Dr Christophe Dugave

Dr Bernard

Dr Patrick Berthault

Céline Boutin

David Buisson

Céline Chollet

Dr Damien Clarisse

Dr Isabelle George

Dr Alaric Desmarchelier

Dr Marielle Tamigney Kenfack

Emilie Nehlig

Olivia Carvalho

Dr Je Remercie

Fréderic Taran

Dr Davide Audisio

Dr Karen Hinsinger

Dr Valérie Pons

Dr Hajer

Remerciement

Je voudrais remercier ici toutes ces personnes qui m'ont beaucoup aidé dans ces trois ans de thèse. Avec vous, j'ai passé non seulement une très belle aventure en chimie, mais aussi des moments sympathiques et mémorables.

Je tiens tout d'abord à exprimer mes sincères remerciements au Dr. Thierry Brotin, au Dr.

Jean-Claude Chambron, au Pr. Cyrille Kouklovski et au Pr. Olivia Reinaud, membres du jury qui ont accepté de juger mon travail de thèse et avec lesquels j'ai eu des échanges très agréables et intéressants.

J'adresse mes remerciements les plus chaleureux au

Résumé en Français

L'imagerie médicale, qui a pris un essor considérable ce dernier siècle, se tourne petit à petit vers l'imagerie moléculaire. La détection plus fiable de nombreuses cibles permettrait une meilleure compréhension et un diagnostic plus précoce de pathologies. Les cryptophanes, dont la structure générale est présentée ci-dessous, sont des molécules cages constituées de deux unités de type cyclotribenzylène reliées entre elles par trois chaînes pontantes (Figure I). Ils ont été synthétisés pour la première fois par l'équipe d'A. Collet au Collège de France au début des années 1981. 2 En 2001, l'équipe d'A. Pines à Berkeley a mis au point la première biosonde en RMN 129 Xe en détectant la complexe biotine-avidine à partir d'un cryptophane décoré par une molécule de biotine. 3 Depuis, de nombreux scientifiques se sont intéressés à la synthèse de nouvelles biosondes à base de cryptophanes pour réaliser leur application in vivo. Pour ce but, nous avons étudié de nouvelles voies chimiques menant à des bioconjugués à base de cryptophane avec une bonne efficacité ainsi que des quantités satisfaisantes.

Figure I: structure générale des cryptophanes

Dans ce contexte, l'objective de cette thèse est designer des nouveaux cryptophanes qui peuvent être utilisé comme une plateforme moléculaire pour construire des biosondes en IRM de 129 Xe utilisable dans l'imagerie in vivo. Pour répondre à cette demande, deux grandes difficultés doivent être résolues. Tout d'abord, les biosondes synthétisés sont peu solubles dans l'eau en raison de la forte hydrophobicité du cryptophane. En milieu aqueux, les cryptophanes sont enclins à former des systèmes auto-organisés qui tendent à favoriser une interaction non spécifique avec les membranes lipidiques et l'incorporation de la sonde à l'intérieur de la membrane cellulaire.

Pour éviter ce problème préjudiciable à l'application de ces biosondes in vivo, le groupe polyéthylèneglycol (PEG) est utilisé pour développer une synthèse efficace de cryptophanes hydrosolubles utilisés potentiellement comme plateformes universelles pour la construction des biosondes de 129 Xe IRM.

Deuxièmement, la haute symétrie des cryptophanes s'est avérée être un inconvénient important pour contrôler la réactivité d'une position particulière par rapport à d'autres sites équivalents. Des mélanges statistiques sont rapportés dans tous les types de voies synthétiques existantes utilisées pour désymétriser la molécule, compliquant les étapes de purification et gênant la production de grandes quantités de cryptophanes nécessaires à la synthèse de biocapteurs. Pour cette raison, il y a une discussion systématique sur la façon de casser la symétrie des cryptophanes et différentes tentatives ont été faites pour synthétiser les cryptophanes mono-fonctionnalisés.

La discussion de stratégie est basée sur la « méthode de template », qui est une méthode largement utilisé dans la synthèse des cryptophanes. Dans cette voie synthétique, 5 stratégies potentielles sont proposé pour casser la symétrie des cryptophanes comme montré ci-dessous (2) Fonctionnalisation des CTVs avec des dérivés d'alcool benzylique différents.

(3) Mono-halogénation des CTVs.

(4) Synthèse des cryptophanes basée sur des CTVs mono-protégé.

(5) Cyclotrimérisation avec des dérivés d'alcool benzylique différents pour former des CTVs asymétriques.

Dans cette thèse, nous avons concentré nos efforts sur les stratégies différentes pour réaliser la synthèse des cryptophanes PEGylés mono-fonctionnalisés. Suivi ces stratégies, beaucoup de cryptophanes et CTVs ont été synthétisés, montrant des applications potentielles très intéressantes dans la construction des biosondes de IRM du xénon in vivo. The imaging techniques are getting more and more attention thanks to their capacity to realize a fine detection and real-time monitoring for the therapy of many diseases. With a reliable detection of numerous targets, which permits a better comprehension and an earlier diagnosis of pathology, the anatomical and functional medical imaging has turned little by little to molecular imaging in the last decade.

