | A Water-Soluble Xe@cryptophane-111 Complex Exhibits Very High Thermodynamic Stability and a Peculiar 129 Xe NMR Chemical Shift[END_REF] Cryptophanes functionalized with hexa-carboxylic groups like cryptophane-A hexa-acid (Ka~6800 M -1 ), [223] (OCH2CO2H)6 (Ka~2200 M -1 ), [233] (OCH2CO2H)6 (Ka~2200 M -1 ) and finally cryptophane-E (OCH2CO2H)6 (Ka~1000 M -1 ) show higher binding constants compared to their congeners in 1,1,2,2-tetrachloroethane, but still following the same decreasing trend in terms of increasing cavity volume. [START_REF] Huber | Water Soluble Cryptophanes Showing Unprecedented Affinity for Xenon: Candidates as NMR-Based Biosensors[END_REF] Tri-functionalized water-soluble cages, based on cryptophane-A derivative, were also described to effectively bind xenon showing incomparable high binding constants up to Ka~ 42000 M -1 , Figure 23. It is worth mentioning that these values are obtained by ITC and not by xenon NMR spectroscopy, making the comparison between the previously mentioned values difficult. [START_REF] Hill | Thermodynamics of Xenon Binding to Cryptophane in Water and Human Plasma[END_REF]112,113 Figure 23: structure of water-soluble tri-functionalized cryptophanes cages bearing different functionalities displaying different xenon binding constants.

Substituents on the cryptophanes rims, also affect the values of binding constants as illustrated in the previous example. A clearer effect can be seen with the tri-functionalized cryptophanes [111] shown in Figure 24, where adding three methoxy or three bromine atoms drops the binding constants to Ka~10 M -1 , this can be directly explained by the packing coefficient, which is approximately equal to 0.79 larger than the one described for the non-functionalized

129 Xe@Cryptophane Chemical shift:

Xenon polarizability is clearly reflected by the ultimate sensitivity of its surrounding environment, which has been illustrated in different examples. For instance, in an original example, a series of deuterated cryptophanes were prepared and their 129 Xe complexes were studied. The 129 Xe NMR spectroscopy revealed that the encapsulation of xenon in completely deuterated cryptophane-A derivative leads to an upfield shielding of its signal by 1.19 ppm compared to the one non-deuterated cage. This example manifests the extraordinary sensitivity of this element to discriminate between the non-deuterated and deuterated cryptophane cages. 115 In addition, a mixture of diastereomers may split a xenon signal into two, each corresponds to a different isomer. Cl6. In this innovative example, the chemical shift of the water soluble cage is completely reversed and it appears at 301 ppm, downfield shifted by about 277 ppm compared to the organic version appearing at 31 ppm. [START_REF] Fairchild | A Water-Soluble Xe@cryptophane-111 Complex Exhibits Very High Thermodynamic Stability and a Peculiar 129 Xe NMR Chemical Shift[END_REF] These examples show that even a small structural change of the cryptophane skeleton induces large chemical shift variations of the 129 Xe signal. This effect is mainly a consequence of the ring current modification that takes place upon these changes. In turn the modification in ring current modifies the magnetic properties of the xenon nucleus, Figure 25.

Figure 25: schematic presentation of 129 Xe@cryptophanes chemical shifts in water and in organic media. The important sensitivity gain delivered in this example is due to the use of hyperpolarized (HP) 129 Xe compared to the much less sensitive thermally polarized gas. Hyperpolarization is defined as a transient imbalance in the nuclear spin repartition between energy levels, which initially follows the Boltzmann distribution. [START_REF] Mari | 129 Xe NMR-Based Sensors: Biological Applications and Recent Methods[END_REF] Perturbing Boltzmann populations of nuclear spins will increase the polarization consequently increasing the global magnetization. Typically, this will enhance the signal by four to five times orders in magnitude depending on strength of the magnetic field B0. Hyperpolarization is achievable by different methods like Brute force, dynamic nuclear polarization (DNP) but Spin Exchange Optical Pumping (SEOP) remains the most convenient and rapid method to produce small amount of hyperpolarized 129 Xe, Figure Technically speaking, SEOP is a two-step process polarization. The first step involves the polarization of electronic spins of the gaseous alkali metal (Rubidium), followed by a transfer of this polarization to the nuclear spins of the noble gas by Fermi-contact interactions. Usually, another gas (nitrogen) is added to the cell to favor this process and to quench the fluorescence of the rubidium atoms. In the first step, the presence of a magnetic field renders the magnetic moments (mg= +/-1/2) of both the ground ( 2 S1/2) and excited states ( 2 P1/2) non-degenerate (different in energy). Circularly polarized laser tuned at the D1 transition of Rb, i.e. at 794.7 nm, is applied leading to the excitation of spin population from ground state (mg = -1/2) to the excited state (mg = + 1/2). In other words, the angular momentum of the photon is transferred to the electron of the rubidium atom. The relaxation however takes place by the accumulation of spin population in the (mg = + 1/2) state rather than (mg = -1/2) of the ground state thus leading to electronic spins hyperpolarization of the rubidium atoms. In the second step, the electronically polarized Rb steam collides with the noble gas ( 129 Xe) leading to a spin exchange consequently forming a nuclear spin polarized 129 Xe, Figure 28. Hyperpolarized, 129 Xe can be stored at low temperature (77 K) in the solid state in a magnetic field of 0.1 T, where it is possible to maintain its polarization for a long time depending on the storage conditions. 122,123 Great care has to be taken to avoid the gas depolarization by using for example special cells coated with silane-or siloxane coatings.

Xe@Cryptophane Biosensors for NMR and MRI Applications

Figure 28: SEOP cell is irradiated by circularly polarized laser light that can be absorbed by the Rb atoms. The first step involves a photon absorption resulting in selective population depletion from one Rb ground electronic state leading to its hyperpolarization. In the second step, gas-phase collisions allow spin order to be transferred from the Rb atom electrons to the noble gas nuclei by Fermi contact hyperfine interactions, thus hyperpolarizing the noble gas.

Over the last ten years, many efforts have been made to detect very low concentrations of xenoncomplexes. For example, Pines and co-workers have shown that hyper-CEST (Chemical Exchange Saturation Transfer) experiments can be used to gain more sensitivity. 124 This technique relies on the indirect detection of the biosensor, which are usually present in micromolar concentrations via observing the variations at the bulk level, which is present in millimolar concentrations. 125 In this experiment, caged xenon is first irradiated by a saturation pulse to depolarize it. The depolarized caged nucleus is then exchanged with the bulk by another (HP) one. Upon its encapsulation, (HP) 129 Xe will be depolarized and exchanged again. The process is repeated many times leading to the accumulation of depolarized 129 Xe in the bulk, which in turn decreases the intensity of free 129 Xe, (on-resonance spectrum in Figure 29).

Observing the difference between on/off resonance spectra allows the signal enhancement by three times order in magnitude consequently decreasing the detection threshold to picomolar range, Figure 29. This technique is currently used to record magnetic resonance images of some (HP) 129 Xe tracers.

Some of these tracers will be discussed in the next section.

129 Xe@Cryptophane Sensors:

With the increasing number of Xenon@Cryptophane sensors, one can distinguish between two types, smart or density based sensors. [START_REF] Mari | 129 Xe NMR-Based Sensors: Biological Applications and Recent Methods[END_REF] In the former, an interaction with the biological target either generates or shifts the existing 129 Xe signal, in the latter, a variation in signal intensity as a function of local environment is observed. In the next paragraphs, we will present briefly an overview of some xenon-based sensors. The biosensors will be arranged based on the contribution of different groups working in the field and the sensors derived from cryptophane-A will be referred to as cryptophane-[222] derivatives.

Pines and Co-workers:

The first milestone example described by Pines and coworkers established the basics of this field, and opened the gate for further developments at structural and technical levels. In parallel to the synthesis of biosensors, Pines and co-workers have developed new NMR techniques to enhance the (HP) 129 Xe NMR sensitivity or to spatially map it. For example, spectrally resolved magnetic resonance images of a biotinylated sensor, Figure 30, were achieved. Cages were first immobilized on an avidin bearing surface thanks to the strong affinity between avidin and biotin (Ka = 10 15 M -1 ). Then, a current flow of (HP) 129 Xe saturated water was passed through the sample. Thanks to the high chemical shift difference among the xenon pool and the cryptophanebiotin complex, a successful detection of the bound xenon with almost zero background noise was performed, Figure 30. 126 As previously illustrated, (HP) 129 Xe is sufficiently sensitive to distinguish between diastereomeric mixtures of cryptophanes. To avoid this problem, the group proposed a new approach based on simply constructing supramolecular assemblies of cryptophane- [222] monoacid and amine-bearing dendrimers. These assemblies are stabilized by acid-base and hydrophobic interactions. A single assembly bears more than one cryptophane cage, which in turn leads to an amplification of the (HP) 129 Xe signal intensity. 127 In another example, the group utilized a mono-acid cryptophane-[222] derivative (structure b in figure 37) and attached it to a viral capsid. With almost hundred twenty-five cages grafted on the capsid surface, enhancement of sensitivity to picomolar (0.7 pM) level has been realized. 128 In another example, the grafting of several cryptophane-[222] derivatives (structure c in figure 37) to EGPR-targeted bacteriophages for cancerous cell detection was also done. The detection of these cancerous cells was performed with high specificity. This was successfully demonstrated by using in-cellulo 129 In 2011, the same group reported the first internalization (endocytosis) of a cryptophane sensor to a cell, by grafting cryptophane cages to the transferrin protein (a cell penetrating peptide). A dual detection by 129 Xe NMR and fluorescence imaging assured the internalization. Unfortunately, the hydrophobic nature of the cage led to non-specific interactions with cell membranes, which were also revealed by 129 Xe NMR, Figure 31. 131 These results highlighted the hydrophobicity of cryptophanes and suggested that a modification of the cage's solubility has to be considered. The problem can be solved for instance by introducing water-solubilizing substituents on the cryptophane skeleton, which is crucial to ensure a good solubility in physiological media for invivo 129 Xe MRI experiments. A bimodal biosensor incorporating a cryptophane-[222] hexacarboxylate and a biarsenical compound CrAsH for fluorescent detection of peptides was also prepared (structure e in figure 37) by this group. Upon binding with an optimized tailored peptide sequence, the CrAsH probe enhanced significantly its fluorescence properties. The tracer also displayed a modified 129 Xe NMR chemical shift that can reach 9.2 ppm. 132,133 The same group showed that the couple xenon-cryptophane complex could be used as a pH 134 In a second novel approach for pH sensing, the group employed two pH-sensitive cryptophane-[222] derivatives bearing respectively three and six carboxylic acid moieties. In this example, the pH is accessed by measuring the difference in chemical shifts between the two cages (ΔδAB) upon pH variation. This approach is rather more convenient and accurate than observing the difference in chemical shifts between the free (HP)

129 Xe and encapsulated one, because the latter can be affected by several parameters like the nature of a buffer, temperature or the presence of ions in solution, Figure 32. 129 Xe NMR was performed at that time. 137 Starting from the same cryptophane derivative and using the same strategy, a folate-targeted biosensor (structure g in figure 37) was also prepared.

The sensor bears water solubilizing peptide chain, Cy3 fluorophore and a folate interacting unit.

Confocal microscopy and (HP) 129 Xe NMR spectroscopy proved the endocytosis of the sensor selectively by folate membrane receptors. 138 Protein targeted cryptophanes were also developed. Using the same strategy ([3+2] cycloaddition), the tri-propargyl cryptophane-[222] derivative (structure h in figure 37) was functionalized with three p-benzenesulfonamide, varying in linker's length. These biosensors target human carbonic anhydrase protein and they distinguished between the two isozymes of carbonic anhydrase with a chemical shift difference of about 4.3 ppm. 139,140 Calcium-activated calmodulin-targeted cryptophane off-on biosensor was also developed using a similar strategy. In the absence of activated calmodulin, an intramolecular interaction between the cryptophane-[222] derivative and its corresponding peptide chain prevents xenon binding, which is revealed by the absence of the characteristic 129 Xe@Cryptophane signal. In contrast, adding the activated protein restores the ability of the cage to bind xenon, hence displaying a characteristic signal by (HP) 129 Xe NMR. 141 Using the same technique, this group was able to nicely target metabolically labeled glycans (glycopolymers species) located at the cell membranes and cannot be easily reached with conventional 1 H MRI. The strategy is based on an immediate attachment of the biosensor on the labeled glycan surface via a covalent bond (structure k in figure 37). Particularly, the glycans were labeled by an azide-bearing synthetic sugar, which is then incorporated as an azidefunctionalized sialic acid at the terminal of the glycans. Taking the advantage of this terminal azide function, the group engineered an alkyne bearing cryptophane-[222] cage. This cage was allowed to react with the azide function by a copper catalyzed "click" reaction. By this, a successful multimodal (fluorescent probe and (HP) 129 Xe sensor) labeling of the cell was achieved. Fluorescence imaging was employed to assess the efficiency of the binding. Then, (HP) 129 Xe NMR experiments were also conducted to image these labeled cells. 146 Interestingly, the reaction between the two molecules by 1-4 addition resulted in a change of the 129 Xe NMR chemical shift approximatively by 1.5 ppm (structures m and n in figure 37). 147,148 In another example, Zhou and co-workers described the synthesis of a cryptophane for detection of H2S (structure o in figure 37) in organic solution. In this example, the cage was also decorated with azido-bearing weakly fluorescent molecule. Upon H2S addition, immediate reduction of the azido group to an aniline takes place leading to fluorescence enhancement combined with a change in (HP) 129 Xe NMR chemical shift. 149

In a third example, this group also reported the synthesis of cryptophane dimer for mercury detection. As exemplified in Figure 36 It is worth mentioning that the above mentioned summary does not fully cover all examples described in literature. We tried only to select the most relevant examples of each group without going into exhaustive details. Some other groups also contributed in this field like Freund and coworker who described the interaction between cryptophane-[222] derivative bearing a specific peptide ligand with the major histocompatibility complexes protein (structure i in figure 37). 151 Meersmann and co-workers also described a new sensing strategy based on the modulation of (HP) 129 Xe relaxation times by grafting a gadolinium (III)-DOTA unit on the cryptophane- [222] monoacid core. This sensor could be turned off by performing a selective reaction to remove the gadolinium (III) from DOTA unit thus increasing the (HP) 129 Xe relaxation times. 152 . Long T1 relaxation is also crucial to be able to manipulate xenon without losing polarization (i.e. transfer of the hyperpolarized gas, mixing and measurement). The xenon carrier itself has to be water soluble to avoid self-aggregation or interaction with cells membrane. In addition it must be prepared in fair quantities and easily modified in order to introduce a sensing unit. Finally, the carrier has to be non-toxic and its bio-distribution has to be assessed prior invivo MRI experiments. These two requirements can only be evaluated at the end of the synthesis. 77

Objectives

Cryptophane derivatives have been heavily used to build xenon biosensors because they meet most of the requirements listed above. As seen previously, the overwhelming majority of designed biosensors use a cryptophane-222 core, where the tethering unit or units are usually grafted on the aromatic core of the cage. Two main types of these cores are used, the cryptophanol-[222] and the cryptophane-[222] hexacarboxylate derived from cryptophane-A. The former cage is mono-functionalizable via the only available hydroxyl group and thus no symmetry problem is encountered and the platform can be prepared in gram scale. However, with five methoxy groups, the cage suffers from limited water solubility and it tends either to aggregate or to interact with the cell membranes. Cryptophane-[222] hexacarboxylate shows a higher water solubility, but the cage is D3-symmetric thus its mono-functionalization and purification is tough because statistical mixture of byproducts are inevitably obtained. Other cryptophane-[222] cages are sometimes used but they also suffer from similar problems.

In this regard, development of a new platform bearing two orthogonal reactive functions could be seen as a solution of the formerly mentioned problems. Hence, in addition to the six identical functions on the aromatic core, a chemical function can be introduced on the cryptophane backbone. This allows the grafting of a tethering unit and hydrophilic groups selectively and separately.

Cryptophane-[223] core appears to be the optimal cage of choice for such a design. Structurally speaking, the cage possesses two ethylenedioxy and a propylenedioxy arms, where the latter can be selectively functionalized, and six identical functionalities at the aromatic rims, Figure 38.

Xenon complexation by cryptophane-[223] has already been assessed in water and this carrier offers convenient characteristics (in-out exchange, binding constants…) suitable for xenon-based sensing applications. In 2015, our group has successfully reported a first generation of cryptophane-[223]-type molecules, which proved satisfying water solubility at neutral pH and suitable xenon complexation properties. Our results have revealed, however, that these molecules showed little reactivity towards introducing a sensing unit as it will be detailed in the next chapter.

In this thesis, a developed version of mono-functionalized cryptophane-[223] with enhanced reactivity has been prepared and their xenon complexation properties have been also assessed. To demonstrate the originality of our approach, we have grafted on this skeleton several sensing units. The detailed synthetic pathways leading to these platforms, along with their successful functionalization will be described in the following chapter. (HP) 129 Xenon NMR studies of the obtained sensors will be also investigated in chapter 3. The advantages and disadvantages of our strategy will be then discussed in details. With this platform in hands, the gate was opened to produce a complete generation of dually functionalized cages. For example, demethylation of the six methoxy groups of compound 8

Sensing unit

provided the hexa-phenol cage with central secondary alcohol 12. This cage is soluble in LiOH (0.1 M), and it showed high affinity towards thallium and cesium cations with respective binding constants of 10 9 and 10 8 M -1 at 298 K, respectively, Figure 41. 64 Introduction of six water-solubilizing substituents (carboxylic acid precursors or six PEG groups)

was done under smooth conditions by selective alkylation of the six phenols on the CTB rims of compound 12 offering compounds 13 and 14. The hydrolysis of the 13 to the corresponding hexa-carboxylic acid cage was also performed. These conditions leave the secondary alcohol function unreacted, Figure 42. These cages show acceptable solubility at neutral pH at moderate concentrations.

Figure 42: structure of water-soluble or pre-water soluble dually functionalized cages, bearing either six methyl ester groups 13 or six PEG groups on the aromatic rims, 14.

To test the reactivity of the central hydroxyl group on compound 13, it was first converted into an electrophile via mesylation reaction to give compound 15 and then allowed to react with different nucleophiles like sodium azide and ethanethiolate. These experiments have shown that there was no possibility to perform any nucleophilic substitution at this position due to steric hindrance.

Under different reaction conditions (thermal or microwave assisted reactions), we observed that the mesylated derivative was either recovered as an imploded form or it was hydrolyzed to the initial alcohol, compound 13. In addition, attempts to exploit the nucleophilicity of the hydroxyl group, in the presence of six ester functionalities also failed due to selectivity issues. Strong bases were always needed to deprotonate the hydroxyl group. This in turn led to the deprotonation of the -protons of the ester groups leading to the formation of side-products, Figure 43. Oxidation of the secondary alcohol in compound 13 to the corresponding ketone was also achieved by using Dess martin periodinane reagent in dichloromethane to give cryptophane 16.

The yield obtained is modest (34%) and an optimization is required to get the cage in decent amounts. This functionality can be exploited in performing imine condensation or Wittig type reactions to graft a spacer and a sensing unit, Figure 44.

Retrosynthetic Analysis of Modified Cryptophane-[223] Derivatives

The The two other envisioned synthetic routes (strategies II and III) employ more or less the same steps discussed in route I, namely cyclization and alkylation, but differ in the origin of the primary alcohol. In route II for example, the hydroxyl group on the cryptophane 27 is a result of a reduction of the methyl ester group. Route III, however, represents the simplest way since it requires less synthetic steps. In this route the primary alcohol function is introduced at the very beginning of the synthesis. It is worth mentioning that all the chiral synthons shown in Figure 46 have been isolated as racemic mixtures. In addition, most of these synthetic steps involve different protection at a certain stage of the synthesis.

Synthesis of Cryptophane 27

A modified template method has been used to prepare cryptophane 27. First, a CTB cap is synthesized, followed by its elaboration with properly functionalized benzyl alcohols and finally their cyclization to offer the cage of interest.

The CTB cap, 4 was synthesized in three linear steps, based on a known procedure starting from the commercially available 4-(hydroxymethyl)-2-methoxyphenol 1. [START_REF] Collet | Cyclotriveratrylenes and Cryptophanes[END_REF] Vanillic alcohol 1 was first protected by a reaction with allyl bromide under basic conditions (potassium carbonate) to give the allyl protected 2 in 75%. This protection step is necessary because the cyclisation of unprotected vanillic alcohol produces side-products in large quantities. 153 Compound 2 was then cyclized under acidic conditions using perchloric acid/methanol by performing simultaneously three Friedel-Crafts alkylations to offer the tri-allyl CTB 3 in a moderate yield (57%). The latter was then de-protected by using palladium acetate, triphenylphosphine and trimethylamine in THF/water mixture to offer CTB 4 in 79% yield, Figure 48. The second important step involves the synthesis of the substituted vanillic alcohol arm bearing either the primary alcohol or its precursors. The simplest thing to be tried was to introduce a primary alcohol unit, 3-bromo-2-bromomethyl-1-propanol, 17, on the vanillic alcohol 1 by an alkylation step under basic conditions (potassium carbonate). Attempts to make compound 20 failed, producing a lot of side-products that could not be easily identified by NMR spectroscopy or mass spectrometry. The second possibility was to alkylate the vanillic alcohol 1 by an ester substituted group (3-bromo-2-bromomethyl-1-methyl propanoate) 18. This can be eventually reduced to the corresponding alcohol derivative 20. Based on this, the same reaction conditions were used and they gave rise to the desired alkylated compound followed by an elimination reaction to offer the byproduct 21. Finally, the use 3-chloro-2-chloromethyl-1-propene, 19, in excess provided the desired product, 22 in a decent yield (65%), Figure 49. Having synthesized these two parts, it is time now to assemble them by an alkylation step in DMF under smooth basic conditions. In this step, a statistical mixture of three products is unavoidable thus leading to the formation of the desired compound in a moderate yield. Herein, protection of the benzyl alcohol is not necessary to assure a good separation of the three main products. The desired product 23 was isolated as a racemic mixture in 40% yield, Figure 50. Double alkylation of the mono-alkylated compound 23 by the two ethylenedioxy arms 24 was also performed under basic condition using cesium carbonate in DMF to offer the desired product 25 in 84% yield. The two arms have to be protected in the form of OTHP acetal to decrease the polarity of the cryptophane precursor and facilitate its separation on silica gel, Figure 51.

Unfortunately, addition of THP groups produces several diastereomeric compounds that cannot be separated by column chromatography and it complicates the interpretation of the 1 H NMR spectra. Nevertheless, some characteristic protons signals can be clearly identified. For instance, the 1 H NMR spectrum of compound 25 shows fifteen protons in the aromatic regions ranging between 6.65 and 7.00 ppm and two protons characteristic of the alkene group at 5.3 ppm. In The formed tri-alkylated template 25 was successfully cyclized under diluted concentrations (10 -3 M) in a mixture of formic acid and chloroform (50/50: v/v) at 60 °C for about 6 hours. This reaction involves rapid deprotection of the OTHP groups and it allows three Friedel-Crafts alkylations affording the desired anti-cryptophane 26 in 34% yield. At this stage, the anticonfiguration has been adopted based on previous results obtained with cryptophane-[223] derivatives. [START_REF] Chapellet | Experimental and Theoretical Study of the Complexation of Cesium and Thallium Cations by a Water-Soluble Cryptophane[END_REF] As usual, this cryptophane is obtained as a racemic mixture and it has a C2symmetry. This symmetry is clearly revealed by 1 H NMR spectrum that displays only six singlets in the aromatic region between 6.6 and 6.7 ppm corresponding to twelve protons. In addition, the two alkenyl protons are shown at 5.58 ppm and appear as a singlet. Finally, the six methoxy groups grafted on the cryptophane skeleton are revealed as three sets of singlet, Figure 52. At this stage, we can take advantage of the presence of the double bond to perform subsequent reactions like metathesis or addition reactions for instance.

It is noteworthy that employing an alkene function during the synthetic pathway saves a lot of protection steps, which would be required if we used a more reactive primary alcohol function instead. For instance, we can assume that the latter function will be surely dehydrated under the acidic conditions used for the cyclization step. To introduce a primary alcohol on the alkene arm of cryptophane 26 several reactions can be imagined but a hydroboration-oxidation step seems to us the best way to afford the desired product 27. To do so, we have used 9-BBN (0.5 M in THF), NaOH (3 M) and H2O2 in a mixture of THF and CH2Cl2 (80/20). Based on the analysis of the crude product, the reaction gave rise to exclusively cryptophane 27 and the second regioisomer was not detected. Purification of the compound afforded cryptophane 27 in an excellent yield (94%). The low solubility of starting material led us to change the usual conditions. Thus, such type of boron-based oxidation is carried only in THF, where it acts as both a solvent and a boron stabilizing ligand. In our case, using these conditions resulted in a decrease of the reaction yield (56%). Addition of CH2Cl2 was thus mandatory to completely dissolve the starting material and to drive the reaction to completion. Other peroxide-based conditions (Oxone) were also tried to perform the oxidation of the double bond. Unfortunately, these conditions led to a complete decomposition of the starting material. It is noteworthy that oxidation of the double bond led to a decrease of the molecular symmetry. Thus, anti-compound 27 has a C1-symmetry and it was isolated as a racemic compound. The proton NMR spectrum of the cage also reflects the lowering of the symmetry since the aromatic protons now appear as twelve singlets. The proton signal corresponding to the alkene group is no longer present and it is replaced by a new downfield shifted signal at 2.4 ppm that corresponds to the β hydroxyl proton (shown in red in Figure 54). Fortunately, we succeeded to obtain X-ray quality crystal of compound 27 from a chloroform/ethanol mixture and to get its X-ray structure. An ORTEP representation allowed us to assure the anti-configuration as assumed before. The cavity volume of the host was estimated to be 108 Å 3 with an electron count of 30, which is compatible with the presence of an ethanol molecule, Figure 53.

Reactivity of the Primary Alcohol on Cryptophane 27

Attachment of a primary alcohol on the middle of the propylenedioxy arm offers new routes for further functionalizations. For example, we can imagine converting the hydroxyl group into a nucleophile under basic conditions. It might be also transformed into an electrophile by performing a mesylation reaction for instance. Finally, we can perform an oxidation reaction to convert the alcohol group into an aldehyde or a carboxylic acid. These different functionalizations are needed to introduce a spacer that will carry the sensing arm.

Electrophilic Activation and Nucleophilic Substitution of the Primary Alcohol:

To invert the reactivity of the primary alcohol to an electrophile, it is necessary to convert it into a good leaving group. This can be achieved by the reaction of the alcohol function of compound 27 with mesyl-chloride or tosyl-chloride for instance. Surprisingly, the transformation of the primary alcohol to the corresponding tosylate group via a reaction with p-toluenesulfonyl chloride in pyridine failed and the starting material was recovered. This lack of reactivity is probably due to the bulkiness of the tosylate group. Using excess of the less bulky methanesulfonyl chloride in a pyridine/dichloromethane at 60 °C afforded the expected mesylated compound 28 in a good yield (82%), Figure 55. For this compound, the 1 H NMR spectrum shows a deshielding of the β hydroxyl proton by about 0.4 ppm with respect to the starting material. In addition, a singlet integrating for three protons at 3.07 ppm corresponding to the methyl group of the mesylate is also clearly visible on the spectrum. With the mesylated cryptophane many promising applications as a peroxide sensor or as a precursor to build-up new cryptophane derivatives.

Interestingly, this reaction did not afford only new cryptophane derivatives, but also it demonstrated the higher accessibility and electrophilic reactivity of the cage compared to its secondary alcohol congener. The thorough investigations performed on this derivative revealed some interesting features (self-encapsulation phenomena) that will be discussed in more details in the second part.

Oxidation of Cryptophane 27:

Beside the successful electrophilic activation of the cage, oxidation of the primary alcohol to the corresponding aldehyde and carboxylic acid appears a promising approach to attach new functionalities by a wide range of reactions like Wittig type reactions, reductive imination, aldolic condensation or amide coupling reactions.

The partial oxidation of the primary alcohol 27 to the aldehyde 30 was performed using Dess-Martin periodinane in freshly distilled CH2Cl2 at room temperature. It is worth mentioning that the use of freshly distilled CH2Cl2 is mandatory to render the reaction complete, Figure 56. Other conditions were also tried like BAIB/ TEMPO reagents in CH2Cl2 at room temperature offered exactly the same results. 155 The 1 H NMR spectrum of the crude aldehyde 30 shows a complete conversion of the alcohol, which is revealed by the appearance of the characteristic aldehyde peak at 9.92 ppm. A deshielding of the β hydroxyl proton by about 1 ppm (3.22 ppm) is also observed. Other impurities corresponding to Dess Martin monoiodinane byproduct are detected on this spectrum. Unfortunately, attempts to purify the obtained product by column chromatography on silica gel led to transformation of this derivative. 1 H NMR spectrum showing two additional signals between 4.0 to 5.0 ppm confirms the transformation of this derivative. This might be explained by the formation of the enol tautomer but the integration does not assure this assumption. A partial decomposition of the compound can be also envisaged to explain these results. Thus to avoid this problem, the aldehyde derivative was immediately used in the next oxidation step without further purification. The second oxidation to carboxylic acid 31 was carried using sodium chlorite in acetonitrile/water mixture at room temperature to offer the desired product 31 in a good yield (70%). 156 This cage was stable enough and it was purified by two different methods, either by precipitation from chloroform/diethyl ether mixture or by column chromatography on silica gel. In the latter case, the acid derivative was converted into the corresponding benzyl ester 32 that is more easily purified by chromatography. This step was followed by a hydrogenolysis reaction with a palladium catalyst in a dichloromethane/methanol mixture to give rise to the acid derivative 31 in an excellent yield (95%). However, the first purification method was preferred and it offers the compound in a higher yield (70%), Figure 56. Direct oxidation of the primary alcohol 27 to the carboxylic acid 31 using trichloro-cyanuric acid failed showing a complete decomposition of the starting materials. 157 The above described derivatives constitute new family of functionalizable cryptophane- [223] type cages, which will be further elaborated by other chemical transformations.

Synthesis of Water-Soluble Platforms for Xenon-Based Sensors

The introduction of water-solubilizing substituent is another key transformation that has to be considered. Interestingly, our cryptophane skeleton possesses several functionalities that can be selectively modified to introduce these substituents. The following paragraph is dedicated to the different strategies used to introduce the water solubilizing groups.

Demethylation Reaction:

Prior to the introduction of water-solubilizing groups on the cryptophane rims, a demethylation step of the six available methoxy groups is required. Demethylation of these groups using lithium diphenylphosphide (1 M) is well established on this type of molecular host.

The synthesis of lithium diphenylphosphide was described by E. Vejeds 158 and used by A. Collet to produce cryptophane-[222] (OH)6 and optimized by Brotin and co-workers. This method is selective for methyl ether deprotection, due to the steric hindrance of diphenylphosphide, and it leaves unreacted the three bridges. Even though this reagent can be kept for weeks in a fridge, we recommend using freshly prepared solution of lithium diphenylphosphide to obtain reliable results.

Introducing water solubilizing precursors at the very beginning of the synthesis can be done by demethylation of the methoxy groups of compound 26, Figure 57. Attempts to perform this reaction with freshly prepared LiPPh2, at 60 °C in THF produced the expected demethylated product but it showed also the disappearance of the NMR signal corresponding to the double bond. The appearance of several byproducts indicates the decomposition of the starting material under these conditions. We switched to another strategy, which is the demethylation of cryptophane 27 bearing a primary alcohol function followed by a selective alkylation step. Our previous experience with cryptophane 12 revealed that secondary alcohol was reactive towards the phenol alkylation reaction when methyl bromoacetate is used as an electrophile. As our alcohol 27 is less hindered and even less basic than that in cryptophane 12, a protection step is hence necessary to selectively functionalize the six phenols. As mentioned previously, the demethylation step is performed under basic conditions and an acidic workup is usually needed to recover the demethylated product. Keeping this information in mind, we must choose an acid and base tolerant protecting group. Based on our knowledge, we believe that a tetrahydropyranyl group would be a good candidate to resist both basic and mild acidic conditions. So we first protected the cage 27 as an acetal under mild acidic conditions using PPTS/DHP reagents. Compound 34 was thus obtained in a good yield (75%), Figure 58. Adding a new stereocenter on the cage led to the formation of a mixture of diastereomers, which are not separable by column chromatography but they can be easily distinguished by [START_REF] Moran | Synthetic Molecular Vessels[END_REF] C NMR spectroscopy.

Introducing Water-Soluble Groups:

Cryptophane rims can be decorated with different solubilizing functions like carboxylic acid derivatives, polyethylene glycol (PEG) or sulfonyl groups. In our case, we focused on grafting selectively six ester groups on the cryptophane 35 rims as precursors to six carboxylic acid functions. We chose to introduce two types of ester group, methyl or benzyl esters to access the carboxylic acid derivative by hydrolysis under basic condition or hydrogenolysis with a palladium catalyst.

These cryptophanes can be obtained by performing an SN2-type reaction on an appropriate alkylating reagent. To do so, the cage was dissolved in DMF in presence of cesium carbonate.

Then alkylation with methyl or benzyl bromoacetate afforded the expected derivatives 36 and 37 in 54% and 70% yields, respectively. These two precursors were also isolated as diastereomeric mixtures but their purification was tedious owing to their high lipophilicity. It is worth mentioning that unlike cryptophane 37, benzyl protected cryptophane 36 tends to implode easily as ascertained by its 1 H NMR spectrum. This imploded form seems to be reversed back to its initial (CC) conformer after the OTHP deprotection. Consequently, the unprotected cage 38 was obtained in an excellent yield (93%). The inversion to the initial (CC) conformer is mostly due to the used reaction conditions (CH2Cl2 at 40 °C). Under these conditions, the cage will be surely filled back with dichloromethane thus restoring the initial conformation. Same conditions were used to obtain the unprotected derivative 39 and it was also obtained in almost quantitative yield (93%), Figure 60.

Successful selective grafting of water-solubilizing precursors on the compound 35 afforded us two new platforms, 38 and 39 for 129 Xe-based biosensing applications. These two derivatives can be either used immediately to graft a sensing unit or can be subjected to subsequent reactions.

Chemical Transformations of Cryptophanes 38 and 39:

One of the strategies used to functionalize the primary alcohol of methyl protected cage 39,

was to simply alkylate it under basic conditions. In this reaction two main requirements have to be considered, the reaction temperature and the used base. In order to avoid implosion of the cryptophane skeleton, a relatively low reaction temperature has to be employed (between 25 and 40 °C). Additionally, mild bases have to be used because strong ones will deprotonate the α-ester protons leading to the formation of many side-products.

We chose to introduce a protected carboxylic acid arm via the alkylation of the hydroxyl group by benzyl bromoacetate to form derivative 40, Figure 61. Taken the above considerations into account, we chose either potassium tert-butoxide or DBU as bases due to their bulky structure and matching pKa. In both cases, the cage was dissolved in DMF in the presence of the base, and then the alkylating agent was added at room temperature. None of these conditions worked, and these reactions resulted in the formation of many side-products.

Functionalization of Cryptophane-[223] Derivatives by Sensing Units

In the first chapter we presented a wide range of 129 Xe-based biosensors, which are mostly derived from cryptophane-[222] monoacid. The attachment of the sensing unit to this cryptophane core is usually achieved by an amide coupling. In our case, we used the same approach to link different amine or aniline-bearing sensing units to cryptophane derivatives 43 and 44. This step is followed by the cleavage of the protecting groups to offer the water soluble cryptophane-[223] molecular sensors. In the next paragraphs, we will present the different sensing units grafted to the cryptophane- [223] cores and the problems encountered during the synthesis, Figure 64. Hydroxamic acids coordinate transition metals via the two oxygen atoms as proved by X-ray crystal structures. In contrast, coordination of transition metals via an oxygen-nitrogen coordination mode has never been reported so far, at least for the primary hydroxamic acid derivatives. As established in literature, hydroxamic acids chelates effectively the late transition metals like copper, nickel and zinc with comparable binding constants in the order of 10 5 M -1 . This triggered us to connect this simple chemical function to our dually functionalized cage and study its interaction with different cations. 160 The link between hydroxamic acid and cryptophane 43 was achieved via an amide coupling reaction. Herein, the source of our hydroxamic acid function is the commercially available Obenzyl hydroxylamine. In order to assess the feasibility of the reaction we first tried the amide coupling on cryptophane

31.

The coupling reaction between this cage and the O-benzyl hydroxyl amine was performed in pyridine using phosphorous oxychloride as a coupling reagent, Figure 66. 163 Under these conditions the carboxylic acid is first converted to the acyl chloride and then reacted with the amine function. This reaction offered the coupled compound 45 in 62% yield. The 1 H NMR spectrum of this compound shows a characteristic deshielded proton of the amide group at 9.7 ppm. Interestingly the diastereotopic protons of the benzyl group appear as an AB system at 4.9 ppm. Finally, the latter derivative was deprotected at room temperature using a hydrogenolysis reaction under classical conditions (H2, Pd/C (10%), CH2Cl2/MeOH mixture). These conditions afforded the compound 46 in a good yield (70%). to an aromatic fluorinated reporter. Unexpectedly, this unit showed a higher affinity to calcium compared to magnesium, where its detection limit is in mM range whereas that of the calcium is in the µM range. Several MRI and fluorescent probes were developed based on a chelating APTRA ligand. Development of more sensitive reporters capable to detect calcium in pico-molar range can be achieved by designing a new (HP) 129 Xe@cryptophane derivative functionalized with an APTRA function. 164,165,166,167 Our targeted APTRA derivative 52 is functionalized with an aniline moiety to anchor it to the cryptophane- [223] derivative via an amide bond, Figure 68. This unit was synthesized in three linear steps starting from a commercially available 2-amino phenol 49 based on a slightly modified known experimental procedures. 168,169 It starts with the alkylation of the 49 with methyl bromoacetate using DIPEA as a base to afford compound 50 in a moderate yield (60%). Nitration of compound 50 in a mixture of nitric and acetic acid afforded compound 51 in 80% yield. The last step involved a hydrogenation step using catalytic amount of palladium on charcoal. This reaction gave rise to APTRA derivative 52 in a good yield (90%).

Figure 68: synthesis of the APTRA unit 52.

50 52 51

Here again, the reaction was performed on the organic version 31 to ensure its viability. Coupling of the APTRA derivative 52 with the compound 31 was performed in pyridine and phosphorous oxychloride. This reaction offered compound 53 in low yield (22%) due to partial decomposition of the APTRA moiety, Figure 69. We then extended this reaction to the pre-water soluble version 44. We chose this compound because it has the same protecting groups as derivative 52. The linkage between the two units was also performed by an amide coupling reaction using phosphorous oxychloride and pyridine to give the product 54 as a white solid in 30% yield, Figure 70. The 1 H NMR spectrum of this pre-water soluble precursor 54 displays a characteristic signal at 9.07 ppm corresponding to the amide proton and three additional signals in the aromatic region that belong to the APTRA core.

For the hydrolysis step, several conditions were tried to simultaneously remove the nine methyl groups and all use different hydroxide salts like LiOH, NaOH and KOH as summarized in Table 3. The solvent used for this step was either a mixture of THF/water or with methanol. The starting material was first dissolved in THF then the metal hydroxide solution was added at zero °C then to 25 °C and monitored by TLC. All the tried reactions revealed the disappearance of starting material within only 10 minutes, except for the first entry, where it was left over night at 52 31 53 40 °C. After that, the crude was extracted with CH2Cl2 and then acidified using HCl in ice to offer a white powder. This was then checked by 1 H NMR to reveal either partially hydrolyzed (LiOH) or decomposed byproducts (NaOH) and this was confirmed by HRMS. A last trial with 18 equiv. of KOH (0.3 M) provided the targeted product 55 in about 70% yield. The formation of this product was confirmed by HRMS. It seems that there is a very narrow window between the hydrolysis and decomposition of this compound, where a careful tuning of the chosen base and its equivalence must be done to avoid these problems. Unfortunately, the hydrolyzed compound 55 shows also some light sensitivity issues where it turns brownish when exposed to light. This prevented us from studying its (HP) 129 Xe encapsulation properties because they may give misleading results.

Biotin Functionalized Cryptophane-[223] Cage:

To further illustrate the feasibility of our approach, we turned our attention towards the bioconjugation of cryptophane-[223] by a biotin arm for avidin detection. This was chosen because of the high binding constant, which is estimated to be in the range of K = 10 15 M -1 . Our biotin source is the commercially available biotin-PEG2-amine, which will be linked to the

56

To link these two parts, we simply used the well-established amide coupling conditions i.e.

phosphorous oxychloride/ pyridine. Unfortunately the reaction did not work and the cryptophane was recovered at the end of the synthesis. The usual coupling conditions using EDCI/HOBt in the presence of DIPEA in DMF for 16 hours gave better results and provided compound 56 as a white powder in 71% yield. The purification of this compound is quite tedious due to its high polarity. The 1 H NMR spectrum of 56 shows a clear signature of the biotin arm in the up field region as also demonstrated by the [START_REF] Moran | Synthetic Molecular Vessels[END_REF] C NMR and the HRMS, Figure 72. The production of the water-soluble sensor 57 necessitates an additional hydrogenolysis step.

Hydrogenolysis using the classical conditions (H2, Pd/C in a CH2Cl2/MeOH mixture) revealed the appearance of a benzyl protected new byproduct. The lack of benzyl deprotection might be due to the sulfur atom, which poisons the catalyst. We switched then to the hydrolysis condition using 18 equiv. of KOH (0.3 M) in THF/water mixture for 30 minutes, and this time the product The success of this reaction clearly shows that it is possible to enlarge the scope of reactivity with our platforms. Further experiments will be necessary in the future to improve the grafting of the sensing arm.

Sensors

From a structural point of view, we have been able to construct water-soluble selectively functionalizable cryptophanes, solving by this the solubility and symmetry problems encountered when using other cages. To test if our platforms attain the requirements for the targeted sensing applications, we have studied the (HP) 129 Xe NMR spectroscopy of these cages in collaboration with Dr. Patrick Berthault at the Laboratoire de Structure et Dynamique par Résonance Magnétique (LSDRM) at CEA in Saclay, France. The production of the (HP) 129 Xe is achieved by a batch mode SEOP, with a polarization ranging between 15 to 20%. The hyperpolarized 129 Xe NMR experiments were performed at 11.7 T. In addition to this, isothermal titration calorimetry (ITC) experiments were also conducted to assure the interactions of our sensors with the targeted analytes.

Characterization of Cryptophane 48 Bearing a Hydroxamic Acid Group

Cryptophane derivative 48 is water-soluble over a large range of pH. Two experimental campaigns were launched to see the effect of metals chelation and pH variation by (HP) 129 Xe spectroscopy and ITC.

Effect of pH:

To ascertain the ability of cryptophane 48 to bind xenon, a simple (HP) 129 In the first chapter, we presented the remarkable ability of (HP) 129 Xe@cryptophane complexes to sense the slight variations in the pH. [START_REF] Huber | Unsaturated Cryptophanes: Toward Dual PHIP/Hyperpolarised Xenon Sensors[END_REF]134 We specifically discussed two cages, the cryptophane hexacarboxylate and the cryptophane tri-carboxylate, each bearing different ionizable groups.

Having seven ionizable groups on cryptophane 48 derivative triggered us to investigate the effect of pH change and eventually compare it to the previously reported results.

For this purpose the (HP) 129 Xe NMR spectra of compound 48 (0. Between pH values of 9.9 and 8.2 not much variation in the encapsulated xenon signal is observed (1 ppm) although the pKa value of the hydroxamic acid group is reported to be in this range. 160 This result indicates that the ionization of the hydroxamic acid function grafted on the derivative can be explained by the smaller cavity size of this compound.

Zinc and Nickel Complexation by ITC and (HP) 129 Xe Spectroscopy:

Grafting a hydroxamic acid function on the cage's 48 arm allowed us to study the chelation of different metal cations like zinc and nickel. To assure that our probe chelates these cations we used ITC experiments. This technique allows us to access not only the binding constants but also the thermodynamic parameters of the complexation (G°, H°, S°). To avoid misleading results it was necessary to conduct these experiments in presence of a buffer. Indeed, in absence of buffer, the addition of a small amount of cationic species changes the pH of the solution and in turn it affects the parameter of complexation. It took a while for us to optimize the experimental conditions due to solubility issues of the metal salt. For instance, ZnCl2 was not soluble neither in HEPES, phosphate buffer nor MOPS. It is noteworthy that a cloudy solution was obtained in all cases even though some articles reported these conditions. 170 Fortunately, we found that 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) (20 mM, pH = 7.5) could be used to dissolve zinc chloride.

Free xenon

129 Xe@48 and K2 = 850 M -1 ) corresponding to formation of 1:2 complex. Similarly, zinc binding by our cryptophane chelator reveals a moderate binding constant (Ka = 4.4 ± 0.22×10 4 M -1 ) lower than that reported with acetohydroxamic acid (Ka = 2.8×10 5 M -1 ). In another study, the Zn 2+acetohydroxamic acid complexation reveals the formation of a 1:2 complex of respective binding constants K = 4.3×10 9 M -1 and K = 1.5×10 4 M -1 . These results indicate that the binding mechanism and strength is strongly dependent on the experimental conditions and the structure of hydroxamic acids. 172,173 The fact that we obtain a 1:1 complex rather than a 1:2 one can be explained by the molecular size of the cages used. Thus, the steric hindrance prevents two cages to be in close proximity.

After confirming the chelating ability of our cage, we have investigated the complexation process by (HP) 129 Xe NMR spectroscopy. These studies were performed in the same experimental conditions as the one used for the titration experiments: TRIS buffer (20 mM, pH = 7.5). Here again, the successive addition of zinc cation solution from 1 to 5 equiv. did not induce significant change of the (HP) 129 Xe@cryptophaneZn 2+ complex. Although the zinc cation is very close to portals of the cryptophane's skeleton, only little spectral modifications are observed. This result is a bit disappointing compared to the previously reported biosensor with cryptophane- [222] derivative bearing an NTA arm, Figure 33. The localization of this sensing unit makes a great difference since as previously mentioned; it affects more significantly the ring current of the benzene ring. In turn, it induces larger magnetic modifications on the encapsulated xenon. This result approves also the little change in xenon signal at pH between 8.2 and 9.9.

The little sensitivity of xenon NMR at this position can also be exemplified by studying the cryptophane 34 bearing a THP group. Cage 34 exists as a mixture of two diastereomers but xenon NMR spectroscopy was unable to distinguish two signals corresponding to each of them. This result also contrasts with the reported examples of cryptophane derivatives bearing a mixture of diastereomers. However, it assures our hypothesis concerning the position of the sensing units. 116,117

Complexation Studies of Cryptophane 57 Bearing a Biotin Derivative

After proving that our platforms cannot serve as smart xenon biosensors due to the position of the sensing unit, we wanted to test its ability as density based sensor instead. This requires the grafting of a sensing unit that has a high affinity to the targeted moiety. For this reason, we chose the classical biotin-avidin complex due to the high binding constant reported (Ka = 10 15 M -1 ). The These promising results assure that our platforms can still be used as MRI tracers, if conjugated with biological probes that have a very high affinity to the targeted units. bearing six ester groups and a unique carboxylic acid arm. The presence of a carboxylic acid moiety at the propylenedioxy bridge allows the selective grafting of a spacer and a sensing arm.

To prove the versatility of our cages, we grafted different sensing units like hydroxamic acid for metal chelation, APTRA derivative for calcium detection and finally a biotin arm for avidin binding. This was followed by a hydrolysis or hydrogenolysis step to produce the water soluble sensors. With these platforms, we were able to solve the two main problems encountered with cryptophanes namely the symmetry and water solubility. To study the responsiveness of our platforms, we investigated cryptophane 48 bearing a hydroxamic acid unit for metal chelation.

The ability of this cage to bind metals has been confirmed by ITC experiments. The (HP) 129 Xe NMR spectroscopy of this cage showed a little sensitivity upon metal chelation. These discouraging results can be explained mainly by the position of the sensing arm. Being at the center of the propylenedioxy arm, the sensing unit has less probability to change the benzene ring currents. This in turn induces fewer changes in the magnetic field around the encapsulated cage.

The fact that compound 48 is poorly responsive by 129 Xe NMR, does not prevent its employment as an MRI tracer but this novel strategy does not seem appropriate to design "smart" biosensors as previously defined. However, this study allowed us to assure the importance of the sensing arm position to get "smart" biosensors and we were able to find discrete solutions for this synthetic challenge.

Experimental

ITC Experiments:

Isothermal titration calorimetry (ITC) experiments were performed at 298 K. In a standard experiment, the host solution (0.08 mM) in TRIS (20 mM, pH = 7.5) was placed into the calorimeter cell (1,4 mL) and 28 successive aliquots (10 μL) of guest solution (10 times more concentrated, 1mM) were added via a computer-automated injector at 3 min intervals. Heat changes were recorded after each addition. Heats of dilution were measured by a blank experiment (in absence of host) under the same conditions and they were subtracted from the titration data prior to curve fitting. The first injection was discarded from each data set to remove the effect of guest diffusion across the syringe tip during the equilibration process. Titrations curves were fitted with the one binding site model using origin software.

Production of hyperpolarized xenon:

Hyperpolarized xenon was produced in the batch mode via SEOP (Spin Exchange Optical Pumping) using a home-built setup. Rubidium is used as the alkali metal, and five minutes suffice

Crystallography:

A suitable crystal of Cryptophane 27 was selected and mounted on a Gemini kappa-geometry diffractometer (Agilent Technologies UK Ltd) equipped with an Atlas CCD detector and using Cu radiation (λ= 1.5418 Å). Intensities were collected at 150 K by means of the CrysAlisPro software. Reflection indexing, unit-cell parameters refinement, Lorentz-polarization correction, peak integration and background determination were carried out with the CrysAlisPro software.

An analytical absorption correction was applied using the modeled faces of the crystals. The resulting set of hkl was used for structure solution and refinement. The structures were solved by direct methods with SIR97 and the least-square refinement on F2 was achieved with the CRYSTALS software. All non-hydrogen atoms were refined anisotropically. The hydrogen atoms were all located in a difference map, but those attached to carbon atoms were repositioned geometrically. The H atoms were initially refined with soft restraints on the bond lengths and angles to regularize their geometry (C---H in the range 0.93--0.98 Å, O--H = 0.82 Å) and Uiso(H) (in the range 1.2-1.5 times Ueq of the parent atom), after which the positions were refined with riding constraints. Residual electronic density inside and in between the cryptophane cages was located but could not be modelled. The contribution of the disordered solvent molecules was removed using the SQUEEZE algorithm. The volume of the cryptophane cage was calculated to be 108 Å 3 using the VOID algorithm within the PLATON software. CCDC 1900550 contains the supplementary crystallographic data for cryptophane 27. These data can be obtained free of charge from the Cambridge Crystallographic data Centre via www.ccdc.cam.ac.uk/data request/cif.

Synthesized compounds:

Compound 22: 3-chloro-2-chloromethyl-1-propene or 19 (4.49 ml, 64.8 mmol, 2 equiv.) was added under argon to a mixture of K2CO3 (4.48 g, 32.4 mmol, 1 equiv.) and vanillic alcohol 1 (5 g, 32.4 mmol, 1 equiv.) in acetone (60 mL). The reaction mixture was heated for 16 hours at 80 °C under an argon atmosphere.

Then, the solvent was evaporated under reduced pressure. The solid residue was then dissolved in ethyl acetate (200 mL). The organic layer was washed three times with 0.1 M NaOH solution and finally washed with water. The organic layer was dried over Na2SO4 and the solvent was evaporated under reduced pressure. The crude was then subjected to column chromatography on silica gel (CH2Cl2/AcOEt: 80/20). After evaporating the solvents, compound 22 was collected as a white solid (5.1g, 65%). Mp: 60 °C. 8, 147.4, 140.7, 134.5, 119.3, 117.5, 114.2, 111.06, 69.4, 65.2, 55.9, 45.08 (3.5 g, 8.6 mmol, 1 equiv.) in DMF (120 mL). The mixture was heated up to 80°C for 16 hours under an argon atmosphere.

HRMS
Then, the dark-brown mixture was poured in water (200 mL) and extracted 5 times with ethyl acetate (800 mL). The combined organic layer was then washed 5 times with water (500 mL) and dried over Na2SO4. Evaporation of the solvent under reduced pressure left an oily residue, which was purified on silica gel (AcOEt/petroleum ether: 90/10 3, 147.8, 147.1, 146.1, 144.9, 144.8, 143.7, 143.6, 140.2, 133.8, 132.1 (2C), 131.9, 131.5, 130.9, 118.96, 115.6, 115.2, 115.0, 114.8, 113.3, 113.2, 112.0, 111.8, 110.5, 69.6, 69.4, 64.9, 55.75, 55.7, 55.5, 55.3, 36-35.9 (3C). under an argon atmosphere. Then, 9-BBN (1.81 mL, 0.5M, 0.9 mmol, 3 equiv.) was added dropwise at 0 °C. The solution was stirred at 50 °C for 5 hours. The mixture was then quenched at 0 °C by a dropwise addition of a NaOH solution (0.56 mL, 3M) and then by addition of H2O2 (1.7 mL, 15%). The reaction was stirred overnight at room temperature for 16 hours. Then, the reaction was quenched by a dropwise addition of 20 ml of Na2S2O3 solution at 0 °C. The reaction mixture was poured in CH2Cl2 ( 50ml) and the aqueous layer was washed 5 times with CH2Cl2 (each time 50 ml). The combined organic layers were dried over Na2SO4 and the solvent was evaporated under reduced pressure.