Among other diagnostic techniques such as bioluminescence, single photon emission computerized tomography (SPECT) or positron emission tomography (PET), magnetic resonance imaging (MRI) offers several advantages owing to its low invasiveness, its harmlessness and its spatial in-depth resolution (Figure 1). In USA, more than 25 % of diagnostic imaging is achieved by MRI, which is about 30 million MRI scans performed each year. However, MRI suffers from poor sensitivity. To address this issue, different strategies were proposed. The contrast agents like gadolinium complexes or super-paramagnetic iron oxide nanoparticles are often engaged to improve the quality of imaging and to facilitate the diagnosis. However, despite their use, 1 H MRI still quickly reaches its limit in sensitivity. As a result, the utilization of hyperpolarizable species such as parahydrogen ( 1 H), helium ( 3 He), carbon ( 13 C) or xenon ( 129 Xe) shows a fascinating future by allowing a much better sensitivity.

Despite considerable advantages of hyperpolarized 1 H, 3 He and 13 C which can be inserted into organic or biological molecules without alteration of their structures, 129 Xe is undoubtedly the first choice for MRI of soft tissues which are difficult to image in vivo.

Besides its well-known application in anesthesia, it is an inert gas with a polarizable electronic cloud which leads to an extreme sensitivity to its chemical environment. Moreover, its capacity of being hyperpolarized makes it possible to obtain a significant gain of sensitivity. Nevertheless, xenon has no specificity to any biological target and freely diffuses through biological barriers including the blood-brain barrier. Therefore it needs to be encapsulated and vectorized to reach biomarkers for molecular imaging. Different molecular cages were proposed to encapsulate xenon with a chemical function for covalent conjugation to a molecular vector for specific imaging.

Cryptophanes (Figure 2) are molecular cages composed of two cyclotriveratrylenes moieties connected by alkylenedioxy linkers. They were originally synthesized by the team of A. Collet in Collège de France in 1981. 2 In 2001, Pines and coworkers in Berkeley have developed the first 129 Xe MRI biosensor in detecting biotin-avidin complex based on a functionalized cryptophane. 3 Since then, many scientists have focused on this field in particular to realize the application of these cryptophane-based biosensors in vivo. For this purpose, we investigated new chemical routes leading to cryptophane-based bioconjugates with good efficiency as well as satisfactory quantities. In this context, the objective of this thesis is to design new cryptophanes which can be used as molecular platforms to construct novel 129 Xe MRI biosensors usable for in vivo MRI imaging. To meet this demand, two main difficulties should be resolved.

First of all, the synthesized biosensors are poorly water-soluble due to the high hydrophobicity of the cryptophane poly-aromatic core. In aqueous milieu, cryptophanes are prone to form self-organized systems which tend to promote a non-specific interaction with the lipid bilayers and the subsequent embedding of the probe inside the cell membrane. To avoid this problem which is detrimental for the application of these biosensors in vivo, the polyethylene glycol (PEG) group is used to develop an efficient synthesis of water-soluble cryptophanes working potentially as universal platforms for 129 Xe MRI biosensors.

Secondly, the high symmetry of cryptophanes has been proven to be an important drawback to control the reactivity of a particular position versus other equivalent sites.

Complex statistical mixtures are reported in all kinds of existing synthetic pathways used to desymmetrize the molecule, complicating the purification steps and jeopardizing the production of large quantities of cryptophanes required for the synthesis of biosensors. For this reason, there is a systematic discussion of how to break the symmetry of cryptophanes and different attempts were made to synthesize mono-functionalized cryptophanes.

In this thesis, we focused our efforts on the design of different strategies to achieve the synthesis of water-soluble mono-functionalized cryptophanes.

This PhD work was achieved in the Tritium Laboratory of SCBM in CEA Saclay in collaboration with several other research teams: P. Berthault's group for 129 Xe NMR experiments and the group headed by F. Fenaille for mass spectrometry analysis.

I. The Hyperpolarized 129 Xe MRI and Its

Encapsulating Cages

Medical imaging is a set of non-invasive methods for visualizing biological processes inside the organisms. It's essential to the comprehension of the pathology, in order to achieve a better diagnosis, prognoses as well as to provide an adapted and efficacious treatment. After more than a hundred-year development since the discovery of X rays, the expectation of medical imaging is no longer the anatomic imaging and functional imaging. The new challenge in this field is now to realize a fine molecular imaging with validated biomarkers. In this thesis, we are especially interested in the application of hyperpolarized 129 Xe MRI to routine magnetic resonance imaging diagnosis which is largely used in hospitals.

General presentation of MRI

The MRI is an imaging technique based on the principles of nuclear magnetic resonance (NMR) which studies the response of nuclear magnetic moment in magnetic field. As the NMR reveals the magnetic properties of the detected atoms into the samples, the application of a specific sequence of magnetic field gradient and adapted signal treatment permit an access to in vivo 2D or 3D image of tissues and organs inside of body. It enables to study the soft tissues like lung, brain, and spinal cord and to provide information about the anatomic structure as well as the function of organs.

a. Principles of NMR

In the absence of magnetic field, the magnetic moments of proton spins do not have a particular orientation. Under the effect of a magnetic field B 0 , they will begin to turn around the direction of B 0 at an angular velocity ω 0 = γB 0 , which γ is the gyromagnetic ratio.

Therefore a longitudinal component of the magnetization M z is created on the Z axis. When the magnetic moments of proton spins are submitted to an oscillating electromagnetic wave at the resonance frequency of protons, an incline of the magnetization M to B 0 (in the XY plane) occurs in a perpendicular way, which is the transversal magnetization M xy (Figure 3). The signal of free induction decay (FID) is recorded. And after a Fourier transform, a signal depending on the resonance frequency is thus obtained as the final NMR spectrum.

b. Principles of MRI

To obtain an image by magnetic resonance spectroscopy, the principle is to apply a magnetic field gradient in the two-dimensional or three-dimensional space so that the frequency of resonance of nuclei changes from one point of the object to another for the same nucleus. With a frequency-fixed wave, only one region will resonate and provide a signal. By shifting the detected frequency, a different region will enter in resonance and will permit to techniques can be applied at the same time to complement each other, [START_REF] Pirot | [END_REF] it is important to understand the advantages and drawbacks of each method to realize the earliest diagnosis of diseases as well as the best feedback of treatment for personalized medicine.