HRMS (ESI): m/z

The residue was subjected to column chromatography using silica gel (CH2Cl2/acetone: gradient 90/10 up to 80/20). Compound 27 (0.27 g, 94%) was then isolated as a white solid and washed on a frit with diethyl ether. 5, 132.3, 132.1, 131.5, 131.3, 131.1, 130.8, 130.6, 120.5, 120.4, 120.2, 120.0, 114.3, 114.2, 113.6, 113.2, 112.7, 112.3, 112.1, 111.8, 69.8, 69.5, 68.9, 68.7, 68.5, 67.0, 64.3, 56.3 (2C), 55.5 (2C), 55.4, 55.3, 40.2, 36.4 Compound 31: cryptophane 32 (0.1 g, 0.1 mmol, 1 equiv.) was introduced and dissolved in acetonitrile (3 ml) and water (0.3ml). Then sodium chlorite (0.019 g in 0.2 ml H2O, 0.212, 2 equiv.) was added along with sodium phosphate monobasic (0.0031g in 0.01 ml H2O, 0.0212, 0.2 equiv.) and then hydrogen peroxide (0.102 ml). The mixture was kept for 16 hours and then quenched with sodium thiosulfate (0.2 ml) at 0 °C. The solvent was then evaporated under reduced pressure and the resulting crude was crystalized from CHCl3 and diethyl ether to give white precipitate. These precipitate were filtered on a fritted glass and washed with ether to give the compound 31 (0.07 g) as pure white product in 70% yield.

Compound 31: compound 32 (0.07 g, 0.067 mmol) was dissolved in a mixture of CH2Cl2/MeOH (4ml/2ml) along with catalytic amounts Pd/C (10% by weight). The mixture was kept under hydrogen for 16 hours at room temperature. The obtained crude was then filtered on celite followed by the evaporation of solvent, to afford compound 31 as a white solid (0.059 g, 95%).

1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 6. 77-6.57 (12s, 12 H), 4.63-4.55 (m, 6 H), 4.38-4 (m, 12 H), 3.8-3.75 (6s, 18 H), 3.43-3.36 (m, 6 H), 3.3 (m, 1H).

13 C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): δ (ppm) 172. 7, 149.59, 149.54, 149.47, 149.41, 147.9, 147.7, 146.76, 146.73, 146.67, 146.64, 146.5, 146.3, 134.0, 133.8, 133.7 (2C), 132.6, 132.4, 132.2, 132.1, 131.5, 131.4, 131.1, 131.0, 120.2 (3C) 9, 147.6, 147.4, 147.3, 147.2, 146.8, 146.7, 146.5 (2C), 134.0, 133.95, 133.9, 133.8, 132.4 (2C), 131.4 (4C), 130.6 (2C), 120.5 (4C), 114 (2C), 113. 7, 113.5, 113.0, 112.8, 112.4 (2C), 112.3, 98.4, 69.7, 69.0, 65.5, 65.4, 64.7, 62.6, 61.8, 56.2, 55.8, 55.6 (2C), 55.5, 55.4, 40.5, 36.4 8, 146.7, 146.3, 146.2, 146, 145.9, 145.6, 145.4, 145.06, 145.0, 144.8, 144.7, 134.3, 134.1, 134.0, 133.9, 132.9, 132.8, 131.4, 131.2, 130.9, 130.8, 129.7, 129.6, 120.5, 120.3, 119.3 (2C), 118.7, 118.5, 118.02 (2C), 117.4, 116.9, 115.6, 115.5, 98.9, 69, 68.7 (2C), 67.6, 66.5, 65.9, 62.2, 35.6 (2C), 35.2 (4C), 30.8, 25.5, 19.8, 19.7. 134.7, 134.3, 133.8, 133.5, 133.4, 133.37, 133.2 (2C), 132.9, 132.0, 131.6, 131.3, 128.5-128.8 (30 C), 121.1, 120.7, 120.6, 119.7, 119.4, 119.2, 119.1, 118.8, 118.1, 115.5, 113.5, 113.1, 69.7, 69.1, 68.8, 68.6, 68.1, 68.0, 67.9, 67.9, 67.8, 67.7, 67.2, 67 (2C), 66.9 (2C), 66.8, 66.7, 66.6, 66.3, 63.4, 41.1, 36.2, 35.9, 35.7 (3C).

Mixture of two diastereomers

HRMS (ESI): m/z

HRMS (ESI): m/z

[M+Na] + calcd. for C104H94NaO25 1765.5981 found 1765.5976.

Compound 39: PPTS (0.050 g, 0.2 mmol) in methanol (3 mL) was added under an argon atmosphere to a solution of 37 (0.17 g, 0.12 mmol) in CH2Cl2 (5 mL).

The solution was stirred under argon at 40 °C. The solvents were evaporated under reduced pressure and the oily residue was subjected to column chromatography on silica gel (CH2Cl2/acetone: 80/20). After evaporating the solvents, compound 39 (0.14 g, 93%) was collected on a fritted glass as a white solid and was washed with a small volume of diethyl ether.

1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 6. 82-6.55 (12s, 12 H), 4.66-4.46 (m, 18 H), 4.34-3.78 (m, 32 H), 3.40-3.34 (m, 6H), 2.29 (m, 1H). 8, 169.7 (2C), 169.6, 169.2, 148.3, 147.9, 147.6 (3C), 147.4, 147.2, 147.1 (2C), 147.0, 145.9 (2C), 134.9, 134.8, 134.4, 133.8, 133.5, 133.48, 133.3, 133.2, 133.1, 132.9, 131.8, 131.7, 121.0, 120.8, 120.3, 119.7, 119.3, 119.2, 118.9, 118.4, 118.1, 114.7, 113.5, 113.1, 69.6, 69.2, 68.8 (2C), 68.6, 68.3, 67.7 (2C), 67.5, 67.3, 67.0, 66.5, 66.3, 51.8-52.1 (6C), 41.0, 35.7-36.2 (6C). ) was collected as white solid.

HRMS (ESI): m/z

1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 7. 44-7.30 (m, 30H), 6.62-6.36 (12s, 12H), 5.31-5.09 (s, 12H), 4.55-4.37 (m, 18 H), 4.15-3.95 (m, 12H), 3.19-3.15 (m, 6H), 3.07 (m, 1H).

13 C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): δ (ppm) 172. 5, 169.6, 169.4, 169.3, 169.2 (2C), 169.1, 147.8, 147.7 (2C), 147.6 (2C), 147.4 (2C), 147.3, 147.2 (2C), 146.2, 145.9, 135.2,-135.5 (6C), 134.4 (2C), 133.6 (2C), 133.3, 133.2 (2C), 133.1 (2C), 133.0, 132.4, 132.0, 128.6-128.8 (30C), 120.9 (2C), 120. 6, 120.27 (2C), 119.1 (2C), 118.8, 118.3, 117.5, 114.5, 114.2, 69.4, 69.3, 68.8 (2C), 68.1, 68.0, 67.9 (2C), 67.2 (2C), 67.0-66.9 (6C), 64.8 (2C), 62.8, 45.6, 36.1 (2C), 35.7 (4C).

HRMS (ESI): m/z

[M+Na] + calcd. for C104H92NaO26 1779.5774 found 1779.5769.

Compound 45: Cryptophane 31 was introduced (0.13 g, 0.13 mmol, 1 equiv.) and then dissolved in freshly distilled pyridine (2ml). Then O-benzyl hydroxylamine (excess, 0.04 ml) was added and the solution was brought to -15 °C. POCl3 (excess, 0.05 ml) was introduced drop wisely and the solution was stirred for 30 minutes at -15 °C then for 5 hours at room temperature. The obtained yellowish solution was poured cautiously in ice/water/NaHCO3 at 0 °C in which a lot of precipitates were obtained. The solution was filtered then extracted 5 times with CH2Cl2 and the solvent was evaporated under reduced pressure. The obtained crude was purified on silica (CH2Cl2/Acetone: 90/10) to afford the product as white powder which was further washed with diethyl ether (0.09 g, 62%).

1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 9.70 (s, 1H), 7.42-7.3 (m, 5H), 6.74-6.55 (12s, 12H), 4.96-4.91 (d, j=, 11.5 Hz, 1H), 4.91-4.94 (d, j=11.5, 1H), 4.62-4.55 (m, 6H), 4.26-3.82 (m, 12H), 3.79-3.67 (6s, 18H), 3.4-3.36 (m, 6H), 3.09 (m, 1H).

13 C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): δ (ppm) 169. 7, 149.6, 149.5, 149.4, 149.3, 148.1, 147.86, 146.8, 146.7, 146.6 (2C), 145.4, 135.6, 134.0, 133.9, 133.7, 133.6, 133.2, 132.4, 132.0 (2C), 131.5, 131.48, 131.4, 130.9, 129.2 (2C), 128.6, 128.4 (2C), 120.3 (2C), 120.1, 119.9, 114.7, 114.4, 114.2 (2C), 113.7, 113.5, 112.9, 112.5, 77.2, 69.7, 69.4, 68.9, 68.7, 66.6, 64.5, 56.3, 56.3, 55.69, 55.61, 55.5, 55.4, 46.1, 36.4, 36.1 (4 C). hours. The produced solution was filtered on cellite and washed with CH2Cl2 for several times. It is important to mention that the product was slightly colored (reddish). Following this, the solvent was evaporated under reduced pressure and the obtained solid was washed with diethyl ether to offer pure product 46 (0.02 g 30%).

HRMS (ESI

1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 10.21 (s, 1H), 6.76-6.6 (12s, 12H), 4.56-4.53 (m, 6H), 4.24-3.92 (m, 12H), 3.83-3.74 (6s, 18H), 3.41-3.37 (m, 6H), 3.2 (m, 1H).

HRMS (ESI): m/z

[M+H] + calcd. for C56H57NNaO14 990.3676 found 990.3671.

Compound 47: Excess of POCl3 (0.013 mmol)

was slowly added at -15 °C to a solution of 43 (0.14 g, 0.08 mmol, 1 equiv.) and O-benzyl hydroxylamine (0.020 mL) in pyridine (3 mL).

The solution was stirred for 30 minutes at this temperature hours and then stirred for further 5

hours at room temperature. The solution was quenched by addition of a mixture of ice/water/NaHCO3 at 0 °C. The obtained mixture was washed 5 times with CH2Cl2. Then the combined organic layers were dried over Na2SO4. The solution was filtered and the solvent was removed under pressure to leave a residue. Purification on silica gel (CH2Cl2/acetone: 95/5) gave rise to compound 47 (0.04 g; 26%) as a white solid. This solid was collected on a fritted glass and washed several times with diethyl ether.

1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 9.63 (s, 1H), 7.43-7.26 (m, 35H), 6.59-6.26 (12s, 12H), 5.35-4.94 (m, 14H), 4.65-4.34 (m, 18H), 4.05-3.68 (m, 12H), 3.27-3.19 (m, 6H), 2.89 (m, 1H).

13 C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): δ (ppm) 169. 5, 169.4, 169.3, 169.2, 169.1, 168.7, 168.6, 147.9, 147.7, 147.6, 147.66, 147.5, 147.3 (2C), 147.2 (2C), 146.5, 146.4, 146.3, 135.3-135.7 (6C), 134.6, 134.2, 133.7, 133.6, 133.4 (2C), 133.3, 133.2, 133.1 (2C), 132.6, 132.1, 129.5-129.5 (35C), 121.0, 120.7, 120.0, 119.6, 119.5, 119.1, 118.8, 118 (2C), 115.5 (3C), 115.2, 78.1, 69.7, 69.1, 68.7, 68.4, 68.1, 67.9, 67.8 (2C), 67.3, 66.8-66.9 (6C), 66.5, 66.0, 64.7, 47.3, 36.0, 35.9, 35.7 (4C).

HRMS (ESI): m/z

[M+Na] + calcd. for C111H99NNaO26 1884.6353 found 1884.6347.

Compound 48: Hydrogen gas was bubbled into a solution of cryptophane 47 (0.057 g, 0.03 mmol)

and Pd/C (10%) (0.05 g) in a mixture of CH2Cl2 ( 2mL) and methanol (1 mL). The solution was kept under hydrogen atmosphere for two nights at room temperature. Then, the mixture was filtered on cellite and washed CH2Cl2 and methanol. The solvents were evaporated under reduced pressure to leave a residue. It was then dissolved in aqueous NaOH (0.3 M) and the aqueous layer was washed 3 times whit CH2Cl2. Acidification with few drops of conc. HCl at 0 °C gave rise to a precipitate which was collected on a fritted glass and washed several times with diethyl ether.

Compound 48 (0.015 g, 40%) was obtained as a white solid.

1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 9.69 (s, 1H), 6.94-6.69 (12s, 12H), 4.79-4.29 (m, 30 H), 3.98-3.84 (m, 7H).

13 C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): δ (ppm) 171. 5, 170.4 (3C), 170.1, 170.0, 168.3, 148.5, 148.2, 147.5, 147.4, 146.3 (2C), 146.2, 146.1, 146.0, 145.9, 145.4, 144.8, 134.9, 133.8, 133.7, 132.9, 132.8 (3C), 132.7, 132.6, 132.3, 132.2, 132.0, 123.9, 120.2, 118.8 (2C), 118.6, 118.4, 117.8, 117.4, 117.1, 115.7 (2C), 115.3, 68.9, 68.2, 67.9 (2C), 67.8, 67.2, 66.9, 66.8, 66.0, 65.6, 65.4, 65.1, 43.3, 35.4-35.0 (6C).

HRMS (ESI): m/z

[M+Na] + calcd. for C62H57NNaO26 1254.3066 found 1254.3061. Compound 53: Cryptophane 31 (0.035 g, 0.036 mmol, 1 equiv.) was first introduced and dissolved in freshly distilled pyridine (1ml). Then the aniline 52 (0.0148 g, 0.0435 mmol, 1.2 equiv.) was added and the mixture was brought to -15 °C. After that POCl3 (1.1equiv., 4.03 µl, 0.043 mmol) drop wisely and then stirred at -15 °C for 30 minutes then at room temperature for 5 hours. The obtained solution was quenched by introducing it in H2O/NaHCO3/ Ice at 0 °C. After that the crude was extracted 5 times with CH2Cl2 and then dried (note a lot of precipitate are obtained) over Na2SO4. The solvent was evaporated under reduced pressure and the crude was subjected to column chromatography using SiO2 (CHCl3/Acetone: 70/30) to give 53 as the white product with (0.01 g, 22%).

1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 9.30 (s, 1H), 7.52 (d, 1H), 6.93 (m, 2H), 6.78-6.56 (12s, 12H), 4.7-4.56 (m, 8H), 4.97-3.97 (m, 16 H), 3.84-3.69 (9s, 27 H), 3.46-3.37 (m, 6 H), 3.25 (m, 1H).

13 C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): δ (ppm) 171. 6 (2C), 169.2, 168.8, 150.0, 149.6, 149.5 (2C), 149.3, 147.9, 147.8, 146.88, 146.8, 146.7 (2C), 146.6, 145.5, 135.7, 134.0, 133.9 (2C), 133.8, 133.7, 133.1, 132.28, 132.24, 132.1, 131.6, 131.5, 131.0, 130.9, 120.6 (3C), 120.6, 120.2, 114.21 (2C), 113.9, 113.8, 113.7, 113.6, 113.5, 112.8, 112.5, 107.6, 69.7, 69.4, 68.9, 68.8, 66.0, 65.9, 65.8, 64.5, 56.3, 56.2, 52.8, 55.7, 55.6, 55.5, 53.6, 52.1, 51.7 (2C), 48.4, 36.4, 36.3, 36.1 (4C).

HRMS (ESI):

m/z [M+H + ] + calcd. for C71H74N2O20 1274.4834 found 1275.4908 Compound 54: Compound 44 (0.13 g, 0.09 mmol, 1 equiv.) was introduced and then dissolved in freshly distilled pyridine (1 mL). Then compound 52 was added (0.033 g, 0.09 mmol, 1equiv.). The mixture was stirred at -15 °C, then POCl3 (0.010 mL, 0.1 mmol, 1.2 equiv.) was added drop wisely and the mixture turned red upon addition. The solution was stirred for 30 minutes at -15 °C then stirred for 5 hours at room temperature where it turned orange. The solution was then poured carefully on a mixture of ice/water/NaHCO3. The obtained solution was extracted 5 times with CH2Cl2, then dried over Na2SO4, filtered and the solvent was evaporated under reduced pressure. The brownish crude was subjected to column chromatography using silica gel (CHCl3/ Acetone: 65:35) the desired product as white powder (0.05 g, 30%).

1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 9.07 (s, 1H), 7.44 (m, 1H), 7.04 (m, 1H), 6.84 (m, 1H), 6.82-6.52 (12s, 12H), 4.69-4.45 (m, 20 H), 4.26-4.15 (m, 18 H), 3.84-3.64 (9s, 27H), 3.46-3.34 (m, 6H), 3.12 (m, 1H).

13 C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): δ (ppm) 171. 6 (2C), 170, 169.9, 169.89, 169.8 (2C), 169.5, 169.3, 169.2, 149.8, 147.9, 147.8, 147.7 (3C), 147.5, 147.44, 147.41, 147.2, 147.0, 146.6 (2C), 135.6, 134.9, 134.4, 133.9, 133.6, 133.57, 133.5 (2C), 133.4 (3C), 133.1 (2C), 132.3, 132.2, 121.1, 120.6, 120.4, 120.2, 119.7, 119.4, 118.9, 118.5, 118.0, 116.3, 115.7, 115.4, 1114.0, 107.6, 69.9,69.21, 68.7, 68.5, 67.9, 67.6, 67.5, 67.4, 67.2, 67.8, 66.0 (2C), 53.6 (2C), 52.1, 52.09, 52.05 (2C), 52.026 (2C), 51.9, 51.7 (2C), 49.1, 36.2 (3C), 35.9 (3C). was dissolved in THF (1.6 ml) and then (0.3M) KOH solution (1.6 ml, 18 equiv.) was added drop wisely at 0 °C. The reaction was stirred for 30 minutes at room temperature. The solvent was evaporated under reduced pressure and then the crude was dissolved in a minimum amount of water. The obtained aqueous layer was washed twice with CH2Cl2. The resulting aqueous layer was acidified at 0 °C with diluted HCl solution. The obtained precipitate was collected on a fritted glass and then washed with ether to give compound 57 as a white compound. Optimization of purification is necessary to be done. The formation of 57 is confirmed by mass spectrometry and the mass found for this derivative is 1572.4942. In the introductory chapter of the first part, we presented the main features of cryptophane derivatives. Among these features, we are particularly interested in investigating the symmetry and the inherent chirality of cryptophanes. Cryptophanes symmetry has an obvious impact on their complexation properties. Depending on the adopted configuration, i.e. syn or anti, cryptophanes can be chiral or achiral (see chapter I). For example, the syn derivative is achiral provided that the two CTB units and the three linkers are identical. Otherwise these syncryptophanes exhibit an inherently chiral structure. On the other hand, the anti-derivatives are always chiral regardless the nature of substituents and the linkers. Thus, many different chiral cryptophane derivatives can be prepared and each derivative is expected to exhibit characteristic chiroptical properties.

HRMS (ESI

The chiroptical properties of enantiopure cryptophanes can be exploited to study the cryptophane itself (determination of the absolute configuration, molecular conformation in solution or in the solid state). More importantly, these chiroptical properties can be also used to study their interactions with chiral or achiral guests. Nowadays, chemists possess in hands a lot of chiroptical techniques to investigate these molecules and their complexes. For instance, the determination of the specific optical rotation can be achieved via optical rotation dispersion (ORD) or polarimetric measurement. Electronic and vibrational circular dichroism spectroscopies (ECD, VCD) are additional tools to characterize these molecules. These two techniques are complementary and they possess their own advantages and disadvantages. Recently, Raman Optical Activity (ROA) has emerged as another useful technique to gain information about the absolute configuration of organic molecules such as cryptophanes and their complexes. X-ray crystallography is another tool that can be used to characterize compounds in the solid state providing information about the absolute configuration of these molecules.

In the following sections, we will provide a brief overview describing different examples of some reported chiral cryptophanes. We will mainly discuss the different approaches used to synthesize enantiopure cryptophane derivatives. Finally, we will describe the chiroptical properties of these cages and their complexes, which have been investigated by the different spectroscopic techniques mentioned above.

Examples on Chiral Cryptophanes

To start with the chiral cryptophanes, it is worth first to discuss the anti-cryptophane derivatives, which are numerous, compared to the chiral syn-cryptophanes. The first reported cryptophane cage is the anti-cryptophane-A 58, [START_REF] Gabard | Synthesis of a (D3)-Bis(cyclotriveratrylenyl) Macrocage by Stereospecific Replication of a (C3)-Subunit[END_REF] which possesses a D3-symmetry. This particular arrangement results from a stereospecific replication of the parental CTB unit symmetry. With this example, a clear illustration of the inherent chirality was delivered. Apart from this example, other D3-cryptophane derivatives were also described such as anti-cryptophane-E, which possesses three propylenedioxy linkers and six methoxy groups. So generally, anti-cryptophane derivatives bearing the same CTB substituents and linkers possess a D3-symmetry.

A decrease of the molecular symmetry from D3 to C3 is obtained when one of the two CTB units carry three different substituents. For example, substituting the three methoxy groups of cryptophane-A by three hydrogen atoms on one CTB unit results the anti-cryptophane-C, 59. [START_REF] Canceill | Analytical Optical Resolution of Bromochlorofluoromethane by Enantioselective Inclusion into a Tailor-Made "Cryptophane" and Determination of Its Maximum Rotation[END_REF] Other C3-symmetrical cages were also recently reported based on the cryptophane-A skeleton, In the first chapter of this manuscript, we also reported a chiral C2-cage with a [223] skeleton bearing a central alkenyl moiety on the propylenedioxy arm, compound 26. [START_REF] Huber | Unsaturated Cryptophanes: Toward Dual PHIP/Hyperpolarised Xenon Sensors[END_REF] C1-symmetrical cryptophanes are also numerous and they are obtained if cryptophanes possess different linkers or substituents. Cryptophane-[222] monoacid 63, which is widely used for xenon-based biosensing applications has a C1-symmtery is one example, Figure 89. [START_REF] Darzac | Synthesis and Application of Cryptophanol Hosts: 129 Xe NMR Spectroscopy of a Deuterium-Labeled (Xe)2@bis-Cryptophane Complex[END_REF] It is noteworthy that all the cryptophanes described in the second chapter of the first part possess C1 symmetry except compound 26.

Synthesis of Enantiopure Cryptophane Derivatives

Chiral cryptophane derivatives are usually obtained as a racemic mixture. Therefore, the study of their chiral properties requires the separation of the two enantiomers. Several approaches can be tested to separate these enantiomers. For instance, asymmetric synthesis of these derivatives starting from enantiopure starting materials is one of the readily used approaches. The separation of the two enantiomers via the formation of a diastereomeric mixture can also be employed. Then, the diastereomers can be separated by different techniques such as column chromatography or crystallization. Finally, HPLC using a chiral stationary phase can be used to obtain these enantiopure molecules. All these techniques possess their own advantages and drawbacks as will be discussed in the following sections.

Enantiomerically Enriched Cryptophanes by Asymmetric Synthesis:

Enantiopure CTB units have been prepared to synthesize enantiomerically enriched cryptophanes with high ee. Collet and co-workers have used extensively this approach to prepare enantiopure cryptophane derivatives with D3 or C3-symmetry. This approach appears as an easy way to access these enantiopure molecules. Unfortunately, great care in the choice of the experimental conditions has to be considered since the CTB unit tends to easily racemize above room temperature. Thus, under the experimental conditions required to perform the second ring closing reaction, a partial racemization is inevitable. Consequently, this approach only allows getting enantiomerically enriched cryptophane derivatives with a moderate to high ee (65 -95%).

Enantiopure CTB units, themselves can be synthesized from the trimerization of the properly functionalized enantiopure starting materials bearing a chiral auxiliary. This trimerization step results in the formation of two diastereomeric CTB units, which can be readily separated on silica gel. Recovery of the desired enantiopure CTB is then achieved by applying multistep reactions.

For example, Collet and co-workers reported the synthesis of the two enantiopure cyclotriguaïcylene (CTG) (M and P), 72 units using α-bromo-propanoic acid as the chiral auxiliary. 177 The first step involved the cyclization of the optically pure vanillic alcohol derivative 67, in HClO4 (65%). This step afforded a diastereomeric mixture of the two CTB units, compounds 68. An additional esterification step using diazomethane allowed the access of the two separated diastereomers, 69. These two diastereomers were subjected to subsequent reactions to give the desired enantiopure CTG, 72, Figure 91. The second strategy overlies on reacting a readily trimerized CTB with a chiral auxiliary to form two diastereomers, which are separated by chromatography. For example, chiral auxiliaries like Ѡ-camphanic acid or R-(+)-2-phenoxypropanoic acids were employed and they were mildly removed after the separation. 178,154 Exploiting the resolved CTB units, enantiomerically enriched cryptophanes were synthesized, like the cryptophane-A, 58. The synthesis of PP-58 starts with the preparation of the M-CTG, 72

followed by a substitution reaction to give the P-CTB template. Unlike the parental CTG units, cryptophanes are resistant to racemization at high temperature. Other enantiomerically enriched cryptophane derivatives with D3-symmetry were also reported, like cryptophane-E or cryptophane derivatives bearing unsaturated linkers like compounds 73, 74 and 75 shown in Figure 92. [START_REF] Gabard | Synthesis of a (D3)-Bis(cyclotriveratrylenyl) Macrocage by Stereospecific Replication of a (C3)-Subunit[END_REF][START_REF] Gabard | New D3 and C3h Cryptophanes with Ethelynic Bridges Structural Assignments, Absolute Confugrations and Circular Dichriosm of D3 Isomers[END_REF]34 Recently, Crassous and co-workers have used this strategy to synthesize enantiomerically enriched thio-substituted cryptophanes 79, Figure 94. These compounds have been obtained from the corresponding functionalized enantiopure CTB units. It is noteworthy that in this case, the second ring closing reaction gives rise to a significant racemization since the two MM and PP cryptophane enantiomers were obtained in an ee 81.6 and 65.6 %, respectively. This cryptophane was later used to determine the ee of enantioenriched chlorofluoroiodomethane derivative. The recognition process was studied by [START_REF] Sherman | Syntheses and Properties of Soluble Carceplexes[END_REF] F and 1 H NMR spectroscopy. 179,180 Figure 94: structure of the enantioenriched compound 79. It is worth to mention that the resolved cryptophanes obtained by enantioenriched CTB units have an ee between 65 and 99%. It is important to be aware for the unavoidable racemization of the enantiopure CTB units, which constitutes the main disadvantage of this method.

Unlike the CTB units, tribenzotriquinacenes or the so-called TBTQ possess a more rigid skeleton.