Optical imaging has unparalleled sensitivity, but it is not capable to go deep into the tissues.

The positron emission tomography (PET) allows to probe all deep inside the human body, but do not possess good spatial and temporal resolution. [START_REF] Meikle | Drug Discovery Today : Technologies[END_REF] The application of computed tomography (CT) is often limited to bones and solid tumors. In addition, the utilization of these two techniques should be moderated due to the significant doses of ionizing radiations delivered during the examination which could be harmful for the patient. Ultrasound is an inexpensive technique but with a poor sensitivity. 7 The most relevant characteristics of these current imaging methods are compared in the following table (

How to improve the sensibility of MRI

The poor sensitivity of MRI results from the intrinsic poor sensitivity of NMR detection.

The signal intensity is proportional to the polarization which depends on the difference of populations of spins between the two energy states (Equation 1).

Equation 1

For a particle with spin I = 1/2, N + and N -represent the number of spins in the two states, B 0 is the applied magnetic field, γ is the gyromagnetic ratio , k B is the Boltzmann constant and T is the absolute temperature.

At the thermodynamic equilibrium, there is no large difference in the population of spins on the two energy levels, therefore causing a low P nuclear polarization. Different strategies can be used to resolve this problem in particular the use of contrast agents or hyperpolarized species.

a. The MRI contrast agents

Currently, the contrast agents are delivered to the patients to improve the signal/noise ratio of MRI. They aim to accelerate the magnetic relaxation rate 1/T1 or 1/T2 of the protons in water. This leads to the shortening of the time necessary for a protons spin to return to its original state after the excitation by the radiofrequency wave. It should be noticed that the contrast agents are not visualized in MRI but that they influence the water molecule protons located around the contrast agents.

With 7 lone pair electrons in its 4f orbit, the lanthanide ion Gd 3+ is the most widely-used paramagnetic ion as a contrast agent in MRI. This kind of paramagnetic metallic ions with a large number of free unpaired electrons (e.g. Gd 3+ , Mn 2+ ), which are also called positive contrast agent, have mainly the effect of reducing the longitudinal relaxation time T1, resulting an increase of the intensity of the MRI signal. However, due to its strong toxicity as a competitor with calcium in all the calcium-dependent processes inside the human body, 8 gadolinium cannot be directly administered and needs to be chelated in order to limit adverse effects. Nowadays there are eight kinds of gadolinium-based contrast agents clinically approved (Figure 6). Unlike the gadolinium-based compounds, another category of contrast agents reduces the transversal relaxation time T2 of protons in water and thus generates a negative contrast by decreasing the intensity of the MRI signal. This kind of contrast agent, also known as negative contrast agent, is basically the super-paramagnetic materials such as superparamagnetic iron oxide. 10 It is called "super-paramagnetic" because of the presence of individual magnetic multi-domains within the iron oxide crystals. When a magnetic field is applied, all these micro-domains are oriented parallel to the field creating a very high magnetization within the particle (Figure 7). This magnetization will increase the relaxation of the protons in water by an order of magnitude. Normally, they will be captured by macrophages in the liver and are generally used for hepatic imaging (detection of small-size hepatic lesion). On the other hand, USPIOs have even smaller size (less than 50 nm) and will be captured by other phagocytic cells such as ganglion cells (Lymph-MRI).

Besides these two kinds of most-widely used contrast agents, there exist other potential candidates for contrast, like super-paramagnetic iron platinum particles and manganese-based nanoparticles. However, no matter which type of contrast agent is used, MRI is still not sensitive enough to achieve images at the air/liquid interface (e.g. lung epithelium) due to the presence of very few detectable atoms. To overcome this problem, several research teams focused on the use of hyperpolarizable atoms such as 129 Xe, 13 C and 3 He.

b. Hyperpolarization

As we can see in Equation 1, the best way to improve the sensitivity of MRI is to increase the polarization P, which depends on the difference in populations of spins between the two Core of the iron oxide energy states. At thermodynamic equilibrium, the Boltzmann's distribution is preponderant and consequently there is almost no difference between the two states. After an artificial modification like a polarization by laser, the difference in populations of spins is obviously augmented (Figure 8) leading to hyperpolarization. The hyperpolarized isotopes that are potentially usable for MRI are parahydrogen, 3 He, 129 Xe, 13 C and 15 N. The choice of these stable isotopes is substantially conditioned by the possibility of obtaining a longitudinal relaxation time longer than the duration of the polarization process.