Although structurally similar, TBTQ are resistant to temperature-induced racemization practiced by the CTB units. This difference is due to the additional methyl bridge fusing the three methylene bridges together. Resolving the two enantiomers of TBTQ derivatives can be achieved by several strategies. One strategy reported is based on breaking their C3v-symmetry by monofunctionalizing one or the three arene rings leading to either a C1 or C3 symmetrical molecule, respectively. These inherently chiral molecules can eventually be used to build chiral selfassemblies. For example, the TBTQ derivative 80 bearing one functionalized aromatic ring was optically resolved using (R)-BINOL. The two obtained diastereomers (81 is one of them) were separated by column chromatography in 34% and 33% yields. The saponification of these esters allows the recovery of the enantiopure TBTQ, 80 with 97% and 95%, Figure 95. The chiroptical properties of these two derivatives were studied by polarimetry and ECD spectroscopy. [START_REF] Niu | Synthesis and Optical Resolution of Inherently Chiral Difunctionalized Tribenzotriquinacenes[END_REF] Figure 95: diastereoselective optical resolution of TBTQ derivative using R-BINOL.

Stereoselective and enantioselective synthesis of TBTQ was also achieved by employing biocatalysis. Starting from four isomers of alcohol functionalized TBTQ 82, one pair of diastereomers (M,R) and (P,R) was selectively acylated using commercially available Candida Antarctica lipase to give two diastereomeric ester derivatives 83. These compounds were then easily separated by column chromatography with a total yield of 48%. Enantioselective hydrolysis of the esters using lipase PS afforded the P-TBTQ derivative whereas using Candida Antarctica lipase the M-derivative was selectively hydrolyzed, Figure 96. 181 In the first chapter, we have also described another method to obtain these compounds in their enantiopure form. Based on a previous work reported by Warmuth and co-workers, Kuck and coworkers synthesized a TBTQ based cryptophane by thermodynamic dynamic resolution approach.

Thus, reacting racemic TBTQ trialdehydes with (S,S)-diaminocyclohexane offered three couples of cryptophane diastereomers (P,P,S,S,S) (M,M,S,S,S) (P,M,S,S,S) in 29%, 40% and 25% yields, respectively. These diastereomers were separated by chromatography and then hydrolyzed to give enantiopure TBTQ building blocks. Some derivatives of these molecules were used to build enantiomerically self-sorted nanotubes indicating their versatility to construct highly organized systems. [START_REF] Wang | C3-Symmetrical Tribenzotriquinacene Derivatives: Optical Resolution through Cryptophane Synthesis and Supramolecular Self-Assembly into Nanotubes[END_REF] Therefore, these compounds offer many interesting possibilities to design enantiopure cages. However, their synthesis and functionalization are tedious; hence these compounds cannot be easily used in their enantiopure form to design MRI tracers for 129 Xe NMR applications.

Synthesis of Cryptophanes by Dynamic Resolution:

Using a thermodynamic dynamic resolution approach, Warmuth and co-workers have reported the resolution of CTB derivative 84 with high ee. The imination of a racemic mixture of CTB derivative functionalized by three aldehydes with two equivalents of (R,R)diaminocyclohexane, 85 offered a unique (P,P,R,R,R)-cryptophane enantiomer 86 in an excellent 92% yield. In fact, the high reaction temperature and thermodynamic sink of the obtained cryptophane enantiomer were the main reasons behind this complete inversion. The hydrolysis of compound 86 in a mixture of trifluoroactetic acid and water gave the enantiopure (P)-84 in 92%

with a high ee (99%), Figure 97. 182

Figure 97: synthesis of (P,P,R,R,R)-cryptophane.

Enantiopure Cryptophanes by Separation of Diastereomers:

Synthesis of enantiopure cryptophane derivatives can also be achieved by reacting a cryptophane in its racemic form with a chiral auxiliary. This in turn leads to the formation of a diastereomeric mixture that can be separated by different techniques. Brotin and co-workers reported this approach in 2003, where they successfully obtained the two enantiomers of cryptophanol-A, 87. In this synthesis, the only available hydroxyl group on the cryptophane-

[222] skeleton was allowed to react with (1S)-(-)-camphanic acid chloride 88 leading to two 84 85 diastereomeric compound 89. Thanks to the large difference in solubility between these two compounds, the separation of these diastereomers was made possible by crystallization from hot toluene. This allowed to collect the first diastereomer in approximatively 15% yield (ee=98-100%). The second enantiomerically enriched diastereomer was collected and then hydrolyzed under basic conditions. Reacting this compound with (1R)-camphanic acid chloride and applying the same procedure allowed to get the second diastereomer in 10% yield and ee = 98%-100%.

Cleavage of the chiral auxiliary offered enantiopure cryptophanol-A, 87, Figure 98. 183,[START_REF] Cavagnat | Synthesis and Chiroptical Properties of Cryptophanes Having C1-Symmetry[END_REF] Figure 98: resolving cryptophanol-A by forming diastereomers with camphanic acyl chloride.

The enantiopure compound 87 was subjected to various chemical transformations to give new enantiopure cryptophanes. The interest of this approach lies on the fact that no racemization takes place compared to the CTB based method. Thus, new enantiopure cryptophanes with different symmetry can be easily obtained by this way, whatever the experimental conditions (high temperature, solvents…). As an example, we can report the synthesis of some other enantiopure cages starting from PP and MM-cryptophanol-A. For example, enantiopure cryptophane-A was prepared from enantiopure cryptophanol-A. The latter was then demethylated with excess of LiPPh2 to give the enantiopure hexa-hydroxyl cryptophane, 90, with an excellent yield.

89

Subsequent alkylation and hydrolysis allowed the formation of an enantiopure cryptophane with six carboxylic acid functions. 184,185 Following the same approach, Dmochowski and co-workers synthesized enantiopure trisubstituted cryptophane-A derivatives. Racemic mixture of tri-hydroxyl cryptophane-A, 91 was resolved by esterification with (S)-Mosher's acid, 92 to give two diastereomeric compounds 93, which were separated by column chromatography. The overall yield was 70% with 35% yield per each compound, Figure 99. The enantiomeric purity w as assessed by ECD. The chiral auxiliary was then removed by hydrolysis under basic conditions without any loss of optical activity. Further functionalization of the two isomers resulted new cryptophanes of various applications. 186 Although this strategy offers the cryptophane derivatives in excellent enantiomeric excess compared to the CTB-based method, this approach requires more synthetic steps and optimization of the separation step for each diastereomer.

Resolving Cryptophanes by HPLC:

Resolving cryptophanes by HPLC has gained a great attention in the last few years since this approach does not necessarily require chemical transformations. The method solely lies on using a suitable chiral stationary phase to separate the two enantiomers. The main inconvenience of this technique is its limitation to small-scale separation due to the high cost of semi-preparative columns, sensitivity and large volumes of mobile phase. This method also requires optimization of the separation conditions that can be difficult in some cases. Solubility issues of the racemic material have also to be taken into consideration.

Collet and co-workers in 1989 reported the first use of HPLC on chiral stationary phase to separate various CTB and cryptophane derivatives. 187 At that time, the increased applications of cryptophanes as chiral hosts rendered it important to develop a new analytical tool for their optical resolution. Chiralpak OT-(+) was used as the stationary phase to separate these compounds but this stationary phase suffers from low stability over time. Nevertheless, several CTB and cryptophanes were efficiently resolved as pointed previously. Table 4 summarizes the results obtained for few cryptophane derivatives. For instance, cryptophane-A, 58 was efficiently resolved into its enantiomers M-(+) and P-(-) using methanol as an eluent. The two enantiomers of cryptophane-A, PP-cryptophane-A and MM-cryptophane-A appear at 22.1 and 30.0 min, respectively. Chiralpak OT-(+) was also employed to separate enantiopure cryptophane bearing aromatic bridged linkers. Thus, anti-cryptophane 94 bearing three ortho-substituted aromatic linkers was resolved using the latter chiral column. The two enantiomers appeared at 23.7 and 39.4 minutes, respectively. Same conditions were used to get cryptophane 95 with retention times 36.5 and 73.0 minutes, Figure 100. These molecules were used to encapsulate various ammonium salts. [START_REF] Akabori | The Complexing Abilities of Diethyleneoxy-and Xylene-Bridged Cryptophanes with Alkanes[END_REF]188 Figure 100: optically resolved cryptophane bearing benzyl substituted linkers.

Symmetry

More recenly, enantiopure nona, 66 and dodecamethoxy 96 cryptophanes were resolved using new chiral HPLC columns. The anti and syn-isomers of cryptophane 66 show an inherently chiral structure (C3-symmetry). For anti-66-cryptophane, an efficient separation was obtained using

Chiralpak IB with ee ≥ 99.5% for both enantiomers. On the other hand, the two enantiomers of the the syn-diastereomer have been separated by using a Chiralpak IA column chromatography (ee ≥ 98.5%). In the case of cryptophane 96, only the anti-diastereomer is chiral and has a D3

symmetry. The two enantiomers of this derivative were separated by using Chiralpak ID column chromatography. Typically, a separation performed on a semi-preparative column allows to isolate few hundreds of mg of enantiopure compounds, which are sufficient to achieve a thourough study of chiroptical properties. 176 Cryptophane with C2-symmetry have also been separated by chiral column chromatography. For instance, compounds 60 and 61 bearing one and two propylenedioxy linkers have been separated

94 95 with chiralpak IA column. A detailed study of chiroptical properties has been reported by polarimetry, ECD, VCD and ROA and it will be discussed later. 189 Interestingly, using a Chiralpak ID column, cryptophane derivatives with no substituents have been successfully separated. Thus, several hundreds of mg of the two enantiomers of Optical resolution of dually functionalized cryptophane 12 bearing a unique secondary alcohol on its propylenedioxy linker was also reported using chiral HPLC with high ee ≥ 99.5%. This cage showed a high affinity towards Cs + and Tl + cations, Figure 41. [START_REF] Chapellet | Experimental and Theoretical Study of the Complexation of Cesium and Thallium Cations by a Water-Soluble Cryptophane[END_REF] Cryptophane-[222] (OH)5, 99 is an interesting molecular host capable of binding a large range of guest molecules. For this purpose, the two enantiomers of this molecule were separated in order to investigate how the presence of a guest molecule affects its chiroptical properties. Two different approaches have been used to get the two enantiomers of compound 99. The first approach lies on the separation of two diastereomeric compounds as described previously in the above section. This method is time consuming but it allows us to obtain these compounds with high ee and in fair quantities to perform the chiroptical studies. More recently, a simplified time saving approach was used instead, based on chiral HPLC separation of the penta-acetate derivative 100. This derivative, which is the protected version of compound 99, was chosen due to its enhanced solubility. Thus, a Whelk-O1 column was used as the stationary chiral phase to offer the two enantiomers of the compound 100 with excellent ee ≥ 99%. The latter was then subjected to an alkaline hydrolysis to give the targeted enantiopure cryptophane derivative. [START_REF] De Rycke | Enantioselective Complexation of Chiral Oxirane Derivatives by an Enantiopure Cryptophane in Water[END_REF][START_REF] Bouchet | Enantioselective Complexation of Chiral Propylene Oxide by an Enantiopure Water-Soluble Cryptophane[END_REF] It is noteworthy that in this example the descriptors of compound 99 and 100 are inverted. Thus PP-100 gives rise to the MM-99, Figure 102.

Synthesis of Chiral Metallo-Cryptophanes:

The synthesis of multi-dimensional supramolecular structures can be achieved by different methods and coordination driven self-assembly is one of them. A particular interest is given to metallo-cages, which are made up of multi-dentate ligands and metal cations due to their potential applications in catalysis, separation, molecular recognition, and nanoscale vessels. 192,193 The first described self-assembled coordination driven cryptophane cage was reported in 2001 by

Zhong and co-workers. In this paper, racemic pyridyl CTB derivative, 101 was self-assembled into [Pd31012] 6+ as the meso-syn cryptophane, 103 and chiral anti-cryptophanes 104 by the coordination with cis-protected palladium salts, 102. Inter conversion between these two syn and

Chiroptical Properties of Cryptophane Derivatives and Their

Complexes

The access of enantiomerically pure cryptophanes allows us to investigate their chiroptical properties. These properties have been heavily exploited to assign the absolute configuration of cryptophanes and to gain insights about the conformation of these molecules in solution. Finally, the presence of a guest inside the cavity of the host might also modify the overall chiroptical properties of the host molecule. Interestingly, a large range of techniques such as ECD, VCD, ROA and ORD and X-ray crystallography can be used to study the chiroptical properties of these molecules. We will describe in the appendix section briefly the principle of these spectroscopic techniques. Through selected examples, which have been reported in the literature, we would like to show how these chiroptical techniques serve to characterize cryptophanes and their complexes.

ECD spectroscopy is one of the earliest chiroptical methods used to study enantiopure cryptophane derivatives. Collet, Gotarelli and co-workers pioneered this work by studying enantiomerically enriched cryptophanes with D3 and C3-symmetry. Based on the Kuhn-Kirkwood-coupled oscillator model, these authors were able to predict the shape of the ECD spectra of cryptophane-A and its congeners. For instance, in the case of the cryptophane-A derivative with D3-symmetry, this model predicts that each transitions 1 Lb, 1 La and 1 Bb (Platt's notation) visible on the UV-visible spectrum (210-310 nm) can be decomposed into three excited states due to the resulting excitonic coupling. Each of these excited states possesses its own rotational strength that determines the intensity and the sign of the ECD bands. The obtained ECD spectrum is the result of the contribution of all these excited states. The ECD spectrum of Since the work of Collet and co-workers, numerous enantiopure cryptophane derivatives have been studied by ECD spectroscopy both in organic solvents and in aqueous solutions. Thus, numerous cryptophane derivatives having different symmetry have been investigated and in some (+)-Cryptophane-A cases their ECD spectra have been predicted thanks to the use of TD-DFT calculations. [START_REF] Brotin | Chiroptical Properties of Nona-and Dodecamethoxy Cryptophanes[END_REF] From a general point of view, it has been observed that ECD spectra recorded in organic solvents are not very sensitive to a change of the nature of the solvent. 183 Spectral ECD modifications take place mainly in the 1 Lb and 1 Bb regions of the ECD spectrum and a change in the nature of the organic solvent slightly affects the ECD spectra. Moreover, more pronounced changes are sometimes observed with the SOR values in terms of sign and magnitude. At first sight, this may appear surprising since the ECD and SOR values are strongly related. An interesting example is observed with cryptophane-[222], 98 devoid of any substituents whose ECD spectra recorded in several organic solvents are almost identical in shape and intensities. In contrast, the SOR values recorded in the same solvents show large variation in intensities. In addition, the sign of these SOR values can be different depending on the solvent and wavelength. 191 This behavior has been attributed to tiny conformational changes that the molecule can adopt in solution. Interestingly, this unusual example reveals that in the case of cryptophane derivatives, polarimetry or ORD spectroscopy is much more sensitive to reveal small conformational changes in organic solvent.

PP-cryptophane-

Another interesting example will be discussed in the next chapter that describes the unusual behavior of cryptophane derivatives subjected to self-encapsulation.

It is noteworthy that this situation can be different in aqueous solution. For instance, it has been observed that the ECD spectra of enantiopure cryptophanes 90 bearing six phenol groups can be extremely sensitive to the nature of guest molecule and the nature of the aqueous solution. An example is reported in Figure 107 that shows ECD modifications upon complexation in LiOH solution. In Figure 107, it can be noticed that large spectral modifications occur in 1 Lb and the 1 Bb regions of the spectrum depending on the nature of the guest. For instance, in presence of CHCl3, the ECD spectrum shows between 270 and 330 nm a broad negative ECD band whereas several positive and negative Cotton bands are observed in absence of a solvent molecule or in presence of CH2Cl2. Large spectroscopic changes are also observed in the 1 Bb region of the spectrum.

These spectral changes are the consequences of conformational changes induced by the nature of the encapsulated guest. Indeed, the nature of the guest and in particular its molecular volume imposes the host molecule to change its molecular conformation to maximize interactions.

Additional hydrophobic effect may contribute to these changes. 184 It has also been observed that a change of the nature of the solvent had a tremendous effect on the shape of these ECD spectra. Replacing, LiOH by CsOH solution resulted in important spectral changes. For instance, we observed a slight bathochromic shift of the whole ECD spectrum but we also noticed that this ECD spectrum remains unchanged upon addition of CH2Cl2 or CHCl3.

Subsequent investigations have revealed that cationic species such as rubidium, cesium and even thallium are strongly encapsulated. For instance, ITC experiments allow us to measure very high binding constants K = 3.80 × 10 8 M -1 and K = 2.39 × 10 9 M -1 in LiOH (0.1 M) for cesium and thallium cations, respectively.

The strong affinity of host 99 bearing five hydroxyl groups for cesium and thallium cations can be assessed by ECD spectroscopy. Indeed, upon addition of these cationic species a strong change of the whole spectrum takes place. These spectral changes are strongly dependent on the experimental conditions. Figure 108 Besides these studies involving cationic species, water-soluble enantiopure cryptophanes have been used to study the encapsulation of small chiral molecules. Due to their small molecular volume, small oxirane derivatives are well adapted to perform these studies. For instance, it has been shown that methyl oxirane and its congeners fit well the cavity of the penta-hydroxy derivative 99. Interestingly, it has been demonstrated that this cryptophane was able to achieve in basic aqueous solution enantioselective encapsulation. This enantioselective complexation can be easily detected by 1 H NMR spectroscopy thanks to the high-shielding effect induced by the six aromatic rings surrounding the guest molecule. In addition, it has been observed that the different diastereomeric complexes give rise to different ECD spectra. Even though these differences are small they cannot be the consequence of artifact, Figure 109. Another example has been reported with cryptophane 99 and bioxirane. Bioxirane is an interesting example since this compound possesses two stereogenic centers; hence there exist three isomers, the RR, SS and meso-bioxirane. Thus, in presence of enantiopure compound 99, three different diastereomeric complexes can be formed in solution. As for the methyl oxirane complexes, 1 H NMR and ECD spectroscopies allows us to distinguish the three diastreomers formed in solution, Figure 110. [START_REF] De Rycke | Enantioselective Complexation of Chiral Oxirane Derivatives by an Enantiopure Cryptophane in Water[END_REF][START_REF] Bouchet | Enantioselective Complexation of Chiral Propylene Oxide by an Enantiopure Water-Soluble Cryptophane[END_REF] Figure 110: structure of (S,S)@MM-99 complex, ECD spectra of (R,R)-bioxirane@PP-99 (red), (S,S)bioxirane@PP-99 (blue), meso-bioxirane@PP-99 (green), and guest-free PP-99 (black spectrum). ECD spectra of (S,S)-bioxirane@MM-99 (red dashes), (R, R)-bioxirane@MM-99 (blue dashes), meso-bioxirane@MM-99 (green dashes), and guest-free MM-99 (black dashes). The spectra were recorded at 278 K in NaOH/H2O (0.08 M) solution.

[Host] = ( 6 . 199 It is noteworthy that the absolute configuration of this derivative was previously determined indirectly from the knowledge of the absolute configuration of the corresponding CTB unit. 34 Besides, this study allowed us to gain information about the conformation of the linkers in solution. This study was somehow complicated and time demanding considering all the possible conformations that the three linkers can adopt in solution. In this example, our results reveal that the linkers of the CHCl3@cryptophane-A complex adopt mainly an all trans conformation. This conclusion is reasonable considering the large molecular volume of the guest (Vvdw = 72 Å 3 ). Cryptophane- Raman Optical Activity (ROA) is another technique, which has been used extensively to characterize cryptophane derivatives. Readers interested in this chiroptical technique can refer to some selected references. 201,202 ROA spectroscopy allows us to investigate the near infra-red region and typically ROA spectra can be recorded up to 100 cm -1 . This represents an undeniable advantage compared to VCD spectroscopy. In the case of cryptophane derivative, ROA spectroscopy has been used as a complementary technique to determine the absolute configuration of cryptophane derivatives even though DFT calculations are more complicated to perform.

An interesting example has recently been reported with cryptophane-[111], 97. In this example, Buffeteau and co-workers showed that ROA spectroscopy could be used to characterize the xenon@97 complex. Figure 113

Objectives

Cryptophanes chiroptical properties have enormously served to determine their absolute configuration, and study their different conformations. In particular, a better vision of cryptophanes conformations facilitates the understanding of their complexation properties. For example depending on the guests' size and shape cryptophanes' arms can adopt different conformations to maximize host-guest interactions.

During our journey in the development of new cryptophane- [223] type derivatives bearing a functionalized propylenedioxy linker, we have evidenced a new conformation adopted by these cryptophanes. This phenomenon is referred to as "self-encapsulation", in which the molecule tends to fill its cavity by its own functionalized propylenedioxy arm in the absence of a potential guest. This phenomenon is solvent dependent and takes place at room temperature, Figure 114.

An in-depth investigation of the effect of this phenomenon on the cryptophanes chiroptical properties will be delivered using different chiroptical techniques. This will allow evaluating the robustness of these chiroptical techniques in response to this particular conformational change. we have presented the several conformations that cryptophanes' CTB units can take in solution or in the solid state. Most of these conformations form when the molecule is stored for a long time in the solid state or when heated at elevated temperature. These conformations are also easily obtained if the cage has sufficiently long linkers, which imposes higher flexibility. Surprisingly, we have also observed that the presence of bulky substituents (benzyl esters for instance) on the CTB units facilitates the formation of the imploded form known as crown-saddle. Most of cryptophanes conformations are heavily described by means of 1 H NMR and X-ray crystallography, where a clear understanding of this arrangement is delivered, however, none of these studies investigated the impact of these conformational changes on their chiroptical properties. [START_REF] Mough | Isolation and Structure of an "Imploded[END_REF][START_REF] Thorp-Greenwood | Fully Collapsed Imploded Cryptophanes in Solution and in the Solid State[END_REF] Recently, our group reported the synthesis of a cryptophane- [223] derivatives with functionalized propylenedioxy arm for xenon biosensing applications. [START_REF] Chapellet | Synthesis of Cryptophanes with Two Different Reaction Sites: Chemical Platforms for Xenon Biosensing[END_REF] Particularly, a cryptophane- [223] platform bearing seven acetate groups, compound 109, showed an unusual behavior upon xenon complexation. Compared to its structural congener 13, compound 109 showed a broadened 129 Xe NMR signal in 1,1,2,2-tetrachloroethane-d2. This strange behavior is characteristic of a fast in-out exchange kinetics of xenon and can be rationalized by the fact that cryptophane's cavity is less accessible to xenon. This result seems to indicate that the cage is adopting a conformation that makes its cavity less accessible to xenon, Figure 115. Particularly, compound 109 is vulnerable to undergo a self-encapsulation phenomenon, in which its central acetate group enters the cavity in the absence of a potential guest. This selfencapsulation behavior have been previously described for other molecular hosts like cyclodextrins and pillar [5]arene, however it is the first time, we observe this phenomenon in cryptophane derivatives. 203,204,205

Synthesis and Separation of Two Enantiomers of Cryptophanes 109, 110, 111 and 112

The synthesis of cryptophane 109 has been previously reported in literature, [START_REF] Chapellet | Synthesis of Cryptophanes with Two Different Reaction Sites: Chemical Platforms for Xenon Biosensing[END_REF] Cryptophane 110 has been obtained by a single acylation reaction of the central hydroxyl group of cryptophane 8 where it was obtained in a good yield (71%). Similar to compound 109, the two enantiomers of cryptophane 110 were also separated using semi-preparative chiral HPLC but this time using (S,S)-Whelk-O1 column as a stationary phase. This provided the [CD(-)254]-110 and [CD(+)254]-110.

Similarly, cryptophane 111 was obtained by an acylation step of compound 27 and it was obtained in a good yield (70%). The two enantiomers of compound 111 were separated (S,S)-Whelk-O1.

Cryptophane 112 which has no central function on its propylenedioxy arm has been prepared using a known procedure and it was obtained in a good yield (71%). The two enantiomers were separated using a (S,S)-Whelk-O1 column. 111 It is worth mentioning that compound 112 tends to partially implode upon separation.

An additional purification step on silica gel was performed to ensure the purity of these compounds.

1 H NMR Analysis of Cryptophanes

The 1 H NMR spectra of cryptophanes 109, 110, 111, 29 and 112 were recorded in different solvents that can either enter their cavities like CD2Cl2 and CDCl3 or solvents that have low affinity to cryptophanes like DMSO-d6, benzene-d6 and C2D2Cl4,. In cryptophane 109 for example, the comparison of these spectra revealed large differences specifically in the signal corresponding to the methyl group of the central acetate moiety. For instance, the 1 H NMR spectrum in CD2Cl2 clearly reveals a sharp signal at 2.08 ppm corresponding to the central acetate group grafted on the propylenedioxy arm. In CDCl3, however, this signal is replaced by another broadened signal, which can be barely seen.

Interestingly in solvents that do not fit the cavity, like DMSO-d6, C2D2Cl4 the signal appearing at 2.08 ppm is replaced by another shielded signal, which appears at 0.18 and -0.15 ppm, respectively with a chemical shift difference of about 2.59 ppm.

Compounds 110, 111 and 29 showed a similar behavior, but interestingly, the signal appearing at almost 2 ppm is now more shielded (~3.9 ppm difference in CD2Cl2 and C2D2Cl4) and it appears at -1.74, -1.35 and -1.1 ppm, respectively for each compound. In Figure 117, we have specifically shown the 1 H spectra of compound 110 in different solvents to clearly illustrate the chemical shift differences as a function of solvents.

Figure 117: 1 H NMR spectra of compound 110 in different organic solvents.

The 1 H NMR spectra of compound 112 do not show any modification upon varying the solvents, this is because the molecule does not carry a central acetate moiety and thus does not seems to undergo a self-encapsulation phenomenon.

Polarimetry and ECD spectroscopy

The specific optical rotation (SOR) values of compounds 109, 110, 111 and 112 were recorded by polarimetry in different solvents (CD2Cl2, CDCl3, DMSO-d6, C2D2Cl4) at five different wavelengths (589, 577, 546, 435, and 365 nm). Similarly, the two enantiomers of compound 112 have the same sign of SOR values whatever the nature of the solvent. The magnitude of these values however is comparable to that observed with compound 109, Figure 119. The ECD spectra of the enantiopure compounds 109, 110, 111 and 112 were recorded in different solvents like CH3CN, DMSO, C2H2Cl4, THF, CH2Cl2 and CHCl3. These molecules showed a good solubility in all solvents except CH3CN. Recording ECD spectra in CH3CN and THF allows us to access a large spectral range of the absorption spectrum reaching 210 nm, which includes the 1 Bb, 1 La and 1 Lb transitions. In CH2Cl2 and CHCl3 the cut-off is around 230 nm, which allows the detection of the 1 La and 1 Lb transitions. However, in DMSO and C2H2Cl4 only the 1 Lb transition is observed.