i. Parahydrogen

Molecular hydrogen actually exists as four nuclear spin isomers. Orthohydrogen is used to describe hydrogen molecules that exist with one of the 3-fold degenerate spin state terms α α, αβ + βα, and β β, whereas parahydrogen molecules exist solely asαββαspin isomers. The ortho and para isomers exist in different rotational states, and their proportion is temperature-dependent when their normally forbidden interconversion is facilitated. 11,12 If pure parahydrogen is desired, running hydrogen gas through a copper block containing silica/FeCl 3 catalyst at 20 K is sufficient. 13 Moreover once formed and removed from the interconversion catalyst, parahydrogen is relatively stable to spin-reequilibration. Therefore, the depolarization time is sufficiently long to enable the synthesis of molecules which can be monitored by hyperpolarized NMR methods. 14 Usually, the application of parahydrogen in

i. Hyperpolarized 3 He

The hyperpolarized 3 He atom is basically used for lung imaging. The first image of lung with a hyperpolarized gas was obtained with 129 Xe. 15 However, helium quickly replaced xenon thanks to a larger gyromagnetic ratio as well as a better hyperpolarization yield. 16 Thus, most current human lung images are obtained with 3 He. However, researchers progressively come back to replace the helium by xenon. In fact, despite its intrinsic advantages, helium severely suffers from its rareness and expensive price. Moreover, unlike 129 Xe, 3 He is not very soluble in biological milieu, a drawback which drastically limits its application for molecular imaging.

ii. Hyperpolarized 13 C

A major advantage of using 13 C as the hyperpolarized atom is that 13 C naturally exists in small amount in numerous metabolites of living organisms. By following the evolution of the 13 C-labeled metabolites in enzyme-catalyzed reactions, precious information about the metabolism can be obtained. 17 In practice, the metabolites are hyperpolarized by dynamic nuclear polarization (DNP): the hyperpolarization of the 13 C nuclei is realized through a transfer of polarization from the lone-pair electrons of a radical species to the 13 C nucleus under the effect of a microwave irradiation. 18 Then the labeled metabolites are injected into the patient and their evolution is monitored by MRI. 19 The example of 13 C 1 -pyruvate can illustrate this assertion. 20 Because pyruvate is at a crossroad of the major energy generating metabolic pathways, its metabolism differs in cancer and normal cells. 21 After the injection into the patient, the 13 C labeled pyruvate will be metabolized to give by-products such as 13 C 1 -lactate and 13 C 1 -alanine. It is known that in cancer cells, pyruvate is predominantly transformed into lactate, and therefore there will be an evident augmentation of the concentration in 13 C 1 -lactate as well as a diminution in the concentration of 13 C 1 -alanine. By measuring the ratio of each hyperpolarized metabolite signals in MRI (Figure 9), a mapping of the metabolic pattern can help the oncologist to trace and identify the tumor and to get a convenient treatment feedback. 22 Besides, the measure of the concentration ratio between 13 CO 2 and H 13 CO 3 -makes possible to measure the extracellular pH which is particularly interesting for localizing certain tumors like lymphatic tumors. Inside the tumor tissues, the pH is more acidic than in normal tissues.

This study has been done in vitro and in vivo, and recently the clinical research on patients has been approved in order to study the prostate cancer. 23,24 Now the challenge is focused on the technical development of hyperpolarization tools in order to lower MRI costs.

Hyperpolarized 129 Xe

a. General presentation of xenon

Xenon is a noble gas, which is inert, odorless and colorless. Xenon has shown no noticeable toxicity for living organism. In the human body, it inhibits glutamatergic receptors N-methyl-D-aspartate (NMDA), which accounts for its anesthetic properties. At lower concentrations, it is a neuroprotective agent by interacting with globular cytosolic proteins. 26 Only a slight dizziness and an euphoria were reported as side-effects. 27 Moreover, as mentioned before, xenon has a very good solubility in biological milieu which enables further application in vivo.

Practically, for in vivo experiments, xenon can be administrated either by inhalation or by injection. The inhalation is the simplest method to bring xenon inside body either by intubation of anesthetized animals or by an inhalation by human volunteers. It is thus possible to observe the lung 11 or even the brain 28 with the help of hyperpolarized xenon. On the other side, the injection is an invasive method but more direct than inhalation. Xenon gas should be dissolved in a biocompatible solvent for optimal solubility and limited relaxation rate. Until now, xenon was injected in saline solutions, the aqueous suspensions of lipid vesicles 29,30 , the emulsions of perfluorocarbons 31,32,33 and liposomes 34 .

More noticeably, xenon possesses a large highly polarizable electron cloud, which makes it extremely sensitive to its chemical environment, resulting in a wide range of NMR chemical shifts (Figure 10). 35,36,37

b. Hyperpolarization with optical pumping

Hyperpolarization enables an increase of sensitivity by several orders of magnitude.

Generally, there are 3 methods for hyperpolarizing nuclei:

1) Dynamic nuclear polarization (DNP) is the most currently used method. Basically, the transfer of polarization from the single electrons to the nuclear spins can hyperpolarize a large variety of atoms in a molecule. For example, polarization of 15 N in the urea and 89 Y in the chelates of yttrium (III) has all been succeeded using DNP. However, as discussed previously, only polarized 13 C could be used for in vivo MRI applications, in particular thanks to its higher sensitivity. 38,39 2) Parahydrogen-induced Polarization (PHIP) is a technique which involves a chemical reaction in liquid phase or gas phase in which polarized para-hydrogen (antiparallel nuclear spins) is transferred to the molecule of interest by hydrogenation. 40,41 Therefore, the two transferred protons become magnetically non-equivalent with a net polarization which can be transferred to neighboring nuclei by scalar coupling (spin-spin interaction). Despite the efficiency of this method, the major drawback arises from the chemical modification of the product of interest, and as a result, a limited selection of hyperpolarizable molecules. 42,43 3) Optical pumping is the currently-used method to hyperpolarize xenon gas, which will be introduced more in detail further.

A. Kastler presented firstly the principles of optical pumping in 1950. 44 This method enables to change the distribution of spin populations on the two energy states through an irradiation of atoms by polarized light in the presence of a magnetic field. Once the stationary state is reached, a strong augmentation of the electronic polarization is obtained. In 1959, the group of M. Bouchiat shown that alkali atoms with an increased electronic polarization could transfer their polarization to the nuclear spin of a noble gas by simple contacts in the gas phase. 45 The hyperpolarization of 129 Xe is thus achieved through optical pumping of rubidium (Rb) followed by a transfer of the polarization of electrons to xenon nuclear spin, a process which clearly implies two separated steps (Figure 11). The first step is the optical pumping of rubidium. 47,48 A chamber containing a mixture of rubidium steam, nitrogen and xenon preheated to 100 °C is illuminated by a laser beam at 794 nm. In the presence of a magnetic field, the only permitted electronic transition of rubidium is the one between the state spin -½ and the first excited state spin +½. Successive collisions between rubidium atoms will lead the populations in the excited states to return to the ground state (Figure 12). However, thanks to the excitation rate which is greater than the relaxation rate, a high concentration of rubidium at the state + ½ is obtained. The second step is the transfer of polarization to xenon nucleus. It involves multiple collisions between rubidium and xenon gas. These collisions will result in a dipolar coupling between electron spins of the metal and the nuclear spins of the gas (Figure 13). Thus, a high concentration in hyperpolarized xenon is obtained in a very short time.

c. Encapsulating structures of xenon

An important limitation for the application of xenon to in vivo molecular imaging is it does not enable to image a specific molecular target since it has no predictable specificity. To overcome this problem, it is therefore necessary to encapsulate the hyperpolarized xenon atom inside a biosensor.

A biosensor is a molecule composed of 1) a molecular cage used to encapsulate the gas with a particular chemical environment, 2) a linker, and 3) an antenna of recognition to target the molecule of biological interest (Figure 15). As xenon is extremely sensitive to its chemical environment, the chemical shifts in 129 Xe NMR is expected to be significantly different for free xenon gas, encapsulated xenon in a free biosensor and encapsulated xenon in a biosensor binding to its biological target. 2. An appropriate in/out exchanging rate, which should be slow enough to visualize the encapsulation of xenon in NMR, and fast enough to constantly enable the molecular cage to be refilled with freshly hyperpolarized xenon in order to overcome the lost in sensitivity due to time-dependent depolarization. In 1998, J. Rebek showed that there is an optimal ratio between the size of the guest molecule and the size of the host molecule, which is 0.55 ± 0.09. 49 As the van der Waals volume of xenon is 42 Å 3 , the ideal van de Waals volume of the molecular cage should be 76

Å 3 .
The hydrophobic character of the xenon makes it particularly sensitive to London forces, 50 caused by the dipole -dipole interactions. Consequently, the interaction between xenon and the atoms of the molecular cage is also a source of stabilization. 51 In the literature, there are numerous molecules which satisfy more or less to the previous criteria. 52 Here below are given several relevant examples.

ii. Cyclodextrins

Cyclodextrins are cyclic oligosaccharides produced by enzymatic degradation of starch, formed by the assembling of 6 to 12 glucose units connected to each other by 1,4 α bonds (Figure 16). The cavity of the smallest cyclodextrin (α-cyclodextrin, consisting of 6 glucose units) has a volume between 140 Å 3 and 170 Å 3 . Although it has been demonstrated that xenon readily enters this cage, the large volume of its cavity still causes a too fast in/out exchanging rate of xenon. Moreover, its very low affinity constant for xenon (about 20 M -1 at 298K) also limits its application for the design of biosensors. 53 These macrocycles are obtained by condensation of phenol with formaldehyde in a basic milieu. These molecules are amphiphiles with a hydrophilic region around the hydroxyl groups and a hydrophobic cavity between the aromatic rings. However, the in/out exchange rate of xenon in this kind of cage is still too fast. In addition, the affinity constant for xenon is generally too low. For example, the water-soluble Calix [4]arene STCAS (4sulfothiacalix [4]arene sodium salt) presents an affinity constant of 14 M -1 at 298 K in water (Figure 17). The cucurbiturils are obtained by reaction of glycoluril with formaldehyde in acidic milieu.

Their general structure is presented in Figure 17a. The cavity volume of the cucurbit[n]urils family (CB[n]) varies from 82 Å 3 to 870 Å 3 according to its size (n = 5, 6, 7, 8, 10).

Theoretically, the smallest cucurbituril CB [5] was calculated to be the best candidate for encapsulating xenon. However, the rigidity of the molecule and the entrance gate defined by the oxygen in carbonyl groups are not favorable for the encapsulation of xenon. 56 Until now, the most-adapted cucurbituril for xenon encapsulation is the water-soluble CB* [6] synthesized by the group of K. Kim. It possesses a good affinity constant for xenon in the 3000 M -1 range (Figure 19).