The ECD spectra of [CD(+)254]-109 in CH2Cl2 revealed two negative-positive bisignate signals from low to high wavelength (Figure 120,A). The first bisignate corresponds to the 1 La transition, which lies between 230 and 260 nm and the second lies between 260 and 310 corresponds to the 1 Lb transition. In THF, an ECD band of the 1 Bb transition is observed as a negative Cotton band. Upon varying the nature of the solvent, no spectral modifications were observed and the spectra are nearly superimposable except in the case of C2H2Cl4, where a decrease in the intensity of the ECD component at 290 nm is detected, Figure 120,B. Similarly, no detectable variation was observed in the ECD spectra of compound 112 in different solvents.

IR and VCD spectroscopy

The chiroptical properties of cryptophanes 109, 110 and 112 were also studied by VCD spectroscopy in different solvents, along with the infrared spectroscopy, which offered insight data about the self-encapsulation phenomenon.

The IR spectra recorded in different solvents are relevant to that reported previously with cryptophane-[223] derivatives. 111,[START_REF] Chapellet | Experimental and Theoretical Study of the Complexation of Cesium and Thallium Cations by a Water-Soluble Cryptophane[END_REF] These spectra displayed at 1750 and 1160 cm -1 reflect the νC=O stretching vibration and the ester asymmetric and symmetric νC-O-C stretching vibrations.

MM-110

B A C D
The νC=O stretching vibration is of particular interest because it reflects the environment and the behavior of the carbonyl groups in the molecules in different solvents. For example, the intensity of this signal is dependent on the number of the acetate groups in the molecule. In compounds 109 and 112, for instance, this peak is more intense because they bear seven and six acetate groups, respectively. A closer look at this spectral region informs us a lot about the selfencapsulation phenomenon. For example, the νC=O signal of compound 109 and 112 are equal to 1762 cm -1 in CHCl3 and 1758 cm -1 in C2H2Cl4. Thus, these peaks correspond to the six acetate groups grafted on the CTB units. In compound 109, however an additional shoulder is observed at 1746 cm -1 that might correspond to the central acetate group. In this compound, it is hard to extract data about self-encapsulation due to the contribution of the other six acetate available moieties, Figure 121, A and C.

Interestingly, in compound 110 which bears a unique central acetate group, more information about the self-encapsulation process can be extracted from the νC=O IR band. In CHCl3 and

CH2Cl2 for example, this stretching vibration appears as a single peak around 1740 cm -1 . In C2H2Cl4 where self-encapsulation takes place, a new signal appears at higher energy, 1752 cm -1 in addition to the initial signal appearing at 1738 cm -1 . This new peak can be assigned to the acetate group pointing inside the cavity. It is worth mentioning that the new signal is double in intensity compared to the initially observed signal at 1739 cm -1 . The new observed peak has a higher energy and this indicates that it interacts less with the surrounding environment. To confirm this result, additional experiment was performed in C2H2Cl4 with xenon, which is a competitive guest. Upon xenon addition, the intensity of the two peaks are reversed, in which the peak observed at 1739 cm -1 that corresponds to the acetate group out the cavity, is now predominant. This result can be explained by the fact that once xenon enters the cavity it expels the acetate group out of it and hence clearly illustrates the self-encapsulation phenomenon, Figure 121,B. wavenumbers which is referred to as an excitonic coupling of the acetate groups. Specifically it arises from the coupling pair of oriented C═O dipoles and has been already observed for cryptophane derivative bearing six carboxylic acid groups Figure 122, A andC.

Self-Encapsulation Confirmation by 1 H NMR and IR

Among the different spectroscopic methods used to characterize the self-encapsulation process, 1 H NMR and IR confirmed with confidence this conformation. For instance, 1 H NMR spectra revealed a clear chemical shift change of the central acetate or thio-acetate group in solvents that fill or do not fill the cavity. In large solvents like DMSO and C2H2Cl4, the central acetate group tends to fill cryptophanes cavity leading to high shielding. In the presence of a potential guest like CHCl3 and CH2Cl2, the acetate group will be expelled out and the guest molecule will occupy the cavity.

Compound 109 shows a shielding of about 2.58 ppm in C2H2Cl4, this value suggests that the acetate group is not completely encapsulated. For compounds 110, 111 and 29, the acetate group is shielded by about 4 ppm. This value is comparable to that observed with cryptophane guests and it suggests a better complexation event. 206,[START_REF] Canceill | Structure and Properties of the Cryptophane-E/CHCl3, Complex, a Stable van Der Waals Molecule[END_REF] This can be explained by the fact that the acetate groups bordering cryptophane-109 skeleton are now replaced by less hindered methoxy groups which in turn enhance the flexibility of the cage allowing better conformational rearrangement.

Infrared spectroscopy has also served to confirm this phenomenon, specifically in compound 110 that bears only one acetate group. For example, the peak observed at 1752 cm -1 in C2H2Cl4 can be clearly associated with the acetate group pointing inward the cavity. This stretching vibration appears at higher wavenumber compared to the acetate group pointing outward (1739 cm -1 ) because it interacts less with surrounding solvent environment. DFT calculations have also served to reproduce these IR spectra, in which an increase of the stretching vibration wavenumber by about 13 cm -1 is calculated for acetate pointing inward the cavity. This has been also confirmed by a competition experiment with xenon that expels the acetate group, thus restores the initial signal which appears at 1739 cm -1 and decrease the one at 1752 cm -1 .

It is worth mentioning, that self-encapsulation is a new conformation that is used by cryptophanes to fill their cavities in the absence of a guest. Normally, cryptophanes use imploded conformation as an alternative tool to occupy their cavities, but this conformation is energetically demanding or it forms when the cryptophane is stored for a long time in the solid state. The latter case is observed for compound 112 which even implodes in solution.

Effect of Self-Encapsulation on Chiroptical Properties

Self-encapsulation induces large conformational changes of cryptophane linkers. These changes were readily confirmed by 1 HNMR and IR spectroscopy (see above). Different chiroptical tools were also employed to characterize this phenomenon. Despite the fact that compounds 109, 110 and 111 undergo self-encapsulation, they have completely different chiroptical properties in different solvents. Investigating the chiroptical properties by ECD spectroscopy revealed little variations in different solvents. 183 This result is in agreement with our previously reported results, which show that the Cotton bands are less responsive to conformational changes in organic solvents. This lack of sensitivity can be explained in part because the benzene chromophores are inserted into two independent rigid structures. Thus, the chromophores and their relative orientation seem to be moderately affected by the conformational rearrangement of the linkers. The ECD spectral results of enantiopure compound 109 are quite surprising, because the SOR are strongly modified under the same experimental conditions. It is also noteworthy that ECD and SOR (ORD) are related by Kramers-Kronigs relationships (see Appendix). Consequently, a change of the SOR (ORD) values must have an impact on the overall ECD spectrum.

To explain this lack of sensitivity several hypotheses can be proposed. One of the proposed hypothesis is the contribution of high-energy excited states, which cannot be accessed in our case by conventional ECD spectrometer ( 220 nm). These high-energy excited states can contribute to the SOR values recorded for compound 109. This assumption is however difficult to check experimentally because we do not have access to the whole ECD spectrum of compound 109.

Synchrotron Radiation Circular Dichroism (SRCD) allows extending the spectral range, but even in this case, it will be difficult to confirm this hypothesis. Such a study has been performed in the case of cryptophane-[222], 98, that shows a large variation of the SOR (ORD) values as a function of the nature of the solvent. 191 Another assumption to explain this unusual behavior will be to consider a participation of the solvent. Indeed, it has been shown in some cases that the solvent may contribute to the sign of the SOR values measured. 207 Thus, a contribution of the solvent cannot be totally excluded, but this effect is also difficult to verify experimentally or by quantum calculations since the use of an explicit model would require large computation times, considering the molecular size of compound 109, the number of possible conformations adopted and the number of solvent molecules surrounding the host molecule. Nevertheless, this surprising effect cannot be solely attributed to the self-encapsulation process since other compounds such as 110 and 111 show a different behavior.

Compared to ECD, VCD spectroscopy revealed a higher sensitivity to conformational changes.

For example, the VCD spectra of [CD(+)254]-109 and [CD(+)254]-110 enantiomers are dependent on the fact that the solvent can enter (CDCl3 or CD2Cl2) or not (C2D2Cl4) the cavity of the host.

Particularly the spectral regions lying between 1480 and 1450 cm -1 associated with the δCH2 bending vibration and in the 1300 to 1250 cm -1 related to the wagging and twisting modes of methylene groups are mostly affected by the nature of the solvent. However, these spectral modifications do not give any clue about self-encapsulation since they are also observed with compound 112, which does not undergo self-encapsulation.

As observed with many different enantiopure cryptophane derivatives studied by our group, polarimetry appears to be the most sensitive technique to detect small conformational changes, when recorded in organic solvent. In contrast, ECD spectroscopy appears much less sensitive in studying these conformational changes due to the particular arrangement of the six benzene chromophores. VCD spectroscopy can also be useful to detect such type of conformational variations, with an additional advantage of being easily calculated using quantum mechanics equations. In addition, VCD spectroscopy combined with DFT calculations remain the methods of choice to determine, with confidence, the absolute configuration of these derivatives. This technique was proved to be efficient in the case of compound 109, where the direct determination of the absolute configuration was found complicated.

Conclusion

Through the run of this part, we have delivered a non-exhaustive description of some chiral cryptophane derivatives. The different chiroptical properties of these cages were also discussed to offer a better understanding of cryptophanes' configurations and conformational variations of cryptophanes and their complexes. Importantly, we also highlighted the main chiroptical tools used in this domain and described the major functions of each tool, their advantages and inconveniences. Exploiting these tools along with 1 H NMR spectroscopy, we were particularly interested in studying the behavior of the cryptophanes-[223] derivatives subjected to "self-encapsulation". This phenomenon is characterized by a conformational change of the central group lying on the propylenedioxy arm, which points inward the inner cavity of the host in the absence of a competitor guest. The chiroptical properties of these cages revealed that compound 109 has a unique behavior, since its SOR values invert depending on the nature of the solvent. To our knowledge, this family of compounds gathers the first examples of cryptophane derivatives that give rise to a self-encapsulation phenomenon. The self-encapsulation process appears as a convenient way to fill the cavity in absence of guest molecules. This process occurs spontaneously in solution, and it requires less energy than the CTB implosion that is usually observed upon heating the compound in solution or in the solid state. In contrast, compound 112 that does not possess appropriate substituent on the linker gives more easily the imploded form.

Experimental Part

DFT calculations

The geometry optimizations, vibrational frequencies and absorption intensities were calculated by the Gaussian 09 program on the DELL cluster of the MCIA computing centre of the Bordeaux University. Calculations of the optimized geometry of PP-109 and MM-110 enantiomers were performed at the density functional theory (DFT) level using B3LYP functional and 6-31G** basis set with the use of IEFPCM model of solvent (C2H2Cl4 described with parameters ε = 7.096 and ε∞ = 2.21). First, a conformational search of the two compounds was performed at the molecular mechanics level (MMFF94 force fields incorporated in compute VOA software, 8 kcal mol -1 limit) on the optimized structure of cryptophane-[223] as a starting point, on which acetate substituents were added. Two structures with, respectively, trans, trans, and gauche-gauche conformations for the two ethylenedioxy and propylenedioxy linkers, with the acetate group grafted on the latter pointing inward and outward the cavity, were optimized. Vibrational frequencies and IR intensities were calculated at the same level of theory. For comparison with experiments, the calculated frequencies were scaled by 0.968 and the calculated intensities were converted to Lorentzian bands with a half-width of 7 cm -1 . Synthesis of cryptophane 110: Acetic anhydride (0.7 mL, 7.4 mmol, 10 equiv.) was added dropwise at 0 °C to a solution of cryptophane 8 (0.7 g, 0.75 mmol) in freshly distilled CH2Cl2 (23 mL) and pyridine (3.5 mL). The reaction mixture was stirred for 48 hours at room temperature. The solution was then quenched at 0 °C with concentrated NaHCO3 (10 ml) for 10 minutes and then poured in 20 mL of H2O. The aqueous layer was extracted 5 times with CH2Cl2 and then dried over Na2SO4. After filtration the solvent was evaporated under reduced pressure.

The crude product was subjected to column chromatography on silica gel (CH2Cl2/acetone: 85/15) to give rise to compound 110 as a white product (0.52 g, 71%).

1 H NMR (400 MHz, CDCl3, 25 °C): 6.63-6.79 (12 s, 12 H), 4.58-4.62 (m, 6H), 4.02-4.2 (m, 11H), 3.93 (m, 1 H), 3.73-3.8 (6s, 18 H), 3.47 (m, 1H), 3.36-3.41 (m, 6 H), 2.17 (s, 3H).

13 C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): 170. 6, 149.6 (2C), 149.55, 149.4, 148.8, 148.1, 146.8, 146.7 (2 C), 146.6, 146.5 (2 C), 134.1, 134.1, 133.8, 133.8, 133.3, 132.8, 132.1, 131.9, 131.5, 131.4, 131.35, 131.3, 120.3, 120.2 (3 C), 117.2, 115.1, 114.2 (2 C), 113.9, 113.7, 113.35, 113.2, 70.8, 69.6, 69.5, 69.1, 68.9 (2 C), 66.6, 56.3, 56.3, 55.7, 55.65(2 C), 55.6, 36.5, 36.4; 36.2 (4 C), 21.2.

HRMS(ESI-TOF) m/z [M + Na]

+ calcd for C57H58O14Na 989.3724, found 989.3719.

Synthesis of cryptophane 111:

Acetic anhydride (0.3 mL, 3.1 mmol, 5 equiv.) was added dropwise at 0 °C to a solution of cryptophane 27 (0.6 g, 0.63 mmol) in freshly distilled pyridine (8 mL). The reaction mixture was stirred for 48 hours at room temperature. The solution was then quenched at 0 °C with concentrated NaHCO3 (10 ml) for 10 minutes and then poured in 20 mL of H2O. The aqueous layer was extracted 5 times with CH2Cl2 and then dried over Na2SO4. After filtration the solvent was evaporated under reduced pressure.

The crude product was subjected to column chromatography on silica gel (CH2Cl2/acetone: 95/5) to give rise to compound 111 as a white product (0.57 g, 70%).

1 H NMR (400 MHz, CDCl3, 25 °C): 6.76-6.52 (12 s, 12 H), 4.62-4.55 (m, 6H), 4.45-3.95 (m, 11H), 3.79 (1s, 9 H), 3.76 (1s, 3H), 3.74 (1s, 3H), 3.44-3.35 (m, 6 H), 2.63 (s, 1H), 2.07 (s, 3H). 5, 149.4 (2C), 149.3, 147.8, 147.5, 147.0, 146.9, 146.7, 146.56, 146.54, 133.9 (2C), 133.8 (3C), 132.3, 132.2, 131.6, 131.5, 131.4, 130 (2C), 120.4 (2C), 120.38, 120.1, 113.9 (2C), 113.6, 113.4, 112.9, 112.9, 112.3, 112.2, 69.65, 69.5, 68.9 (2C), 64.92, 64.2, 62.56, 56.19, 55.7, 55.52 (2C), 55.4, 39.6 (2C), 36 (2C), 35.9 (2C), 20.9. was added dropwise at 0 °C to a solution of cryptophane223 (OH)6 (0.35g, 0.42 mmol) in Pyridine (5 mL). The reaction was stirred at room temperature for 16 hours under an argon atmosphere. Then, the reaction was quenched with a solution of sodium bicarbonate at 0 °C.

HRMS(ESI-TOF

The solution was then extracted 5 times with CH2Cl2. The combined organic layers were dried over sodium sulfate. A filtration, followed by evaporation of the solvents under reduced pressure, gives rise to compound 112 as a white product. The solid was then washed on a frit with an Et2O/CH2Cl2: 75/25 mixture to give compound 112 as a clean white solid (0.31g, 71%). (2 H), 4.65-4.58 (m, 6 H), 4.28-4.286 (2 H, m), 4.16-4.15 (m, 2 H), 4.04 -3.8 (m, 8 H), 3.5-3.4 (m, 6H) 21 -60.1 -62.3 -70.3 -123.4 -190.9 CHCl3 0.23 -25.4 -28.3 -32.1 -48.5 -59 31 -148.8 -148.8 -180.6 -355.4 -639.1 CHCl3 0.24 -190.4 -200.0 --230.2 -420.2 -775.4 DMSO 0.31 -208.5 -218.5 -252.4 -463.6 - 26 -66.0 -70.1 -78.7 -131.6 -191.3 CHCl3 0.24 -38.6 -40.9 -47.3 -74.5 -90.4 DMSO 0.25 -50.0 -52.9 -60.5 -101.5 -145.0 DMF 0.27 -53.9 -54.8 -61.8 -102.6 -142.3 C2H2Cl4 0.30 -38.9 -38.3 -45.7 -75.6 -112.7 Specific This interesting phenomenon induces large conformational changes, which were successfully proved by 1 H NMR and IR spectroscopy. The chiroptical study of these enantiopure cages, which were separated by our collaborator at Aix Marseilles University, Dr. Nicolas Vanthuyne, revealed that the overall chiroptical properties of these cages were modified under the effect of selfencapsulation. Specifically, polarimetry appears to be the most sensitive technique to small conformational variations in organic solvents. Other spectroscopic techniques like ECD and VCD combined with DFT calculation, which were performed with our collaborators Dr. Thierry Buffeteau and Dr. Nicolas Daugey in Bordeaux, have mainly served in the determination of the AC of cryptophanes, where the direct determination of AC is hard to assess.

MM-110

PP-112

Appendix 1. Nuclear Magnetic Resonance (NMR) Spectroscopy Principles

Any nucleus with an odd number of protons and/or neutrons has a charge with a characteristic spin (I) of (mI) different orientations of (2I+1) values varying from -I, 2I+1 to +I states, all of which are degenerate (have the same energy). A proton, for example, has an (I) =1/2 which means, it has two allowed spin states (mI) of 1/2 and -1/2. The spinning of this nucleus creates a characteristic magnetic field with a magnetic moment (µ) which is related to (I) by an intrinsic constant, gyromagnetic ratio (γ), characteristic to each nucleus (eq. 1), Figure 156.

µ= ℏ γ I eq. 1

ℏ: reduced planks constant

In the presence of an external uniform magnetic field B0, the randomly orientated nuclei will align themselves in the direction of the applied magnetic field and they will lose their degeneracy due to the interaction of their magnetic moments with external applied field (Zeeman energy levels). Nuclei with lower energy, α state, orient themselves with the direction of the field, whereas nuclei with higher energy align in the opposite direction, β state. The energy values of each state are linear values to B0 and it is given in (eq. 2).

E= -ℏ γ mIB0

eq. 2

The thermal population distribution between the two states is based on Boltzmann distribution coefficient given in (eq.3) where N represents the number of nuclei residing in the lower and higher energy states, Nα and Nβ, respectively, K is the Boltzmann constant and T is temperature.

Nβ/ Nα= e ΔE/KT = e (-ℏ γ B0) /KT eq.3 For protons in 1.41 T magnet, the distribution coefficient between the two states is equal to 0.9999382 which means that there is a slight excess on nuclei in the lower energy state directed parallel to the magnetic field. The difference in population between the two states is referred to as polarization (P) and it is expressed in (eq. 4).

P = Nα -Nβ/ Nα +Nβ ≈ (ℏ γ B0) /2KT

eq.4

This will be expressed as a net longitudinal magnetization M0 in the direction of the applied field B0, (eq. 5) where Ns represents the excess of population residing in the α state.

M0= (NS ℏ γ P)/2

eq.5

The nuclei at this stage are known to precess in the direction of B0 because they cannot be really oriented parallel or anti-parallel to it. The precession frequency is the known Larmor frequency Ѡ0 and it is expressed in (eq. 6).

Ѡ0= γB0

eq. 6

Once a radio frequency pulse is applied, excess of nuclei in the lower energy will be excited to a higher energy sate, and they will coherently precess, creating a transverse magnetization along characterizes each enantiopure compound. Since 589 is also dependent on the temperature and concentration effect, these parameters have to be carefully mentioned. Concentration C is usually reported in g/100 mL of solvent and l' is cell length which is usually equal to 1 dm.

Electronic Circular Dichroism ECD Spectroscopy:

Circular dichroism results from a differential absorption of the left and right circularly polarized light by chiral molecules. In the classical approach, this difference affects the respective intensities of these two components creating an elliptical polarized light whose ellipticity is described by an angle θ which is characteristic of the chiral material studied. In an ECD spectrum, the difference in the absorptivity of the left and right circularly polarized light (Δɛ = ɛL -ɛR) is traced as a function of the wavelength. Each excited state visible on the ECD spectrum has a characteristic Rotational Strength Rij that determines its intensity and sign. The shape of the ECD spectrum is the result of the summation of all contributed rotational strengths of each electronic transition. It is worth mentioning that the sum of the rotational strengths extended to all the excited states is equal to zero. Nevertheless, it is important to note that the experimentalists do not have access to the whole ECD spectrum. Usually, the spectral range that is accessible depends on many different factors: the molecule it-self, the solvent, the concentration, and the spectrophotometer used. For instance, it is now possible to extend the spectral range by using Synchrotron Radiation circular Dichroism Spectroscopy.

It is worth to note that ORD and ECD are strongly related via Kramers-Kronig relationship. 211 Thus, theoretically these two spectra can be deduced from each other but practically this is difficult to achieve since we cannot have access to the whole ECD or ORD spectra. In recent years, numerous studies have been undertaken to predict ORD curves from partial ECD spectra, Figure 159. ECD spectra can be reproduced by quantum calculations by using TD-DFT. However, these theoretical spectra are quite difficult to reproduce because we need to consider many excited states. These simulated spectra give better results if we consider the role of the solvent (implicit or explicit model). However, this increases the complexity of such simulation and considerably increases the computational time.

Vibrational Circular Dichroism VCD Spectroscopy:

Similar to the above described ECD spectroscopy, vibrational circular dichroism is the measurement of the differential absorption of left and right circularly polarized light in the infrared region. The obtained VCD spectra of two enantiomers are equal in magnitude but opposite in signs just like ECD spectra. In contrast to ECD spectroscopy, VCD spectra are more complicated in shape since each vibrational mode possess its own rotational strength. Thus numerous transitions can be detected on the VCD spectra of organic molecules. Nevertheless, solvent absorption often decreases the spectral window accessible. Thus the choice of the organic solvent is critical to record such spectra. Usually, CD2Cl2, CDCl3, 1,1,2,2-terachloroethane-d2 or even DMSO-d6 can be used to record spectra between 1800 cm -1 and 1000 cm -1 . Despite the strong absorption of water, VCD experiments can also be performed in deuterated water but these experiments usually require small path-length compared to organic solvents. This spectral region contains numerous bands and that can be useful to get information about the absolute configuration of the molecule of interest and its conformation in solution. Interestingly, this technique gives rise to VCD spectra, which are the sum of all conformations adopted by the molecule in solution. This may complicate the analysis of these spectra in the case of highly flexible molecules. VCD spectra can be reproduced by DFT calculation. A comparison of the experimental and calculated spectra allows the determination of the absolute configuration of the molecule. In addition it gives insights about the conformation adopted by the molecule in solution. Sometimes, a weighted sum of theoretical VCD spectra is necessary to reproduce the experimental VCD spectrum. VCD spectroscopy only requires calculation performed on the ground state contrary to ECD spectroscopy. This represents an undeniable advantage to perform these calculations. VCD spectra are more difficult to record than ECD spectra. Consequently,

  List of Abbreviations and Symbols ............................................................................................... Résumé de la Thèse: Les cryptophanes constituent une classe de composés macrocycliques uniques caractérisée par la présence d'une cavité lipophile dont la taille peut varier selon la nature du cryptophane. En solution, cette cavité est susceptible d'accueillir divers substrats comme de petites molécules ou des atomes (ions, gaz rare) dont les propriétés physiques s'en trouvent alors modifiées. Depuis la première description de ces composés en 1981 par André Collet, les cryptophanes ont connu un regain d'intérêt au début des années 2000 avec la mise en évidence de la complexation du xénon par des cryptophanes de petites tailles. Cette découverte a depuis été exploitée par plusieurs équipes internationales pour préparer des bio-sondes moléculaires pouvant présenter un intérêt en imagerie de résonance magnétique nucléaire (IRM) du xénon hyperpolarisé. Au contraire des autres techniques IRM développées jusqu'ici, l'IRM du xénon hyperpolarisé des complexes xénon-cryptophane repose sur une technique de détection basée sur le déplacement chimique. Les complexes xénon-cryptophane présentent ainsi une signature spécifique qui peut être facilement identifiée et exploitée pour produire une image IRM. La très grande sensibilité de la technique combinée aux propriétés exceptionnelles des complexes xénoncryptophanes rendent cette approche extrêmement intéressante pour concevoir de nouveaux traceurs pour l'imagerie RMN du xénon hyperpolarisé.

Figure 4 :

 4 Figure 4: CTB unit symmetry and helical chirality.

C

Figure 5 :

 5 Figure 5: possible cryptophanes' configurations.

Figure 6 :

 6 Figure 6: inter-conversion between CTB crown conformers passing by a relatively non-stable saddle conformer.

Figure 8 :

 8 Figure 8: possible conformers of cryptophanes.

Figure 9 :

 9 Figure 9: m-xylyl bridged cryptophane capable of forming imploded form on the left. Imploded form of the cryptophane as determined by the single-crystal structure on the right.

Figure 11 :

 11 Figure 11: representative plot of the raw ITC data for the titration of cryptophane-[222] (OH)5 (29.9 µM) with Tl + (401 µM) in competition with Rb + (299 µM).

  electron rich cavity of cryptophanes renders anion complexation impossible. Coordination of the aromatic rings with electron withdrawing transition metals from their outer rims makes cryptophane's cavity poor in electrons, thus suitable for anion complexation. This has been smartly demonstrated by Holman and co-workers that reported a water soluble cryptophane-[333] (OMe)6 cage with six [Cp*Ru] + moieties. Encapsulation of [CF3SO3] -, [SbF6] -and [PF6] -was then achieved using this cage and they were stabilized by π-anion interactions, Figure13.[START_REF] Fairchild | Selective Anion Encapsulation by a Metalated Cryptophane with a π-Acidic Interior[END_REF]

Figure 13 :

 13 Figure 13: structure of water soluble cryptophane [333] (OMe)6 [Cp*Ru]Cl6. 68

Figure 19 :

 19 Figure 19: access of multiply functionalized cryptophane by template method.

Figure 20 :

 20 Figure 20: family of dually-functionalized cryptophane cages bearing a central hydroxyl group and six modifiable groups on the CTB rims.

Figure 21 :

 21 Figure 21: example on mono-functionalization of cryptophane-[111] by water-solubilizing group.

  the past two decades, a wide variety of cages has been synthesized for this purpose, most interestingly, the cryptophane-[111] cage synthesized by Brotin and Dutasta, showing the highest binding constant of xenon of about 10000 M -1 in 1,1,2,2-tetrachloroethane-d2 due to the perfect volume compatibility. Since then, cryptophane cages have been intensively used for xenon encapsulation in organic and aqueous media. Moreover, cryptophanes are not the only hosts for xenon encapsulation in solution, where several other molecular hosts capable of xenon complexing have been reported in the literature.