vi. Cryptophanes

Cryptophanes are composed of two units of cyclotriveratrylene (CTV) derivatives connected to each other through alkyldioxy bridges (Figure 20). The structure can vary according to the length of the molecular bridges (l, m and n) as well as groups R1 and R2 on the aromatic rings. Their hydrophobic cavity is suitable for encapsulating little neutral molecules like xenon. As a consequence, they have the best affinity constant for xenon ever reported (29,000 M -1 for the water-soluble cryptophane[111] 58 ) and also exchange rates totally adapted to the experiment timescale. According to the calculation of J. Rebek 49 , the smallest cryptophane[111] approaches the optimal ratio of 0.55 between the volume of xenon and its cavity. This partly explains why cryptophane [111] has the best affinity for xenon, independently of the solvent used, either organic solvent tetrachloroethane (K = 10,000 M -1 ) 59 or water (29,000 M -1 for the watersoluble cryptophane[111] 58 ). In fact, the proximity of its aromatic rings with the xenon electronic cloud stabilizes the encapsulated gas. As shown in Table 2, for the organosoluble cryptophanes, the wider the cryptophane cavity (longer alkyldioxy bridges), the less the xenon is stabilized by the van der Waals force related to aromatic rings, and therefore the worse the affinity for the xenon gas is. 61,62,63 When the cavity is large enough (e.g. like in cryptophane [333]) the molecule of solvent can also enter the cage and therefore competes with the solvent. This phenomenon can also explain the poor affinity of xenon for larger cages. and length can be tuned. Considering that the systematic nomenclature of these molecules is particularly long and complex, a simplification was quickly adopted. The first elaborated cryptophanes were named "cryptophane A, B, C…". But for now, as a large diversity of structures in this family has been synthesized, this nomenclature fits no longer the new compounds. A new arbitrary nomenclature is often adopted, defining the number of carbon atoms in each bridging chains and the substituent groups on the aromatic rings. For example, the cryptophane A has been renamed cryptophane[222](OMe) 6 (Figure 22).

Cyptophanes

a. Stereochemistry and symmetry

Stereomeric cryptophanes arise from the chirality of CTV units. When R 1 and R 2 are the identical groups, the CTV derivatives are achiral. And when they are different, the molecule presents a planar chirality with 2 enantiomers with C 3 symmetry (Figure 23).

b. Conformers

In solution, the CTV derivative exists in 3 different forms. Besides the most stable "crown" conformation, which is also the major form, there are also "saddle" and "twisted" conformations. Racemization of the enantiomerically pure CTV derivative through a "crown to crown" interconversion was observed (Figure 25). The intermediate "saddle" and "twisted" conformations, which are usually non-isolated, are less stable than "crown" because they induce repulsive interactions between a methylene group and its opposite aromatic ring. [START_REF] Collet | [END_REF] It is possible to calculate the energy barrier for "crown to crown" inversion which has been evaluated to be about 110 kJ.mol -1 . 69, 70 As a consequence, cryptophanes, composed of two units of CTVs, also possess different conformations (Figure 26). Besides the major "out/out"conformation, the other three conformations, "in-out", "in-in" and "out-saddle", can be formed when there is no molecule inside of the cage. The imploded form ("in-out") of cryptophane is preferred when the molecule of solvent does not penetrate easily into the cavity and its formation is discernable by a series of new specific signals in 1 H NMR. 63,71,72 In addition, longer is the bridging chain, more flexible is the cryptophane, leading to the formation of the imploded conformations. It is possible to interconvert "in-out", "in-in" or "out-saddle" to "out/out" conformation by heating the molecules in a solution whose solvent molecule can enter inside the cage, or adding a gas which can be encapsulated by the cyptophane.

Synthesis

There are 3 strategies for synthesizing cryptophanes, all starting from benzylic alcohol derivatives (Figure 27). Each of them possesses different advantages and particular drawbacks. 66,73 Figure 27: Three methods to synthesize cryptophanes a. The "direct method"

In the "direct method", the benzyl alcohol is firstly converted to a "dimer" and then the dimer is used for cyclotrimerization to form the cryptophane (Figure 28). As the simplest and quickest method to synthesize cryptophanes, the poor yield (less than 5 % for small cryptophanes like cryptophane A, less than 20 % for the larger ones) and the limitation to synthesize only the cryptophanes of C 3h and D 3 symmetry jeopardize its further application. It is also interesting to notice that cryptophane anti is preferentially obtained in this method. 73

b. The "template method"

This approach is a multi-step synthesis which requires the formation of two cyclotriveratrylene units at different stages of the synthesis.

The "template method" starts with the synthesis of the first CTV which acts as a "template" here. In a general way, CTVs are obtained through cyclotrimerisation of benzyl alcohol derivatives. This reaction takes place in acidic milieu (Lewis acid or Brönsted acid). The choice of the acid depends on the substituent groups R 1 and R 2 .

The proposed mechanism is shown in Figure 29. First, the carbocation is obtained from the benzyl alcohol derivative under acidic conditions. Two successive condensations of this carbocation lead to a dimer and then a linear trimer. The final cyclization affords the CTV. 68

Figure 29: Proposed mechanism of cyclotrimerization to form CTVs

It is important to notice that this reaction needs an electron-donating group in position of R 2 of the benzylic alcohol in order to activate its nucleophilic para position on which an electrophilic Friedel-Crafts substitution takes place. The substituent group R 2 can be either -OR, -NRR′, or -SR. The methoxy group is mostly-used in the synthesis of CTVs.

Furthermore, the R 1 position should be protected to avoid a secondary electrophilic substitution on this position.

After the first cylotrimerization, a deprotection step of the CTV 1 is necessary to obtain the free phenols. The generated CTV 2 is then functionalized with three substituted benzyl alcohol to obtain 4, which is an already "pre-organized" intermediate to form the final cryptophane. Then the second cyclotrimerization is done according to the preformed "template" to obtain cryptophane anti 5 and/or syn 6 (Figure 30). The last step requires high dilution condition in the order of 10 -3 M to promote the intramolecular reaction and reduce the formation of polymers.

Figure 30: General synthesis of cryptophanes by template method

Regarding the distribution of syn-and anti-configurations, it is interesting to point out that the major configuration seems to depend on the number of carbons atoms on the bridging chain. When the number of carbon is even, cryptophane anti is obtained as major product (e.g.

for cryptophane A, syn/anti = 0/80). When it is odd, the cryptophane syn is predominant (e.g.

for cryptophane E, syn/anti = 50/21). In a general way, the parity of the number of carbons forming the bridge will determine the mode of cyclization (Figure 31). Compared with the direct method, the template method displays advantages like significantly better yields and the possibility to synthesize cryptophanes with different symmetries. This is particularly important for its application in the construction of biosensors and it is also the reason why the template method is the most widely used strategy for synthesizing cryptophanes.

c. Coupling of CTVs

This method consists in synthesizing cryptophanes by coupling two CTV subunits. It was firstly reported by D. Cram for the preparation of cyptophanes with acetylene groups in the bridging chain. 74 This relatively direct strategy suffers from poor yield due to the formation of by-products like oligomers and polymers during the coupling reaction. Recently, this method was used for synthesizing the small-sized cryptophanes [111] 59,75 and [000] 76 (Figure 32).

Strategies for the hydrosolubilization of cryptophanes

The design of biosensors usable for in vivo molecular imaging implies that the molecular platform has a good solubility in water. After more than 30 years development in cryptophane chemistry, a large number of different chemical functions can be introduced on the aromatic rings as well as the bridging chains. Although cryptophanes are still hydrophobic molecules, different functionalization can be used to improve their water-solubility.

The first strategy based on the introduction of carboxylic acid groups on the aromatic rings. 63 Starting from cryptophane A, the utilization of the lithium base PPh 2 Li allows obtaining the expected cryptophane with six phenol moieties. Then the nucleophilic substitution with methyl bromoacetate leads to the formation of a cryptophane carrying six ester functions, which are finally hydrolyzed to give cryptophane[222] hexa carboxylic acid (Figure 34). 78 , giving rise to the air stable chloride salt [(Cp*Ru) 6 7]Cl 6 , hereafter [8]Cl 6 (Figure 35). Unlike carboxylic acid derivatized cryptophanes, the water solubility of which is attributed to the deprotonation of the carboxyl groups, [8]Cl 6 is highly water-soluble at neutral pH ( >30 mM, 293 K). 58 Moreover, this molecule has shown an affinity for xenon even better than 7 (10,000 for 7, 29,000 for [8]Cl 6 ). Dmochowski. 79 By using the template method, the tripropargyl cryptophane was synthesized in 10 steps with 4 % overall yield. Then the reaction of cryptophane with 3 equivalents of 2-azidoethylamine was carried out via copper(I)-mediated [3 + 2] Huisgen cycloadditions 80 to give the water-soluble tris-(triazole ethylamine) cryptophane (TTEC) (Figure 36). It was reported that the affinity constant for xenon of TTEC is as high as 42,000 M -1 . According to the author, the presence of different substituent groups on the two CTVs causes an axial dipole moment which complementally stabilizes the highly polarizable electron cloud of xenon. 81 However, the measurement of the affinity constant was carried out by isothermal titration calorimetry instead of NMR, thus limiting the comparison with other cryptophanes.

Figure 36: Hydrosolubization of cryptophane[222] by introducing triazole groups

The latest development in this field was made by the group of L. Schröder. Starting from cryptophane A diacid (CrA-da), which was commercially available, a straightforward synthesis of the dendronized cryptophanes was achieved with good yields (60 % for two steps). 82 Conjugation of CrA-da with polyglycerol dendrons by using the standard amide bond formation reaction conditions was followed by the subsequent deprotection under acidic conditions with TFA yielded the final dendronized cage molecules (Figure 37). According to xenon NMR experiments, the presence of dendrons on cryptophanes has no negative impact of the encapsulation of xenon gas. Based on a short, high-yielding, and scalable synthetic route of cryptophane[1.1.1] 7 ( 5 steps, 12 % overall yield) 83 which is also developed by our group, the functionalization leading to the first metal-free water-soluble cryptophane[111] was achieved. After a monobromination of the cryptophane[111] (this step will be discussed more in the part A-II-4), the lithiation of bromine 9 by n-BuLi followed by carboxylation with CO 2 afforded the carboxylic acid 10. Then a Schotten-Baumann reaction was performed between cryptophane 10 and an amino acid-based poly-sulfonated linker 11 to give the desired water-soluble cryptophane 12 as a mixture of two racemic diastereomers (Figure 38). 84 As a metal-free cryptophane, 12 avoided the potential risk in its application in vivo, like a DNA-binding activity of some ruthenium-arene complexes. 85 Its solubility in water (about 1.0 mg/mL) is suitable for xenon NMR experiments. was designed (Figure 39). Starting from the cryptophane[1.1.1] 7, a hexabromination with an excess of NBS was followed by a lithiation with n-BuLi. In this case, the lithium/bromine exchange occurred, but the lithiated cryptophanes were not efficiently trapped by CO 2 .

Conversely, using benzyl chloroformate as an electrophile led to hexa-benzyl ester compounds 14, which was then deprotected by hydrogenolysis over Pd/C, to give cryptophane[111] hexacarboxylate 15 (18 % overall yield for three steps). 86 As expected, compound 15 is soluble in water at a concentration of 2.0 mg/mL.