Figure 24 :

 24 Figure 24: structure of tri-functionalized cryptophane-[111] derivatives.

Figure 26 :

 26 Figure 26: structure of the first Xenon@Cryptophane based sensor and the corresponding HP xenon spectrum, showing at around 70 ppm the signals of cryptophane-encapsulated species. Upon avidin addition, a shift of about 2.3 ppm is observed with a decrease in the intensity of xenon encapsulated in functionalized cage.

27. 121

 121 Nevertheless this technique is not common in laboratories and it requires specific skills to design and operate this kind of apparatus. For this reason our team established an old collaboration with Dr. Patrick Berthault and his co-workers in CEA, (Paris, Saclay), who are specialized in the production of (HP) 129 Xe by SEOP using the apparatus shown in Figure27. In addition this group has succeeded to use (HP)129 Xe as an MRI tracer and developed new NMR sequences and protocols for efficient sensors detection.

Figure 27 :

 27 Figure 27: equipment for production of hyperpolarizing xenon gas of Berthault group at CEA Paris.

Figure 29 :

 29 Figure 29: hyper-CEST detection scheme for encapsulated 129 Xe a) the initial spectrum obtained after a second of acquisition without selective irradiation b) selective radio frequency depolarization of the encapsulated 129 Xe resulting in its depolarization and the accumulation of depolarized gas in the bulk c) the resulting spectrum from selective "on resonance" saturation of the encapsulated 129 Xe showing a drop in its intensity.

Figure 30 :

 30 Figure 30: structure of the water soluble biotinylated biosensor immobilized on avidin bearing agarose beads. On the right are the spectrally resolved xenon-based MR images of the agarose bead. 126

Figure 31 :

 31 Figure 31: a) structure of the cryptophane derivative, b) hyperpolarized xenon spectrum of xenon trapped in transferrin linked cages obtained in one scan, and the sub-spectrum was obtained by accumulation of 256 selective excitations with a repetition time of 122 ms after c) microscopic image of the cells incubated with the biosensor d) fluorescence imaging assuring the presence of the biosensor in the studied cells. 131

  sensor. Two examples have been reported with a modified cryptophane-[222] derivatives bearing respectively three and six carboxylic acid moieties. In these examples, a change of the pH of the a change of the encapsulated xenon chemical shift. For instance, with the cryptophane-[222] hexacarboxylate, a modification of the pH from 3.5 to 10.8 resulted in an upfield shift of 129 Xe NMR signal by about 4 ppm.

40

 40

Figure 32 :

 32 Figure 32: structure of pH-sensitive cryptophanes A and B and the corresponding plot of chemical shift difference of A and B as a function of pH. 40

Figure 33 :

 33 Figure 33: structure of NTA-substituted cryptophane-A hexa-acid, and the corresponding HP 129-xenon NMR spectrum upon different metal cations chelation.135,136

  Concerning129 Xe@Cryptophane complexes sensitivity to their physical environment, Dmochowski and coworkers developed another smart pH sensor for cell labeling. This cage bears a pH sensitive peptide chain that undergoes conformational change upon going from pH = 5.5 to pH = 7.5. The peptide adopts a functional conformation at acidic pH, capable of acid cell labeling, whereas it loses its penetration activity upon neutralization. These conformational variations were successfully studied by tryptophan fluorescence, electronic circular dichroism and xenon NMR spectroscopies. In this example, hyper-CEST proved a high sensitivity of the sensor up to picomolar concentrations. Most importantly, the interaction of acidic cell with this biosensor showed a record breaking chemical shift of about 13.4 ppm relative to the non-bound form at neutral pH, Figure34.142

Figure 34 :

 34 Figure 34: a) structure of the biosensor, hyper-CEST 129 Xe NMR spectra at pH 7.5 b) Xe@cells, red trace (196.3 ppm); Xe@aq, blue trace (192.3 ppm); (c) Xe@sensor (65.0 ppm) and at pH 5.5; (d) Xe@cells, red trace (198.0 ppm); Xe@aq, blue trace (192.0 ppm); (e) Xe@sensor-cells, red trace (78.4 ppm) and Xe@sensor, blue trace (68.0 ppm). Sums are shown as solid black lines. Figure adapted from reference 142.

Figure 35 :

 35 Figure 35: structure of the biosensor bearing a fluorophore, b) 1 H MRI of the two compartment phantom housing cells encapsulated within alginate beads. Blue compartment is filled with CrA-FAM labeled cells and the red one is incubated with unlabeled cells. c) Localization of the two compartments by a single CEST-acquisition (2 min) compared to the more sensitive accumulations of 20 CEST-acquisitions. 145

  Development of cryptophane-[222] derivatives for biothiol detection was achieved in organic media. A mono-functionalized cryptophane-[222] bearing an acrylate moiety aimed to react with biothiols derivatives such as cysteine, homocysteine, or glutathione has been prepared.

Figure 36

 36 Figure 36. 150

Figure 36 :

 36 Figure 36: a) structure of the cryptophane dimer and the conformational change observed upon mercury cation addition, b) HP 129-xenon NMR spectrum with and without the metal ion c) chemical shift image (CSI MRI) of sensor c1) with, and c2) without the addition of cation.

Figure 37 : 2 .

 372 Figure 37: structures of some biosensors developed by different groups.

Figure 38 :

 38 Figure 38: possible dual functionalization of cryptophane-[223] cages.

  In this chapter, before describing in details our synthetic work, we wish to present an overview of the first generation of cryptophane-[223] cages bearing two orthogonal functionalities synthesized by Dr. Laure-Lise Chapellet et al. We will also discuss the main advantages and disadvantages of this strategy.[START_REF] Chapellet | Synthesis of Cryptophanes with Two Different Reaction Sites: Chemical Platforms for Xenon Biosensing[END_REF] Anti-cryptophane-[223] derivative (C2-symmetry) possesses a unique propylenedioxy arm, where a central anchoring unit can be added. This allows us to keep the six hydroxyl groups untouched to introduce other substituents. The addition of functionality at the center of the propylenedioxy arm decreases the molecular symmetry (C1-symmetry). However, it is important to note that no additional stereocenter is created upon this structural change since the two CTB units possess the same absolute configuration (MM or PP). This won't be the case for syn-cryptophane-[223] type molecules, where the two CTB units possess different descriptors (MP/PM). Consequently, introduction of a new functionality at this position does not complicate the synthesis of such constructs since no diastereomeric mixtures are obtained.Chapellet et al. described the first generation of bi-functionalized [223] cages. These cages were decorated with a secondary alcohol grafted on the central carbon of the propylenedioxy arm and six carboxylic acid moieties on the CTB rims, Figure39. This platform was successfully obtained in eight steps starting from the commercially available vanillic alcohol 1.

Figure 39 :

 39 Figure 39: structure of a dually functionalized cryptophane-[223] derivative bearing a central secondary alcohol on the propylenedioxy linker.

Figure 40 :

 40 Figure 40: a) scandium triflate catalyzed cyclization versus b) formic acid catalyzed cyclization to obtain the dually functionalized cage.

Figure 41 :

 41 Figure 41: structure of water soluble hexa-phenol for Tl cation complexation.

13

 13

Figure 43 :

 43 Figure 43: illustration of failed trials to alkylate cryptophane 13.

Figure 44 :

 44 Figure 44: oxidation of the secondary alcohol of cryptophane 13 into a ketone.

Figure 45 :

 45 Figure 45: structure of the second generation of bi-functionalized cages.

  targeted cryptophane-[223] derivatives presented in this manuscript are asymmetric with two types of linkers: two ethylenedioxy and one functionalized propylenedioxy arms. Template method is employed, because it allows a selective introduction of different linkers having various length and substituents. Basically, compound 27 shown in Figure 46 can be obtained after either performing a chemical transformation to an existing properly functionalized cryptophane or directly by cyclizing a precursor molecule containing a primary alcohol. Three routes can be envisioned to obtain compound 27, routes I, II and III. The retrosynthetic analyses of these strategies are shown in Figure 46 and Figure 47. We decided to detail only the successful strategy (synthetic route I) in this manuscript. In route I, compound 27 can be obtained by performing a hydroboration-oxidation step on the terminal alkene of cryptophane 26. This crucial step assures a regioselective insertion of the hydroxyl group on the terminal position. Cryptophane 26 itself is a result of classical cyclization of the tri-substituted template 25 by three consecutive Friedel-Crafts alkylations. In turn, the latter can be obtained after a double alkylation of a mono-substituted derivative 23 by compound 24 whose synthesis has already been reported. 153 The mono-alkylated synthon 23 is a result of an alkylation between 4 154 and an alkenyl substituted vanillic alcohol 22. Compound 22 can be easily prepared by reacting vanillic alcohol 1 with 3-chloro-2-chloromethyl-1-propene 19.

Figure 46 :

 46 Figure 46: retrosynthetic analysis strategy I leading to anti-cryptophane 27.

Figure 47 :

 47 Figure 47: retrosynthetic routes II and III.

Figure 48 :

 48 Figure 48: synthesis of the first CTB 4 cap.

Figure 49 :

 49 Figure 49: tried strategies for adding the primary alcohol moiety or its precursors.

Figure 50 :

 50 Figure 50: scheme showing the synthesis of 23 in racemic form and the corresponding byproducts.

  axial and equatorial protons of the CTB are displayed between 3.4 and 4.6 ppm respectively.

Figure 51 :

 51 Figure 51: synthesis of cryptophane 26 by cyclization using formic acid and the hydroboration-oxidation step to introduce the primary alcohol derivative 27.

Figure 52

 52 Figure 52: 1 H NMR spectrum of cryptophane 26 illustrating the C2-symmetry.

Figure 53 :

 53 Figure 53: ORTEP representation of compound 27. Two different views are shown. Hydrogen atoms have been removed for clarity. The cavity is filled with an ethanol molecule (not shown). The displacement ellipsoids were plotted at 30% probability level.

Figure 54 :

 54 Figure 54: 1 H NMR spectrum of compound 27 reflecting the C1-symmetry and zoomed is the central proton on the propylenedioxy arm (shown in red for illustration).

Figure 55 :

 55 Figure 55: mesylation of cryptophane 27 followed by nucleophilic substitution by potassium thio-acetate to form cryptophane 29.

Figure 56 :

 56 Figure 56: oxidation of cryptophane 27 into an aldehyde 30 and then to the carboxylic acid derivative 31.

Figure 57 :

 57 Figure 57: attempts to demethylate cryptophane 26 to form cryptophane 33.

33

 33

Figure 58 :

 58 Figure 58: protection of cryptophane 27 by a THP group followed by a demethylation step to form cryptophane 35.

Figure 59

 59 Figure 59: 1 H NMR spectrum of cryptophane 35 in 400 MHz spectrometer.

Figure 60 :

 60 Figure 60: alkylation of compound 35 followed by a deprotection step to form compounds 38 and 39.

Figure 61 :

 61 Figure 61: structure of compound 40.

Figure 62 :

 62 Figure 62: oxidation of compounds 38 and 39 to the aldehyde and the corresponding acids 43 and 44.

Figure 63 :

 63 Figure 63: 1 H NMR spectrum of compound 43 in CDCl3 in 400 MHz spectrometer.

Figure 64 :

 64 Figure 64: schematic presentation showing the possible functionalization of compound 43 and 44 by an amide coupling reaction.

  Hydroxamic acids are a family of organic compounds of molecular structure RCC(O)N(RN)OH (RC = alkyl/aryl; RN = alkyl/aryl or H), Figure65. These compounds were first described by H. Lossen in 1869, where he reported the synthesis of oxalohydroxamic acid from diethyl oxalate and hydroxylamine.159 Hydroxamic acids are weak acids that undergo two consecutive deprotonations of the first (N-OH) proton to give the hydroxamato group and a second deprotonation (NH-O) to offer the hydroximato function. The pKa of the first deprotonation ranges between 8.5 and 9.4 in aqueous media.160 Hydroxamic acids are characterized by their ability to chelate a wide range of transition metals. The chelating ability is readily exploited by nature, in which they exist in many bacteria as the so-called siderophore for coordinating dissolved iron-(III). These iron-(III)-siderophore complexes are recognized by cellsurface proteins, which transport them to the cytoplasm where they release their iron-(III) content. Additionally, they act as inhibitors for Ni (II) urease and Zn (II)-containing metalloproteins like matrix metalloprotease (MMP), which are involved in cancerous cell proliferation.161,162

Figure 65 :

 65 Figure 65: structure of hydroxamic acid function and its corresponding (O, O) chelation modes.

Figure 66 :

 66 Figure 66: synthesis of compound 46 bearing a hydroxamic acid function.

31 45 Figure 67 :

 314567 Figure 67: synthesis of water-soluble cryptophane-[223] 48 bearing a central hydroxamic acid function.

  APTRA or (o-aminophenol-N,N,O-triacetic acid) ligand has been first reported by London and coworkers in 1988. It was initially used to detect cytosolic magnesium and it was connected

Figure 69 :

 69 Figure 69: synthesis of organic version of cryptophane-[223] 53 bearing an APTRA derivative.

Figure 70 :

 70 Figure 70: synthesis of water soluble cryptophane 55 functionalized by an APTRA derivative.

  cryptophane-[223] core by amide coupling reaction,Figure 71. 119

Figure 71 :

 71 Figure 71: synthesis of compound 56 by an amide coupling reaction of cryptophane 43 and biotin-PEG 2-amine.

Figure 72 :

 72 Figure 72: 1 H NMR of compound 56 in CDCl3 in 400 MHz spectrometer.

  obtained after an extraction and acidification step. The 1 H NMR spectrum of this derivative shows the full disappearance of the six benzyl groups but the compound is still not pure. The formation of compound 57 was also confirmed by HRMS and xenon NMR experiments. Further work has to be done to optimize this step, Figure73.

Figure 73 :

 73 Figure 73: synthesis of water soluble cryptophane 57.

  Xe NMR was done in D2O at 298 K. This spectrum shows two peaks corresponding to the free xenon in solution at 196 ppm and the caged xenon at 55 ppm, Figure 74. Indeed the latter signal is upfield shifted compared the free xenon due to cryptophane's shielding effect of the six aromatic rings surrounding the encapsulated xenon. The appearance of two signals indicates a slow in-out exchange dynamics of xenon. This behavior has already been observed with cryptophane-[223] type derivatives that show also specific signal for the 129 Xe@Cryptophane-[223] between 50 and

Figure 74 :

 74 Figure 74: one-scan (HP) 129 Xe NMR spectrum of compound 48 in D2O recorded at 25 °C.

  2 mM) was studied at three different pH values (pH= 3.3, 8.2 and 9.9) in D2O at 298 K. The spectra at each pH show two NMR signals. One signal corresponding to xenon dissolved in the bulk at 196 ppm and the upfield shifted signal of the caged one, Figure 75. At pH 9.9, 8.2 and 3.3 the encapsulated xenon peaks are located at 58.4 (green), 59.4 (red) and 61.3 ppm (blue), respectively, Figure 75.

  not "felt" by the encapsulated xenon. This result seems to be very surprising and disappointing because the hydroxamic function is very close to center of the cage.Consequently, we assumed that the close proximity between this function and the encapsulated xenon would result in a greater effect. The lack of sensitivity can be, in part, explained by the localization of the sensing unit. In contrast to the other examples reported in the literature, this sensing unit is located on the linker. Consequently it won't affect the ring current of the CTB units as in the case of other biosensors developed so far.The drop of the pH to acidic conditions (pH = 3.3) resulted a greater chemical shift variation by about 3 ppm. This variation is due to the change of ionization state of the carboxylic acid groups grafted on the CTB rims, which in turn modifies the ring current of the benzene rings. Similar effect was previously observed with cryptophane-[222] hexacarboxylate, where the change in the pH values from 3.5 to 10.8 resulted in a downfield shift by nearly 4 ppm.134 The greater variation observed in case of cryptophane-[222] hexacarboxylate compared to our cryptophane-[223]

Figure 75 :

 75 Figure 75: one-scan 129 Xe NMR spectra recorded at 298 K of compound 48 (0.2 mM) in D2O at pH = 9.9 (green), pH = 8.9 (red) and pH = 3.3 (blue).

Figure 76 :Figure 76 .

 7676 Figure 76: chelation of zinc cation by compound 48.

Figure 77 :

 77 Figure 77: representative plot of the raw ITC data for the titration of 48 with Zn 2+ . Titrations were carried out at 298 K in TRIS buffer (20 mM, pH = 7.6) with 48 (0.08 mM) in the sample cell and 1mM of Zn 2+ in the syringe.

Figure 78 :

 78 Figure 78: representative plot of the raw ITC data for the titration of cryptophane-[222] hexa-acid with Zn 2+ . Titrations were carried out at 298 K in TRIS buffer (20 mM, pH = 7.6) with cryptophane (0.08 mM) in the sample cell and 1mM of Zn 2+ in the syringe.

  hyperpolarized129 Xe NMR experiments of compound 57 revealed two peaks, one corresponds to the free xenon in solution, resonating at 196 ppm and another sharp signal at 52 ppm corresponding to the encapsulated xenon in the cavity of 57. The sharp signal obtained for Xe@57 gives an indication about the purity of our compound because assessing this parameter by conventional spectroscopic techniques was hard. We used only HRMS to assure the formation of our compound. Upon avidin addition, broadening of the initial signal is observed with a shift by about 5 ppm (57.5 ppm). It is worth mentioning that the complex precipitates partially under the used experimental conditions, so an optimization of these conditions is required. Nevertheless, this chemical shift change and broadening have already been observed with the first example of xenon cryptophane biosensor reported by Pines and co-workers. Interestingly, unlike Pines example which shows four signals corresponding to the two cryptophanes diastereomers, only one sharp signal is obtained in our case, thus facilitating the analysis, Figure79.

Figure 79 :

 79 Figure 79: one-scan 129 Xe NMR spectra recorded at 298 K of compound 57 (0.9 mM) in PBS buffer with 10% D2O at pH = 6.4, and upon of one equiv. of avidin (0.2 mM) at pH = 7.4.

  Mass spectra (HRMS) were performed by the Centre de Spectrométrie de Masse, University of Lyon. Analyses were performed with a hybrid quadrupole-time of flight mass spectrometer, microToF QII equipped with an Electrospray ion source. Data Analysis® 4.0 was used for instrument control, data collection, and data treatment. HRMS analyses were performed in full scan MS with a mass range from 50 to 2000 Da at an acquisition rate of 1 Hz. Transfer parameters were: RF Funnel 1, 200 V; RF Funnel 2, 200 V; hexapole, 50 V; transfer time, 70 μs and PrePulse storage time, 1 μs before each acquisition batch, external calibration of the instrument was performed with a sodium formate clusters solution. 1 H and 13 C NMR spectra were recorded at 400 and 100.6 MHz, respectively. Chemical shifts are referenced to Me4Si ( 1 H, 13 C). Column chromatographic separations were carried out over Merck silica gel 60 (0.040-0.063 mm). Analytical thin layer chromatography (TLC) was performed on MERCK silica gel TLC plates F-254. The solvents were distilled prior to use: DMF and CH2Cl2 from CaH2, THF from Na/benzophenone and pyridine from KOH.

  to produce five milliliters of xenon at a polarization ranging from 15 to 20%. Frozen hyperpolarized xenon immersed in a bath of liquid nitrogen and subjected to a magnetic field of 0.3 T provided by a solenoid is transported near the NMR magnet. There it is sublimated in the fringe field of the magnet and transferred thanks to a vacuum line to the NMR tube. A hollow spinner enabled us to condense it in the upper part of the NMR tube (screw capped Wilmad PP-528) without cooling the solution. The hyperpolarized 129 Xe NMR experiments were performed at 11.7 T ( 129 Xe resonance frequency 138.35 MHz) using a 5mm-broadband inverse probe head.

Figure 80 :

 80 Figure 80: 13 C NMR of cryptophane 34 composed of a mixture of diastereomers in CDCl3.

Compound 46 :

 46 ): m/z [M+H] + calcd. for C63H63NaNO14 1080.4146 found 1080.4140. The protected cryptophane 45 (0.07 g, 0.066 mmol) was introduced along with catalytic amount of Pd/C (10% by weight) and then dissolved in CH2Cl2/ Methanol mixture. The solution was purged first with Argon then with hydrogen and placed under hydrogen atmosphere for 24

Figure 83 :

 83 Figure 83: HSQC of compound 48 in DMSO-d6 in a 400 MHz spectrometer. Zoomed is the aromatic region corresponding to twelve proton and their corresponding carbons.

  ): m/z [M+Na] + calcd. for C83H86N2NaO32 1645.5061 found 1645.5056.

Figure 84 :

 84 Figure 84: 1 H NMR spectrum of compound 54 in CDCl3 in a 400 MHz spectrometer.

Figure 85 :Figure 86 :

 8586 Figure 85: hyperpolarized 129 Xe NMR spectrum of compound 34 recorded at 298 K in C2D2Cl4-d2.

Figure 87 :

 87 Figure 87: Hyperpolarized 129-xenon spectra of compound 48 in TRIS buffer (20 mM, pH = 7.5) at 25 °C a) in absence of Zn 2+ (green spectrum). b) in presence of 1.0 equiv. of Zn 2+ (blue spectrum). c) in presence of 5.0 equiv. of Zn 2+ .

  like cryptophane-[222] (OCH2CO2H)3 (OH)3. 174 Further decrease in cryptophanes molecular symmetry from D3 to C2 is achieved by changing one of the three linkers but keeping the same CTB substituents. For example, anti-cryptophanes-[223] 60, [233] 61 or [224] 62 (OMe)6 are all chiral with C2-symmetry. 111 The crystal structure of compound PP-60 assures its anticonfiguration, Figure 88.

Figure 88 :

 88 Figure 88: crystal structure of PP-60.

Figure 89 :Figure 90 :

 8990 Figure 89: symmetry of some anti-cryptophane derivatives.

Figure 91 :

 91 Figure 91: synthesis of enantiopure 72 from optically pure vanillic alcohol derivative 67.

Figure 92 :

 92 Figure 92: structure of enantiomerically enriched MM-anti-cryptophane derivatives having D3-symmetry.

Figure 93 :

 93 Figure 93: synthesis of enantioenriched 59 and 64.

Figure 96 :

 96 Figure 96: bio-catalyzed stereoselective optical resolution of TBTQ.

Figure 99 :

 99 Figure 99: resolving tri-hydroxy cryptophane-[222] by Mosher's acyl chloride.

  Figure 101: structure of cryptophane-[111] 97 and the corresponding retention time of each enantiomer. On the right shown the structure of cryptophane-[222] 98.

Figure 102 :

 102 Figure 102: synthesis of enantiopure hepta-hydroxyl cryptophane-[222].

Figure 104 :

 104 Figure 103: synthesis of Pd-based metallo cryptophanes.

Figure 105 :

 105 Figure 105: nitrile-functionalized CTB unit and the synthesis of metallo anti-cryptophane derivative.

  A has been reported in Figure106. This ECD spectrum shows several Cotton bands with positive or negative signs. The two first bands observed between 260 and 310 nm can be assigned to the Cotton bands of the 1 Lb transition. Then, the negative-positive bisignate (230-260 nm) corresponds to the ECD bands of 1 La transition. It has been observed, by analogy, with other cryptophane-A congeners that this bisignate can be extremely useful to assign the absolute configuration of cryptophane derivative in organic solvents. Indeed, in contrast to the ECD bands of the 1 Lb transitions, which are very sensitive to structural modifications and other external factors, the sign of this bisignate is robust and characteristic of the absolute configuration of the molecule. The negative-positive bisignate observed in Figure106allows us to assign the PP absolute configuration to the (+)-cryptophane-A derivative. Finally, the strong ECD band observed in the region of 220 nm corresponds to the 1 Bb transition.

Figure 106 :

 106 Figure 106: ECD of (+)-cryptophane-A spectrum of 1,4-dioxane.

Figure 107 :

 107 Figure 107: ECD spectra of empty free MM-90 and in the presence of CH2Cl2 and CHCl3 in LiOH (0.1 M) shown on the left. On right is shown the ECD spectra of MM-90 in solutions of LiOH, NaOH, KOH, and CsOH in H2O (0.1m).

  Figure 108: ECD spectra recorded at 293 K of PP-99 (from 6.5 to 8.5 × 10 -5 M): (a) in LiOH/H2O solution (0.1 M) in the presence of different amounts of a CsOH/H2O solution; (b) in a saturated solution of CHCl3 in LiOH/H2O in presence of different amounts of a CsOH/H2O solution. 197

Figure 109 :

 109 Figure 109: structure of (S)-methyl oxirane@MM-cryptophane 99, and the corresponding ECD spectra of MM-99 and PP-99 in the presence of rac-oxirane, (S)-oxirane, and (R)-oxirane in NaOH/H2O solution (0.1 M) at 293 K. The concentration of MM-99 and PP-99 was taken in the range 5×10 -5 and 5×10 -4 M.

  Figure110: structure of (S,S)@MM-99 complex, ECD spectra of (R,R)-bioxirane@PP-99 (red), (S,S)bioxirane@PP-99 (blue), meso-bioxirane@PP-99 (green), and guest-free PP-99 (black spectrum). ECD spectra of (S,S)-bioxirane@MM-99 (red dashes), (R, R)-bioxirane@MM-99 (blue dashes), meso-bioxirane@MM-99 (green dashes), and guest-free MM-99 (black dashes). The spectra were recorded at 278 K in NaOH/H2O (0.08 M) solution.[Host] = (6.2-8.8) × 10 -5 M, [guest] = 0.06 M.

[

  Figure 111: VCD spectrum of cryptophane-A in CDCl3.

Figure 112 :

 112 Figure 112: comparison of the VCD spectra of PP-compound 90 dissolved in D2O in the presence and the absence of a guest molecule.

  shows the ROA spectra of cryptophane-[111] recorded in the absence and in the presence of xenon. It can be noted that these two spectra are superimposable over a very large spectral range except a 190 cm -1 , where a clear difference can be observed. DFT calculations reveal that this vibration mode can be interpreted as the breathing mode of the host molecule. Upon the presence of xenon inside the cavity this mode is altered. This example clearly shows that ROA can be a useful complementary tool to characterize cryptophane molecules and their complexes.190

Figure 113 :

 113 Figure 113: comparison of experimental ROA spectra of [CD(+)254]-97 in CD2Cl2 solution in presence (red spectrum) or not (black spectrum) of xenon.

Figure 114 :

 114 Figure 114: solvent dependent self-encapsulation phenomenon of cryptophane-[223] derivatives.

Figure 115 :

 115 Figure 115: one-scan 129 Xe NMR spectra recorded at 298 K on solutions in C2D2Cl4 of cryptophane 13 (a) and cryptophane 109 (b).

109

  Figure 116: structure of cryptophane derivatives subjected to self-encapsulation phenomenon.

starting from cryptophane 12 ,

 12 where a hepta-acylation step in pyridine provided compound 109 in a good yield (74%). The two enantiomers of cryptophane 109 were then separated on a semi-preparative HPLC on a Chiralpak-IC column. A racemic mixture of 460 mg of cryptophane-109 provided the enantiomer [CD(-)254]-109 first (180 mg) and the [CD(+)254]-109 as a second fraction (200 mg). An enantiomeric excess ee > 99.5% and ee > 99% have been measured for [CD(-)254]-109 and [CD(+)254]-109, respectively.

The

  SOR values of enantiopure compounds [CD(-)254]-109 and [CD(+)254]-109 show a solvent dependent behavior at these wavelength, different from that observed for compounds 110, 111 and 112. For example, the SOR values of [CD(-)254]-109 in CH2Cl2 are positive and similar to CHCl3 but the latter are lower in values. In contrast, a complete inversion of SOR values sign is observed in DMSO and C2H2Cl4, where they show low negative SOR values ([α]589 = -7.6 × 10 -1 deg cm 2 g -1 and [α]589 = -10.4 × 10 -1 deg cm 2 g -1 at 25°C in DMSO and C2H2Cl4, respectively). It is worth mentioning that the [CD(+)254]-109 has the same SOR values with opposite signs, Figure 118.

Figure 118 :

 118 Figure 118: plot of the SOR values of [CD(+)254]-109 and [CD(-)254]-109 as a function of the nature of the solvent: black (CH2Cl2), orange (CHCl3), blue (DMSO), red (C2H2Cl4).

Figure 119 :

 119 Figure 119: plot of the SOR values of [CD(+)254]-110 and [CD(-)254]-112 as a function of the nature of the solvent: black (CH2Cl2), orange (CHCl3), blue (DMSO), red (C2H2Cl4).

  For compounds 110 and 111, the ECD spectra are different from that recorded for compounds 109 and 112. In particular, the 1 Lb transition is now composed of three Cotton bands instead of two. Unlike the first two described compounds, the ECD bands of the 1 Lb transition is more responsive to the change of the nature of the solvent, where modifications in its shape and intensity are observed especially in the ECD components between 285 and 310 nm, Figure120, Cand D.

Figure 120 :

 120 Figure 120: ECD spectra of [CD(+)254]-109 (black spectra) and [CD(-)254]-109 (red spectra) recorded in A) CH2Cl2 B) C2H2Cl4 at 20 °C. ECD spectra of [CD(+)254]-110 (black spectra) and [CD(-)254]-110 (red spectra) recorded in C) CH2Cl2 D) C2H2Cl4 at 20 °C.

Figure 121 :

 121 Figure 121: IR spectra in the ester νC═O spectral range of (A) 109, (B) 110, and (C) 112 in CDCl3 (black spectra), CD2Cl2 (red spectra), and C2D2Cl4 (blue spectra) solvents (15 mM, 250 μm path length). Additional IR spectrum of 110 in C2D2Cl4 in the presence of xenon (green spectrum in B).

Figure 122 :

 122 Figure 122: VCD spectra in the 1850 to 1050 cm -1 spectral range of (A) [CD(+)254]-109, (B) [CD(+)254]-110, and (C) [CD(+)254]-112 in CDCl3 (black spectra), CD2Cl2 (red spectra), and C2D2Cl4 (blue spectra) solvents (15 mM, 250 μm path length).

Figure 123 :

 123 Figure 123: comparison between the PP-cryptophane-A and PP-109.

Figure 124 :

 124 Figure 124: attributed absolute configuration of cryptophanes 110, 111 and 112.

Figure 125 :

 125 Figure 125: Comparison of experimental VCD spectrum of [CD(-)254]-109 in C2D2Cl4 solution (15 mM, 250 μm path length) and VCD spectra of PP-109 calculated at the B3LYP/6-31G** level (IEFPCM = C2H2Cl4) for the trans and gauche-gauche conformations of the ethylenedioxy and propylenedioxy linkers, respectively, and for the acetate group grafted on the propylenedioxy linker pointing toward (blue spectrum) or outward (red spectrum) the cavity.

  ) m/z [M + Na] + calcd for C58H60O14Na 1003.3880, found 1003.3875 Synthesis of cryptophane 112: Acetic anhydride (2.2 mL, 41 mmol)

  ),69.8 (2 C), 69.1 (2 C), 63.7 (2 C), 36.4 (2 C), 36.1 (4 C), 20.7 (4 C), 20.6 (2 C).HRMS (ESI-TOF) m/z [M + Na]+ calcd for C61H56O18Na 1099.3364, found 1099.3359

Figure 126 :

 126 Figure 126: chromatograms (Chiralpak IC, 250 x 4.6 mm, heptane/ethanol/CH2Cl2: 50/10/40, 1 mL/min at 25 °C) of the (rac)-109 before preparative separation on Chiralpak IC (250 x 10 mm, heptane/ethanol/CH2Cl2: 50/10/40, 5 mL/min).

Figure 127 :

 127 Figure 127: chromatograms (Chiralpak IC, 250 x 4.6 mm, heptane/ethanol/CH2Cl2: 50/10/40, 1 mL/min at 25 °C) of the collected [CD(-)254]-109 and [CD(+)254]-109 after preparative separation on Chiralpak IC (250 x 10 mm, heptane/ethanol/CH2Cl2: 50/10/40, 5 mL/min). Detection performed by UV-Vis spectroscopy at 230 nm (black chromatograms) and CD spectroscopy at 254 nm (red chromatograms).

Figure 128 :

 128 Figure 128: chromatograms ((S,S)-Whelk-O1, 250 x 4.6 mm, heptane/ethanol/CH2Cl2: 20/40/40, 1 mL/min at 25 °C) of the (rac)-110 before preparative separation on (S,S)-Whelk-O1 (250 x 10 mm, heptane/ethanol/CH2Cl2: 20/40/40, 5 mL/min).

Figure 133 :

 133 Figure 133: chromatograms ((S,S)-Whelk-O1, 250 x 4.6 mm, ethanol/CH2Cl2: 50/50, 1 mL/min at 25 °C) of the collected [CD(-)254]-112 and [CD(+)254]-112 after preparative separation on (S,S)-Whelk-O1 (250 x 10 mm, ethanol/CH2Cl2: 50/50, 5 mL/min). Detection performed by UV-Vis spectroscopy at 230 nm (black chromatograms) and CD spectroscopy at 254 nm (red chromatograms).

Figure 134

 134 Figure 134: 1 H NMR spectrum (300 MHz) of (rac)-109 recorded in CD2Cl2 at 25 °C. The letters A,B,C,D,E show protons signals related to the imploded form.

Figure 135

 135 Figure 135: 1 H NMR spectrum (300 MHz) of (rac)-109 recorded in CDCl3 at 25 °C. The letters A,B,C,D show protons signals related to the imploded form.

Figure 142

 142 Figure 142: 1 H NMR spectrum (300 MHz) of (rac)-111 recorded in different solvents at 25 °C.

6 Figure 143

 6143 Figure 143: 1 H NMR spectrum (300 MHz) of (rac)-29 recorded in different solvents at 25 °C.

Figure 144 :

 144 Figure 144: plot of the SOR (10 -1 deg cm 2 g -1 ) values versus l (nm) for the [CD(+)254]-109 enantiomer in CH2Cl2 (black), CHCl3 (orange), DMSO (blue) and C2H2Cl4 (red) at 25 °C.

Figure 145 :

 145 Figure 145: plot of the SOR (10 -1 deg cm 2 g -1 ) values versus l (nm) for the [CD(-)254]-110 enantiomer in CH2Cl2 (black), CHCl3 (orange), DMSO (blue) and C2H2Cl4 (red) at 25 °C.

  values measured for [CD(-)254]-111 in different solvents at 25 °C.

Figure 146 :Figure 147 :

 146147 Figure 146: plot of the SOR (10 -1 deg cm 2 g -1 ) values versus l (nm) for the [CD(+)254]-112 enantiomer.

Figure 148 :

 148 Figure 148: ECD spectra of [CD(+)254]-110 (black spectra) and [CD(-)254]-110 (red spectra) recorded in CH2Cl2, CHCl3, THF, CH3CN, DMSO and C2H2Cl4 at 20 °C. (c = 10 -4 -10 -5 M). UV-visible spectrum of 110 recorded in THF at 25 °C (c = 1.02 10 -5 M).

Figure 149 : 1 Figure 150 :

 1491150 Figure 149: 1 Lb transition of the ECD spectra of [CD(+)254]-111 (black spectra) and [CD(-)254]-111 (red spectra) recorded in CH2Cl2, CHCl3, THF, CH3CN, DMSO and C2H2Cl4 at 20 °C. (c = 10 -4 -10 -5 M).

Figure 151 :

 151 Figure 151: IR spectra of a) [CD(+)254]-109, b) [CD(+)254]-110 and [CD(+)254]-112 c) in CDCl3 (black spectra), CD2Cl2 (red spectra) and C2D2Cl4 (blue spectra) solvents (0.015 mM, 250 µm path length).

C

  Figure 152: VCD spectra of [CD(+)254]-109 (black spectra) and [CD(-)254]-109 (red spectra) in a) CDCl3, b) CD2Cl2, and c) C2D2Cl4 solvents (0.015 mM, 250 µm path length).

C

  Figure 153: VCD spectra of [CD(+)254]-110 (black spectra) and [CD(-)254]-110 (red spectra) in a) CDCl3, b) CD2Cl2, and c) C2D2Cl4 solvents (0.015 mM, 250 µm path length).

Figure 154 :Figure 155 :

 154155 Figure 154: VCD spectra of [CD(+)254]-112 (black spectra) and [CD(-)254]-112 (red spectra) in a) CD2Cl2 and b) C2D2Cl4 solvents (0.015 mM, 250 µm path length).

Figure 156 :

 156 Figure 156: population distribution of spins at equilibrium state.

  Another way to measure SOR values as a function of the wavelength is the Optical Rotation Dispersion (ORD) and it can be extended to the UV-visible spectral regions (on-resonance). Two different parts of the ORD spectrum can be discussed. In the off-resonant spectral region (the molecule does not absorb light), the ORD curves tend to show a monotonous behavior. In addition the SOR values tend to increase negatively or positively as we get close to an absorption wavelength. In some other cases a change of the sign of the SOR values can be sometimes observed. In the on-resonant spectral region (the molecule absorbs light), the ORD spectrum shows a dispersion band (bisignate). This effect is called the anomalous effect (AORD). This effect is translated by the appearance of maximal peaks and minimal troughs. A quantum theoretical description of ORD spectroscopy can be found in some reviews and articles, Figure158.209, 210

Figure 158 :

 158 Figure 158: plain and anomalous ORD curves.

Figure 160 :

 160 Figure 160: Differential absorption of left and right polarized light by the exciton coupled chromophores and the corresponding excitonic couplets (black curve) which is a summation of the positive (blue curve) and negative (red curve) Cotton effects.

  [START_REF] Canceill | Analytical Optical Resolution of Bromochlorofluoromethane by Enantioselective Inclusion into a Tailor-Made "Cryptophane" and Determination of Its Maximum Rotation[END_REF]

  [START_REF] Cherubini | Hyperpolarised Xenon in Biology[END_REF]

Table 2 :

 2 solubility of xenon in different media.

	Liquid	Ostwald Solubility
	Hexane	4.8
	Oil	1.9
	Fat Tissue	1.3
	DMSO	0.66
	Water	0.11
	Blood	0.14
	Plasma	0.1
	Saline	0.09

  arene derivative, an organic guest and xenon were also studied, showing a variation in the xenon chemical shift upon adding the organic guest like hexane.104 Better association properties were recorded with cucurbit[n]urils CB, which are composed of n glycoluril units and have a hydrophobic cavity accessible through two identical portals surrounded by polar carbonyl groups. Specifically, water-soluble CB*[6] derivative has a high xenon binding constant in water

of 3000 M -1 obtained by ITC. The appearance of two signals for the free (190 ppm) and encapsulated xenon

(97 ppm)

indicates a slow exchange between the bound and unbound species at the NMR time scale. It is worth mentioning that in CB xenon's in-out exchange rate can be modulated by adding cations that interact with the polar carbonyl groups, hindering by this the kinetics of exchange, Figure

  It seems that these substituents hinder xenon binding by preventing the cage to adopt a syn periplanar expanded conformation.114 Up to now, rim functionalization is only limited to oxygen derived substituents. However, adding new original functions or different heteroatoms like sulfur or nitrogen is quite complicated. Thus the trend predicted of binding constants is restricted to what have been synthesized.

	cryptophane-[111] cages.
	Ka= 420000 M -1	Ka= 33000 M -1	Ka= 17000 M -1

  of the encapsulated129 Xe increases linearly with a rate of 0.1 to 0.3 ppm/K. This change is a result of increase in both, the cryptophane's conformational flexibility and the accessibility of xenon to new cryptophane's regions. The temperature variations were successfully mapped by MRI, facilitating the detection of temperature changes up to 0.1 °C.

	To talk about the effect of cavity volume on xenon's chemical shift, a series of cryptophanes
	[222], [223], [233], [333] bearing identical substituents ((OMe)6 or (CH2CO2H)6), have been
	studied in 1,1,2,2-tetrachloethane-d2 or water. The study showed a subsequent decrease in the
	chemical shifts by almost 10 ppm from smallest to largest cages. Although this trend seems to be
	directly linked to cavity volume, in reality it is likely to be a consequence of many different
	parameters like cage's shielding effect and most importantly the conformation adopted by the
	linkers upon xenon encapsulation. 111
	In general, for cryptophanes-[222] type molecules, the chemical shift varies from 62-78 ppm in
	both aqueous and organic media. Contrary to the formerly mentioned trend, the smallest
	cryptophane-[111] has a chemical shift of 31 ppm in 1,1,2,2-tetrachloroethane-d2. In this cage,
	possible modulation of the chemical shifts in cryptophane-[111] is achieved by grafting electron
	donating or withdrawing groups of different sizes and orientations on the cage rims. For instance,
	Xe@tri-methoxy or Xe@tri-bromo functionalized cages show chemical shifts of 39 and 80 ppm,
	respectively. 114 Additionally, going from electron-poor hexa-boronic acid cryptophane to the
	electron-rich hexa-hydroxy cage by oxidation triggers a shielding in chemical shifts by almost 62
	ppm. 118 This fact is also impressively illustrated in water soluble cryptophane-[111] (RuCp*)6

116,117

Variations in solvent, temperature, pressure, pH or structure of the host induce a detectable change in 129 Xe chemical shift. Pines and co-workers have demonstrated the use of 129 Xe@cryptophane-[222] derivative for MRI thermometry. Upon temperature change, the chemical shift

111 222 223 233 333 or 220 In organic solvents In water 222 60

	tail was specifically prepared for this purpose. Upon biotin-avidin complexation, a change in the
	cage environment i.e. rotational and vibrational motions is excellently translated by a
	hyperpolarized 129 Xe chemical shift variation. In this example, an unambiguous assignment of
	free, caged and caged xenon recognizing the protein was done, showing a downfield shift by
	about 2.3 ppm upon avidin complexation, Figure 26. 119 Later on, a detailed description of the
	obtained signals was performed, showing the sensitivity of the caged xenon to different
	diastereomers present in solution. 116 Several versions of this biosensor bearing linkers of variable
	flexibility and length were synthesized, in an attempt to optimize its design to get better
	301
	responsiveness (structure a in figure 37). 120
	196	120	100	80	40	31	20	0
		129 Xe chemical shift (ppm)
	Combining the exceptional properties of cryptophanes (encapsulation and modulation of
	chemical structure) and xenon (magnetic properties and hyperpolarization) paves the way to
	develop a new class of ultrasensitive non-invasive molecular MRI tracers. The principle of these
	NMR biosensors relies on varying the resonance frequency of the encapsulated xenon, compared
	to the bulk, which can be exploited to monitor changes at the molecular level. This concept has
	been first demonstrated in 2001 by Pines and co-workers, where they described the first
	129 Xe@Cryptophane biosensor for avidin in-vitro detection. A cryptophane-[222] monoacid,
	functionalized with a water solubilizing linker consisting of amino-acid chain, and a biotinylated

.2. Berthault and Co-workers:

	specific cell-surface binding with minimal cell internalization of the targeted bacteriophage with
	Liquid flow
	cancerous cells. 129	Agarose bead sample with immobilized biosensor
	Spectrally resolved MR image of the sample 4.1Among the different contributions of this group in the field, we chose here to discuss only
	some major examples. Similar to Pines, Berthault group selected to work with cryptophane-[222]
	Splitting of the image between the monoacid cage and functionalized it with short DNA strand (structure d in figure 37). It is
		xenon in the bead and xenon in water.
	noteworthy that in this example an enantiopure cryptophane derivative was used for the design of
	the final molecule. Detection of the hybridization of this strand with its complementary sequence
	was made possible via simple (HP) 129 Xe signal and a moderate downfield shift by 0.5 ppm was
	observed at low concentration (10 µM). At higher concentration, the authors observed micelle
	formation, which in turn resulted in the appearance of multiple signals. Interestingly, introducing
	a non-complementary sequence did not change the initial chemical shift. This type of
	experiments is rather complicated by 1 H NMR especially at low concentrations. 130
		Xe NMR
	spectroscopy assisted with hyper-CEST experiments. Fluorescence imaging also assured the

  [START_REF] Berthault | Accurate PH Sensing via Hyperpolarized Xe NMR[END_REF]

  These two formerly presented cryptophane-[223] derivatives set up a new family of dually functionalizable platforms. These platforms can be immediately used for building different xenon-carriers for MRI applications. The synthesis of these compounds constitutes a very important result as it considerably simplifies the introduction of the sensing unit. It is noteworthy that this could not be achieved with the water-soluble cryptophane hexa-carboxylate. In addition, the presence of a carboxylic acid function at the cage's propylenedioxy linker is of special interest because it is widely used in biochemistry to elaborate sensors via amide coupling reactions. The cleavage of the six other ester groups can also afford very easily the corresponding water-soluble sensors.

Table 3 :

 3

	52
	44	54	55

different trials to hydrolyze compound 54 to the water soluble cryptophane 55.

1 H} NMR (100.6 MHz, CDCl3, 25 °C)

	1H), 6.77 (s, 1H), 6.73 (s, 1H), 6.69 (s, 1H), 6.67 (s, 1H), 6.58 (m, 1H), 5.40 (s, 1H), 5.37 (s, 1H),
	5.29 (m, 2H), 4.71 (s, 3H), 4.67-4.62 (m, 4H), 4.52 (m, 2H), 3.84 (s, 3H), 3.79 (s, 3H), 3.71 (s,
	3H), 3.65 (s, 3H), 3.52-3.46 (m, 3H), 1.54 (m, 1H).

). Evaporation of the fractions gave rise to compound 23 (2 g, 40%) as a glassy product. 1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 6.92 (s, 1H), 6.87 (s, 1H), 6.84 (s, 1H), 6.79 (s, 13 C { : δ (ppm) (100.6 MHz, CDCl3) 149.

1 H} NMR (100.6 MHz, CDCl3, 25 °C):

  [M+Na] + calcd. for C36H38NaO9 637.2413 found 637.2408.

	Compound 25: 2-((4-(2-bromoethoxy)-3-methoxybenzyl)-Cryptophane 26: CTB 25 (0.70 g, 0.62 mmol) was dissolved in a Cryptophane 27: Derivative 26 (0.28 g, 0.304 mmol) was
	oxy)tetrahydro-2H-pyran 24 (4.106 g, 11 mmol, 2 equiv.) was mixture of chloroform (300 ml) and formic acid (300 ml). The dissolved in a mixture of THF (20 mL) and CH2Cl2 (5 mL)
	added under an argon atmosphere to a stirring mixture of solution was then stirred for 6 hours at 55 -60 °C. The solvent was
	Cs2CO3 (7.69 g, 23 mmol, 2 equiv.) and CTB 23 (3.65 g, 5.94 then evaporated under reduced pressure to leave a yellow residue. It
	mmol, 1 equiv.) in DMF (140 mL). The reaction mixture was was then purified by column chromatography on silica gel
	heated to 80°C for 16 hours under an argon atmosphere. Then, (CH2Cl2/Acetone: gradient 97/3 up to 95/5). The white solid was
	the yellowish mixture was poured in water (200 mL) and ethyl acetate (200 mL). The aqueous then crystallized in a mixture of CH2Cl2 and ethanol. The crystals were collected on a fritted glass
	layer was extracted 5 times with ethyl acetate (500 mL). The combined organic layer was then and washed with diethyl ether. Compound 26 (0.19 g, 34%) was obtained as a white solid.
	washed 5 times with water (500 mL) and dried over Na2SO4. The solvent was removed under
	reduced pressure to leave a glassy solid, which was purified on silica gel (AcOEt/petroleum
	4.73-4.63 (m, 10H), 4.53 (m, 2H), 4.43-4.32 (m, 8H), 3.90 (m, 2H), 3.86 (2s, 6H), 3.74-3.71 (3s, 13 C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): δ (ppm) 149.5 (2C), 149.4 (2C), 147.7 (2C),
	9H), 3.61 (s, 3H), 3.48-3.53 (m, 5H), 1.85-1.59 (m, 12H). 147.3(2C), 146.6 (2C), 146.4 (2C), 140.0 (1C), 133.8 (4C), 132.2 (2C), 131.9 (2C), 131.3 (2C),
	δ (ppm) 149.8, 149.7 (2C), 148.6, 148.5, 148.3, 130.6 (2C), 120.7, 120.4 (2C), 120.3 (2C), 113.9 (2C), 113.3 (2C), 113.2 (2C), 112.2 (2C), 69.6
	147.6 (2C), 147.5, 146.9 (2C), 146.8, 147.5, 140.6, 134.5, 133.1 (2C), 132.5, 131.9 (2C), 131.8 (2C), 68.9 (2C), 68.8 (2C), 56.1 (2C), 55.5 (2C), 55.4 (2C), 36.4 (2C), 36.08 (2C), 36.04 (2C).
	(3C), 120.5 (3C), 119.2, 117.0, 116.9, 116.1, 115.1, 113.8-114.3 (8C), 112.1 (2C), 111.0, 97.7 HRMS (ESI): m/z [M+Na] + calcd. for C56H56NaO12 943.3669 found 943.3664.
	(3C), 70.1, 69.9, 68.6 (2C), 68.4, 67.93, 65.1, 62.2 (2C), 55.7-56.2 (6C), 36.3 (2C), 30.5 (2C),
	25.4 (2C), 19.4 (2C).
	HRMS (ESI): m/z [M+Na] + calcd. for C66H78NaO17 1165.5136 found 1165.5131.

ether: 90/10) to give rise to compound 25

(5.63 g, 84%)

as a white glassy solid. 1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 7.01-6.65 (15s, 15H), 5.30 (s, 1H), 5.29 (s, 1H), 13 C { 1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 6.77 (s, 2H), 6.72 (s, 2H), 6.65 (s, 2H), 6.655 (s, 2H),

6.64 (s, 2H), 6.62 (s, 2H), 5.58 (s, 2H), 4.63-4.56 (m, 6H), 4.46 (d, J=10.7 Hz, 2H), 4.35 (d, J=10.7 Hz, 2H), 4.29-4.09 (m, 8H), 3.80 (s, 6 H), 3.79 (s, 6H), 3.75 (s, 6H), 3.43-3.36 (m, 6H)

.

1 H NMR (400 MHz, CDCl3, 25 °C): δ

	6.79-6.61 (m, 10 H), 6.51 (s, 2H), 4.62-4.53 (m, 6H),
	4.25-3.93 (m, 14 H), 3.87-3.77 (4s, 12 H), 3.74 (s, 3H), 3.71 (s, 3H), 3.43-3.34 (m, 6H), 2.40 (m,
	1H).
	13 C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): δ (ppm) 149.6, 149.5 (2C), 149.4, 147.5, 147.4,
	147.3, 146.7, 146.6 (2C), 146.63, 146.3, 134 (2C), 133.8 (2C), 132.

1 H} NMR (100.6 MHz, CDCl3, 25 °C):

	Cryptophane 28: Excess of methanesulfonyl chloride (1.3 Cryptophane 29: A mixture of cryptophane 28 (0.15 g, Compound 30: Compound 27 (0.26 g, 0.20 mmol, 1 equiv.) was
	mL, 16.8 mmol) was added at 0 °C to a mixture of 0.150 mmol, 1 equiv.), potassium thioacetate (0.1 g, 0.85 introduced and dissolved in freshly distilled CH2Cl2 (26 ml).
	cryptophane 27 (0.5 g, 0.53 mmol), CH2Cl2 (25 mL) and mmol, 5 equiv.), DMF (2 mL) and CH2Cl2 (0.5 mL) was Excess of Dess martin periodonane (15% wt solution in CH2Cl2
	pyridine (2.3 mL, 28.5 mmol). The mixture was stirred for 16 stirred for 3 days at 100 °C before the addition of CH2Cl2 (20 2.6 ml) was then added. The reaction mixture was stirred under
	hours at 70 °C before the addition of methanol (25 mL) at mL) and water (20 mL) at room temperature. The aqueous argon for 16 hours at room temperature. The next day crude was
	room temperature. The solvents were evaporated and the layer was extracted by CH2Cl2 (3 × 20 mL). The combined filtered on a fritted glass and the filtrate was collected and
	resulting solid dissolved in CH2Cl2 (100 mL). The organic layer was washed twice with water (50 organic layers were washed by water (4 × 20 mL), NaCl (20 mL), dried over Na2SO4 and evaporated under reduced pressure. The resulting crude was crystalized from chloroform diethyl
	mL) and brine (50 mL), dried over Na2SO4 and evaporated. Silica gel chromatography evaporated. Silica gel chromatography (CH2Cl2/acetone: 94/6) and washing over a fritted glass ether to give beige precipitate that were collected on a fritted glass and washed with ether. The
	(CH2Cl2/acetone: gradient 95/5 up to 90/10) afforded pure cryptophane 28 (0.42 g, 82%) as a with diethyl ether afforded pure cryptophane 29 (0.065 g, 43%) as a white solid.
	white solid.
	6H), 4.56-4.48 (m, 2H), 4.34-4.21 (m, 2H), 3.92-3.22 (m, 10H), 3.88 -3.74 (6s, 18H), 3.49-3.33
	(m, 6H), 3.07 (s, 3H), 2.84-2.68 (m, 1H). 13 C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): δ (ppm) 195.7, 149.6 (2C), 149.5 (2C), 147.6,
	δ (ppm) 149.6 (2C), 149.5, 148.0, 147.8, 146.9, 147.1, 146.9, 146.8, 146.7 (2C), 134.2, 134.1, 134.0, 133.9, 132.6, 132.5, 148.0, 131.7 (2C),
	146.8 (2C), 146.7 (2C), 146.5 (2C), 134.1, 134.0, 133.9 (2C), 132.4 (3C), 132.2, 131.6 (2C), 131.6 (2C), 130.7 (2C), 120.5 (2C), 120.7, 120.4, 114.4 (2C), 113.8, 113.7, 112.6, 112.5, 112.4,
	130.9, 130.8, 120.5, 120.4 (2C), 120.3, 114.2 (2C), 113.8, 113.6, 113.3, 112.8, 112.6, 69.7, 69.1, 69.8 (2C), 69.5, 69.1, 69.0, 66.4, 65.4, δ 64.8, 56.4 (2C), 56.0, 55.8, 55.7, 55.6, 40.3, 36.6 (2C),
	69.0, 67.8, 64.9, 64.0, 56.4, 55.8 (2C), 55.7 (2C), 55.6 (2C), 53.6, 39.7, 37.1 (2C), 36.5 (2C), 36.2 (2C), 36.1 (2C), 30.7, 27.9.
	36.3, 36.2, 31.1. HRMS (ESI): m/z [M+Na] + calcd. for C58H60NaO13S 1019.3652 found 1019.3647
	HRMS (ESI): m/z [M+Na] + calcd. for C57H60 NaO15S 1039.3550 found 1039.3545
	(2C),
	36.1 (3C), 36.

HRMS (ESI

): m/z [M+Na] + calcd. for C56H58NaO13 961.3775 found 961.3770. 1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 6.83-6.62 (m, 10H), 6.56 (s, 2H), 4.76-4.70 (m, 13 C { 1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 6.85-6.62 (m, 10H), 6.54 (s, 2H), 4.

75 -4.50 (m, 6H), 4.37-4.20 (m, 3H), 4.19-3.91 (m, 8H), 3.88-3.73 (6s, 18H), 3.51-3.33 (m, 6H), 3.31-3.10 (m, 1H), 3.21-3.10 (m, 1H), 2.59 -2.44 (m, 1H), 2.37 (s, 3H)

.

produced product was used directly in the next step without further purification on silica because of its sensitivity.

HRMS (ESI, [M+Na] + ) calcd. for C56H56O13 959.37 found 959.35.

1 H} NMR (100.6 MHz, CDCl3, 25 °C):

	Compound 32: Potassium carbonate (0.07g, 0.506 mmol, Cryptophane 34: A solution of PPTS (0.12 g, 0.47 mmol)
	2.5 equiv.) was added along with compound 31 (0.19g, in CH2Cl2 (1 mL) was added to a solution of 27 (0.3 g, 0.31
	0.2 mmol, 1 equiv.) and dissolved in DMF (2.5 ml) for 10 mmol) and 3,4-dihydropyran (0.1 mL, 1.09 mmol) in THF
	minutes. After that benzyl bromide (0.046 ml, 0.38 mmol, (8 mL). The solution was heated up to 60 °C for 48 hours.
	2 equiv.) was added and the mixture was heated for 16 Then, the solvent was evaporated under reduced pressure.
	hours at 50 °C. After 16 hours the crude was washed five The oily residue was subjected to column chromatography
	times with ethyl acetate and the produced organic layer was washed 5 times with water. The on silica gel (CH2Cl2/acetone: 90/10) to give rise to compound 34 as a mixture of two
	resulting organic layer was dried over Na2SO4 and then filtered. The solvent was evaporated diastereomers in the form of white solid (0.23 g, 75%) was collected on a fritted glass and
	under reduced pressure then the crude was subjected to column chromatography using SiO2 washed with diethyl ether.
	(CH2Cl2/ acetone: 90/10) to give it as pure white product (0.1 g, 50%).
	5.27 (d, J=12.5 Hz, 1H), 5.2-5.16 (d, J=12.5 Hz, 1H), 4.62-4.55 (m, 6H), 4.4-3.9 (m, 12 H), 3.79-
	3.69 (6s, 18 H), 3.43 -3.35 (m, 6H), 3.31 ( m, 1H).
	δ (ppm) 170.6, 149.5, 149.49 (2C), 149.4, 148.0,
	147.6, 147.0, 146.8, 146.7 (2C), 146.6 (2C), 135.9, 134.0, 133.9 (2C), 133.4, 132.37, 132.3,
	132.0, 131.8, 131.4, 131.3, 130.87, 130.8, 128.5 (2C), 128.1, 127.8 (2C), 120.4 (3C), 120.2,
	114.1, 114.0, 113.94, 113.61, 113.16 (2C), 113.09, 112.49, 69.67, 69.61, 68.95 (2C), 66.61,
	, 120.1, 114.1 (3C, 113.6 (2C), 113.5 64.79, 64.25, 56.28 (2C), 56.07, 55.56 (2C), 55.5, 46.02, 36.42 (2C), 36.12 (2C), 36.03 (2C).
	(2C) 112.6, 69.6, 69.5, 68.8 (2C), 65.2, 64.8, 56.3 (2C), 55.8, 55.6, 55.5 (2C), 45.3, 36.4 (2C), HRMS (ESI): m/z [M+Na] + calcd. for C63H62NaO14 1065.4037 found 1065.4032.
	36.15 (4C).

HRMS (ESI, [M+Na] + ) calcd. for C56H56NaO14 975.3567 found 975.3562. 1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 7.36-7.29 (m, 5 H), 6.67-6.53 (12 s, 12H), 5.32-13 C { 1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 6.

76-6.55 (12s, 12H), 4.69 (m, 1H), 4.60-4.55 (m, 6H), 4.20-3.97 (13H), 3.88 (m, 2H), 3.78 (3s, 9H), 3.76-3.74 (3s, 9H), 3.59 (m, 1H), 3.38-3.35

(m, 6H), 2.60 (m, 1H), 1.

82-1.50 (m, 6H)

.

1 H} NMR (100.6 MHz, CDCl3, 25 °C)

									Cryptophane 35: (4 mL, 4.1 mmol, 20 equiv.) of lithium
									diphenylphosphide (1 M) was added to a solution of 34
									(0.21 g, 0.21 mmol) in THF (3 mL) under an argon
	MeO	O	O OMe	OMe MeO	O CH *	O	O	atmosphere. The red solution was heated up to 60 °C for 48 hours. Then, the mixture was poured in 20 mL of water and
	O		MeO	O	O	OMe
									then extracted 5 times with CH2Cl2. The aqueous solution
	was acidified at 0 °C with an ammonium chloride solution (10 mL, pH ~ 5). The resulting
									42-8.22 (6s, 3 H), 7.95-7.74 (6s, 3H), 6.65-
	6.48 (m, 12H), 4.66 (m, 1H), 4.47-4.39 (m, 6H), 4.13-3.87 (m, 13H), 3.72 (m, 1H), 3.34 (m, 2H),
	3.22-3.17 (m, 6H), 2.33 (m, 1H), 1.67-1.49 (m, 6H).

precipitate was collected on a fritted glass and washed with water. The dried solid was then washed several times with diethyl ether to give a beige solid. The solid was then purified on column chromatography (silica gel, acetone/DMF: 96/4) to give clean compound 35 as a mixture of two diastereomer (0.16 g, 84%). 1 H NMR (400 MHz, DMSO d6, 25 °C): δ (ppm) 8.

13 C { : δ (ppm) 146.

  [M+Na] + calcd. for C55H54O14Na for 961.3411 found 961.3405.

	Compound 36: Benzyl bromoacetate (0.88 mL, Compound 37: Methyl bromoacetate (0.2 mL, 2.28 Compound 38: PPTS (0.3 g, 1.3 mmol, 5 equiv.) in
	5.5 mmol, 12 equiv.) was added under an argon mmol, 24 equiv.) was added under an argon atmosphere methanol (2 mL) was added in one portion to
	atmosphere to a solution of compound 35 (0.44 to a mixture of cryptophane 35 (0.09 g, 0.095 mmol, 1 cryptophane 36 (0.47 g, 0.25 mmol) in CH2Cl2 (6
	g, 0.46 mmol, 1 equiv.) and Cs2CO3 (1.83 g, 5.5 equiv.) and Cs2CO3 (0.37 g, 1.13 mmol, 12 equiv.) in mL). The solution was heated to 40 °C for 48 hours.
	mmol, 12 equiv.) in DMF (8 mL). The mixture DMF (3 mL) and CH2Cl2 (1 mL). The mixture was heated Then, the solvents were evaporated under reduced
	was heated for 16 hours at 60 °C under argon. up to 50 °C for 16 hours under argon. Then, the mixture pressure and the residue was purified on silica gel
	The mixture was poured in water and then extracted 5 times with AcOEt. The combined organic was poured in water and extracted 5 times with AcOEt. The combined organic layers were then (CH2Cl2/acetone: 96/4). Compound 38 (0.4 g, 93%) was obtained as white glassy solid.
	layers were then washed 5 times with water, dried over Na2SO4. Filtration and evaporation of the
	148.4, 147.9, 147.6, 147.5 (2C), 147.4, 147.2 (4C), 146.0 (3C), 135.6 (2C), 135.5, 135.4 (3C),
	86-6.68 (10 H, 10s), 6.55-6.53 (2H, 2s), 4.68-
	15-7.01 (m, 30 H), 6.42-5.99 (12s, 12H), 5.00-4.48 (18 H, m), 4.44-3.75 (35 H, m), 3.35-3.34 (6H, m), 2.51 (1H, m), 1.83 (m, 6 H).
	4.8 (m, 12 H), 4.36-4.07 (m, 18 H), 3.88-3.49 (m, 13 H), 2.87-2.82 (m, 6H), 1.98 (m, 1H), 1.45
	(m, 6H).
	HRMS (ESI, [M+Na] + ) calcd. for C109H102NaO26 1849.6557 found 1849.6552.

solvent under reduced pressure gave an oily residue, which was subjected to column chromatography on silica gel (CHCl3/acetone: 95/5). Compound 36 was collected as viscous oil (0.43 g, 54%) as a mixture of two diastereomers. This compound was used without further purification for the next step. 1 H NMR (300 MHz, CDCl3, 25 °C): δ (ppm) 7.

washed 5 times with water and dried over Na2SO4. Evaporation of the solvent gave rise to an oily residue, which was subjected to column chromatography on silica gel (CHCl3/acetone: gradient 90/10 to 85/15). Compound 37 was isolated as a white solid as a mixture of two diastereomers (0.090 g, 70%) and used without further purification for the next step. 1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 6.HRMS (ESI): m/z [M+Na] + calcd. for C73H78NaO26 1393.4679 found 1393.4673. 1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 7.

42-7.30 (m, 30H), 6.58-6.39 (12s, 12H), 5.31- 5.00 (m, 12H), 4.60-4.36 (m, 18 H), 4.18-3.83 (m, 14H), 3.26-3.05 (m, 6 H)

, 3.02 (m, 1 H).

13 C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): δ (ppm) 169.

5, 169.3, 169.2 (2C), 169, 168.8,

  [M+Na] + calcd. for C68H70NaO25 1309.4103 found 1309.4098.Then, the solution was filtered on a fritted glass. The filtrate solution was collected and the solvent was removed under reduced pressure to leave a solid residue. Dissolution in a minimum of CH2Cl2 and precipitation with diethyl ether gave rise to compound 41 as white solid. This compound decomposes on silica gel. Thus, 41 was used without further purification for the next step. An HRMS spectrum allows us to ascertain the structure of 41.Then, the solution was filtered on a fritted glass. The filtrate solution was collected and the solvent was removed under reduced pressure to leave a solid residue. Dissolution in a minimum of CH2Cl2 and precipitation with diethyl ether gives rise to compound 42 as white solid. This compound also decomposes on silica gel. Thus, compound 42 was used without further purification for the next step. An HRMS spectrum allows us to ascertain the structure of 42.

	Compound 41: Excess of Dess martin periodinate Compound 42: Excess of Dess martin periodinate (15% wt Compound 43: Sodium chlorite (0.01 g in 0.1 ml
	reagent (15% wt solution in CH2Cl2, 0.9 mL; 0.37 mmol) solution in CH2Cl2) reagent (0.4 mL; 0.16 mmol) was added water, 0.16 mmol, 3 equiv.) was added along with
	was added at room temperature to a solution of 38 (0.1 g, at room temperature to a solution of 39 (0.15 g, 0.11 mmol) sodium monobasic phosphate (99.45 µg in 0.01 ml
	0.057 mmol) in freshly distilled CH2Cl2 (13 mL). The in freshly distilled CH2Cl2 (4 mL). The solution was stirred water, 0.0083 mmol) and H2O2 (1.59 µl) to a solution of
	solution was stirred at room temperature for 16 hours. at room temperature for 16 hours under an argon atmosphere. compound 41 (0.1 g, 0.06 mmol, 1 equiv.), CH3CN (1.5
	mL), H2O (0.15 mL) under an argon atmosphere at
	room temperature. The, solution was stirred for 16 hours at room temperature and the solvent was
	removed under reduced pressure. The resulting residue was subjected to column chromatography
	on silica gel (CH2Cl2/acetone: 75/25). After evaporation of the solvents, compound 43 (0.07 g, 70
	%

HRMS (ESI): m/z

[M+Na] + calcd. for C104H92NaO25 1763.5825 found 1763.5819.

HRMS (ESI): m/z

[M+Na] + calcd. for C68H68NaO25 1307.3947 found 1307.3942.

R Bridges Retention time (min)

	D3	OMe	O(CH2)2O	22.1(+)	30.0(-)
	D3	OMe	O(CH2)3O	27.4(+)	37.6(-)
	D3	OMe	O(CH2)4O	30.3(+)	75.3(-)
	D3	OMe	O(CH2)6O	38.9(+)	54.9(-)

Table 4 :

 4 analysis of D3-cryptophanes by chiral pack-OT (+), the (+) and (-) signs correspond to the SOR sign recorded at 589 nm.

  This interesting finding triggered us to see first the effect of the self-encapsulation on the cryptophane's physical properties in solution by 1 H NMR. Since this cage is chiral, we wanted also to see the effect of this solvent-dependent conformation on the overall chiroptical properties. Investigating other cryptophane-[223] cages with similar structure, by 1 H NMR spectroscopy, like cryptophane 110 bearing six methoxy groups and one central secondary acetate, compound 111 bearing a primary acetate or compound 29 bearing a thioacetate moiety, revealed that these molecules are also capable of performing self-encapsulation, Figure116. It is worth mentioning that the properties of compound 29 has been only studied by 1 H NMR but other compounds were subjected to a full investigation. Having these molecules in hand, we were able then to separate their enantiomers and compare their chiroptical properties

Table 5 :

 5 Table 5 shows the chemical shifts of the central acetate or thio-acetate groups in different solvents. chemical shift variation (ppm; ref: TMS) of the methyl function of the central acetate group in different solvents.

	Solvent	CD2Cl2	CDCl3	DMSO-d6,	C2D2Cl4	Benzene-d6
	109 δ(ppm)	2.08 (s)	2 (v.b)	0.18 (b)	-0.506 (b)	-
	110 δ(ppm)	2.23 (s)	2.16	1.9 (b)	-1.74 (b)	-1.55
	111 δ(ppm)	2.08 (s)	2.07 (s)	2.07 (b)	-1.35	-1.6
	29 δ(ppm)	2.35 (s)	2.34	2.4	-1.1	-1.3

1 H NMR (400 MHz, CDCl3, 25 °C):

	6.94 (s, 2 H), 6.91(2 H), 6.89 (2 H), 6.87 (2 H), 6.86(2 H),
	6.62

  , 2.31 (s, 6 H), 2.29 (s, 6 H), 2.25 (s, 6 H), 2.09 (m, 2 H).

  Specific optical rotation values measured for [CD(+)254]-109 in different solvents at 25 °C.

						.8
	DMSO	0.26	+7.2	+7.0	+8.0 +19.9 +61.4
	C2H2Cl4	0.26	+8.1	+9.1 +12.0 +23.4 +66.3
	Solvent	Conc. []589 []577 []546 []435 []365
	CH2Cl2	0.21 +58.4 +62.0 +70.3 +121.2 +188.2
	CHCl3	0.25 +23.9 +24.9 +28.4 +46.9 +57.3
	DMSO	0.26	-7.6	-8.9	-9.7	-23.4 -66.1
	C2H2Cl4	0.29 -10.4	-9.8	-13.6 -26.2 -64.4

Specific optical rotation values measured for [CD(-)254]-109 in different solvents at 25 °C.

  Specific optical rotation values measured for [CD(+)254]-111 in different solvents at 25 °C.

	Solvent		Conc. []589 []577 []546 []435 []365
	CH2Cl2		0.28 +150.0 +158.4 +183.8 +343.8 +656.2
	CHCl3	2 g -1 )	0.27 +214.4 +202.3 +235.2 +431.4 +804.1
	DMSO		0.25 +215.1 +226.26 +262.7 +480.8 +887.7
	C2H2Cl4		CD (-) 254 -110 0.27 +207.1 +213.6 +251.6 +466.8 +872.7
	Solvent		Conc. []589 []577 []546 []435 []365
	CH2Cl2		0.

Avidin

CDCl 3

Figure 81: 13 C NMR spectrum of compound 35 in DMSO-d6 in 400 MHz spectrometer.

Figure 82: 1 H NMR spectrum in DMSO-d6 of compound 48.

Figure 136: 1 H NMR spectrum (300 MHz) of (rac)-109 recorded in DMSO-d6 at 25 °C.

Figure 137: 1 H NMR spectrum (300 MHz) of (rac)-109 recorded in 1,1,2,2-tetrachloroethane-d2 at 25 °C.

Figure 138: 1 H NMR spectrum (300 MHz) of (rac)-110 recorded in CD2Cl2 at 25 °C.

Figure 139: 1 H NMR spectrum (300 MHz) of (rac)-110 recorded in CDCl3 at 25 °C.

Figure 140: 1 H NMR spectrum (300 MHz) of (rac)-110 recorded in 1,1,2,2-tetrachloroethane-d2 at 25 °C.

Figure 141: 1 H NMR spectrum (300 MHz) of (rac)-110 recorded in benzene-d6 at 25 °C.

Specific optical rotation values measured for [CD(-)254]-112 in different solvents at 25 °C.

Compound 44: Sodium chlorite (0.035 g in 0.35 mL water, 0.38 mmol, 2 equiv.) was added along with sodium monobasic phosphate (0.01g in 0.017 mL water, 0.07 mmol) and H2O2 (0.53 mL) to a solution of compound 42 (0.25 g, 0.19 mmol, 1 equiv.), CH3CN (5.3 mL), H2O (0.15 mL) under an argon atmosphere at room temperature. The, solution was stirred for 16 hours at room temperature and then quenched with sodium thiosulfate (2 mL) at 0°C. The solvent was then removed under reduced pressure. Then, the crude was recrystallized from CH2Cl2 and diethyl ether to give a white precipitate. Filtration on a frit allows us to collected compound 44 (0.19 g, 78%) as a white solid. This solid was washed several times with diethyl ether and dried in air.

1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 6. 81-6.60 (12s, 12 H), 4.66-4.42 (m, 18 H), 4.38-4.13 (m, 12 H), 3.83-3.76 (6s, 18 H), 3.39-3.36 (m, 6 H), 3.24 (m, 1H). [START_REF] Moran | Synthetic Molecular Vessels[END_REF] C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): δ (ppm) 172. 6, 170, 169.9 (4C), 169.8, 147.8, 147.7, 147.7 (2C), 147.6, 147.5 (2C), 147.4, 147.3 (2C), 146.4, 146.2, 134.5 (2C), 133.7, 133.6, 133.5, 133.4 (4C), 133.3, 133.2, 132.9, 132.3, 121.0 (2C), 120.1 (2C), 119.7, 119.3 (2C), 118.2 (2C), 117.4, 115.4, 115.0, 69.5 (2C), 68.8 (2C), 67.7 (2C), 67.6 (2C), 67.2, 67.0, 65.6, 65.2, 52.0-52.2 (6C), 45.9, 36.3 (3C), 35.9 (3C).

HRMS (ESI): m/z

[M+H] + calcd. for C68H69O26 1301.4077 found 1301.4072.

Compound 55: Compound 54 (0.03 g, 0.0184 mmol, 1 equiv.) was dissolved in THF (1.18 ml) and then (0.3 M) KOH solution (1.1 ml, 18 equiv.) was added drop wisely at 0 °C.

The reaction was stirred for 30 minutes at room temperature. The solvent was evaporated under reduced pressure and then the crude was dissolved in a minimum amount of water. The obtained aqueous layer was washed twice with CH2Cl2. The resulting aqueous layer was acidified at 0°C with concentrated HCl solution. The obtained precipitate was collected on a fritted glass and then washed with ether to give compound 55 (0.02 g, ~74%) as a white compound. The compound showed high sensitivity to light and heat and it structure is confirmed by HRMS.

HRMS (ESI): m/z

[M+Na] + calcd. for C74H68N2NaO32 1519.3652 found 1519.3647.

Compound 56: To a solution of 43 (0.07 g, 0.039 mmol, 1equiv.) dissolved in DMF (1.5 ml) was added EDCI.HCl (0.01 g, 0.055 mmol, 1.4 equiv.), HOBt (0.015 g, 0.11 mmol, 0.28 equiv.) and DIPEA (0.11 mmol, 0.02 ml, 3 equiv.) and stirred for 10 minutes.

Then Biotin peg 2-amine (0.014 g, 0.039 mmol, 1 equiv.) dissolved in DMF (0.5 ml) was added and the mixture is stirred for 16 hours. The reaction mixture was extracted 5 times with CH2Cl2 and the resulting organic layer was washed 5 times with brine. The solvent was removed under reduced pressure and the resulting crude was subjected to column chromatography on silica gel (CH2Cl2/MeOH: 95/5). Compound 56 (0.06 g, 71%) was obtained as white solid.

1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 7. 3-7.2 (m, 30H), 6.6-6.3 (12s, 12H), 5.8 (bd s, 1H), 5.3-5 (m, 12 H), 4.5-4.3 (m, 19H), 4.2-3.7 (m, 13 H), 3.47-3.4 (m, 10H), 3.1-3.3 (m, 7H), 3.01 (m, 1H), 2.9 (m, 1H), 2.8 (m, 1H), 2.6 (m, 1H), 2.1 (m, 2H), 1.6 (m, 4H), 1.44 (m, 2H).

13 C { 1 H} NMR (100.6 MHz, CDCl3, 25 °C): δ (ppm) 173. 2, 171.1, 169.6, 169.7 (5C), 163.4, 148, 147.6, 147.4 (3C), 147.2 (4C), 147, 146.4, 135.3 (6C), 134.4, 134.1, 133.5, 133.4 (21C), 133.2 (2C), 133 (2C), 133.7, 132.6, 131.8, 128.6 (30C), 120.8, 120.5, 120.2, 119.7, 119.3, 118.9, 118.6, 118.2, 117.9, 117.5, 115.5, 114.7, 70.1, 69.9, 69.7 (2C), 69.5, 69, 68.6, 68.4, 67.9, 67.8, 67.7, 67.4, 67.2 (4C), 66.8 (3C), 667.7 (3C), 66.3, 65.56, 61.5, 60, 55.2, 47.6, 40.3, 39, 35.9 (2C), 35.7(2C), 35.63 (2C), 30.2, 27.8 (2C), 25.3.

HRMS (ESI): m/z

[M+2Na] 2+ calcd. for C120H120Na2N4O29S 1079.3777 found 1079.3772.

Experimental Details: Polarimetric, UV-Vis, and ECD measurements

Optical rotations of the two enantiomers of 109, 110, 111 and112 were measured in four solvents (CHCl3, CH2Cl2, DMSO, andC2H2Cl4) at 589, 577, 546, 435, and365 Spectra were recorded between 210 and 350 nm (THF, CH3CN), 230 and 350 nm (CH2Cl2 and CHCl3), and 250 and 350 nm (DMSO, C2H2Cl4) with a 0.5 nm increment and a 1 second integration time. Spectra were processed with standard spectrometer software, baseline corrected, and slightly smoothed by using a third-order least square polynomial fit. UV-Vis spectra of compounds 109, 110, 111 and 112 were recorded in THF (210-350 nm) at 25°C with a 1 cm path length quartz cell.

IR and VCD measurements

The IR and VCD spectra were recorded on a FTIR spectrometer equipped with a VCD optical bench. IR absorption and VCD spectra were recorded at a resolution of 4 cm -1 , by coadding 50 Analytical separation for compound 112 The key factor affecting the sensitivity of the NMR signals is the polarization of the nuclei between the two states. Higher polarization results in a higher longitudinal magnetization M0, enhancing thus the sensitivity. Back to (eq.4) enhancing the polarization can be reached by increasing the magnetic field (high costs) or by decreasing sample's temperature (not suitable for biological samples). A last way to improve the sensitivity is to play by the nuclear polarization itself, by disturbing the thermal Boltzmann equilibrium of nuclei between the different states into a non-equilibrated polarization or the so-called hyperpolarization which will be detailed throughout the introductory chapter of the first part.

Optical Activity of Chiral Molecules and the Spectroscopic Tools

The optical activity of chiral molecules originates from their differential interaction with contra-rotational components of the plane polarized light i.e. left and right circularly components.

In other word, chiral molecules tend to affect the respective velocities and intensities of left and right polarized lights. These effects are the origin of what is known as the optical rotation and circular dichroism.

2.1.Optical Rotatory Dispersion ORD and Polarimetry:

When passed through a chiral sample, the two compartments of circularly polarized light are transmitted at different velocities. This will lead to a rotation of their resultant plane polarized light by an angle noted α. The difference in left and right circularly polarized light velocities originates from the difference in the refractive indices of these two components in a chiral sample.

This phenomenon is the origin of optical rotation.

α =

𝟏𝟖𝟎𝒍 (𝒏𝑳-𝒏𝑹)

𝝀

Optical rotation angle α is expressed in the above equation where l is path length, n is the refractive index and λ is the wavelength. This equation shows that α is proportional to the difference in the refractive indices and it is inversely proportional to the wavelength.

This angle can be measured by a polarimeter at a well-defined wavelength. The sodium D-line transition at 589 nm is the most common used. To characterize each sample a correction with respect to the concentration is needed. Thus, the Specific Optical Rotation (SOR) l (nm) The presence of more than one chromophore in cryptophane molecules, in close vicinity to each other and with proper orientations results in the interaction between them. This is called the excitonic coupling. This phenomenon is defined as the delocalization of the electronic excitation along the chromophores array, rather being confined on one chromophore. The process generates coupling interactions of the initial excited states to form new excitonic states, which are slightly shifted in frequency with respect to the initial excited state. Absorption of light in the ECD spectrum results in a bisignate band (positive-negative or negative-positive). For two chromophores interacting with each other, the sign of these ECD bands can be easily interpreted by the excitonic coupling method developed by Nakanishi and co-workers. 212 It is noteworthy that in the case of cryptophane derivatives where six chromophore are involved a direct interpretation of their excitonic coupling is more complicated and can only understood by using quantum calculations, Figure 160.

List of Main Compounds

Synthesized and Their

Corresponding Numbers