Figure 39: Hydrosolubilization of cryptophane[111] by introducing six carboxylic acid groups

As reported for the cryptophane[222], our group has developed the synthesis of a highly water-soluble cryptophane 24 which could be seen as a universal platform for the construction of 129 Xe MRI-based biosensors. 87 This platform contains a poly(ethylene glycol) (PEG) chain as water-soluble moiety: this choice will be discussed further in the part B-I-2. Unlike in the other strategies, the water-soluble moieties can be introduced at the early stage of the synthesis. This not only improves the solubility of the cryptophane intermediates and facilitates the synthesis and purification steps but also decreases the number of steps by avoiding successive protection and deprotection of the vanillyl alcohol. Thus the phenol group of 16 was substituted by the tosyl-functionalized PEG monomethyl ether 17 to give the PEGylated benzyl alcohol derivative 18. After cyclotrimerization, product 19 was engaged in the demethylation with PPh 2 Li. Then last two steps of the reaction were carried out following the classical template method, leading to PEGylated cryptophanes 23 and 24 with different substituent groups (Figure 40). These cryptophanes are soluble up to 50 mg/mL water in a physiologically relevant pH range (pH 5 to 8), which is the highest water solubility ever reported for cryptophane [222]. Moreover, the presence of three propargyl groups in cryptophane 24 makes possible further functionalization.

Strategies of mono-functionalization of cryptophanes

As underlined before most of the previously synthesized cryptophanes possess a high symmetry. In fact, this character can be an important drawback for the synthesis of biosensors for in vivo imaging. The high symmetry makes the control of the reactivity on a particular position difficult versus other equivalent sites. Usually, the cryptophanes used for synthesizing biosensors possess three (e.g. Figure 41) 88 or six (e.g. Figure 42) 89 functionalizable positions while only one is needed to react with the linker. Therefore a complex statistical mixture of different products is expected, complicating the purification steps. As a result, the optimization of yields is difficult and an efficient scale-up is then ruled out. Contradictorily, large quantities of desymmetrized cryptophane are required for the synthesis of biosensor which is often strenuous. Therefore, an optimization of the monofunctionalization of cryptophanes is crucial. The earliest effort in this field focused on the synthesis of the monohydroxy cryptophanol 31, which can be easily obtained through a deprotection from mono-protected cryptophane 30.

As an important intermediate in the synthesis of mono-functionalized cryptophanes, compound 30 can be synthesized using two alternative strategies. The first one, which is still the most widely-used to break the symmetry of cryptophanes, was developed by the group of T. Brotin and J. P. Dutasta. 90 This strategy is based on the mono-functionalization or the bisfunctionalization of CTV 25, which was treated successively with two different benzyl alcohol derivatives 26 and 28. Through a disubstituted intermediate 27, the cryptophane precursor 29 was obtained, latter transformed into the expected cryptophane 30 (Figure 43). 91 This synthesis significantly unlocked the development of cryptophanes usable for in vivo applications, and therefore numerous subsequent biosensors were obtained from this molecule. Simultaneously, another strategy to synthesize cryptophane 30 was developed by the group of A. Pines. 92 It involves the selective deprotection of CTV 32 using Pd[P(C 6 H 5 ) 3 ] 4 as a catalyst and Bu 3 SnH. 83 The monoprotected CTV 33 was then allowed to react with protected vanillyl ether 34 to give the bis-functionalized CTV 35. A second deprotection step followed by the condensation with the allylic containing derivative 37 provided the cryptophane precursor 38. Finally, the last cyclization reaction was carried out in pure formic acid to yield cryptophane 30 (Figure 44). 3 Compared with the previous method, this synthetic route gives better yields but requires several additional steps. Afterwards, a more straightforward synthetic pathway to cryptophanol 31 was developed by the group of T. Brotin 94 . Removal of a single methyl group of cryptophane A was easily achieved by using iodotrimethylsilane, yielding cryptophanol 31 in 37 % yield as well as unreacted cryptophane A which could be reused in another cycle of reactions (Figure 45). Our group has also reported the only existing approach to mono-functionalized cryptophane [111]. 83 Limited by the lack of modifiable functional groups, the functionalization of cryptophane[111] was always difficult. However, since it is composed of phenoxy moieties, it is expected to be iodinated or brominated. After several attempts, the iodination was achieved by using (I 2 , C 6 H 5 I(OAc) 2 ) 97 , while the bromination was achieved with Nbromosuccinimide (NBS) (Figure 47). Starting from these halogenated compounds, a large number of chemical functions can be thus introduced using either organolithium or palladium chemistry, including the carboxylic acid and the trisulfonated linker as presented previously.

Cryptophane-based biosensors for 129 Xe MRI

The first cryptophane-based biosensor for 129 Xe NMR synthesized by the group of Pines in 2001 is capable to detect the formation of the complex biotin/avidin at micromolar concentrations. 3 This biosensor is composed of the hydrophobic core of cryptophane A (the black part), a water-soluble peptide linker (the green part) and an recognition antenna , containing a biotin motif (the blue part) which strongly binds to avidin (Figure 48). The 129 Xe NMR experiments also confirm the recognition and the endocytosis of the biosensor, despite of the presence of an unexpected signal which corresponds to the nonspecific interaction between the biosensor and the lipid membrane of the cells. This problem is undoubtedly due to hydrophobicity of the cryptophane moiety and could be solved by the introduction of the solubilizing groups on the molecular cage.

The first images of hyperpolarized 129 Xe MRI in cellulo were presented by the group of L. Schröder in 2014. 108 This biosensor, which is also based on the cryptophanol motif, bears a fluorescein moiety permitting to monitor its internalization by confocal microscopy (Figure 52-a). MRI experiments with the biosensor-labeled cells were done within a NMR compatible packed-bed bioreactor working under continuous perfusion with a hyperpolarized xenonsaturated milieu. The xenon gas is introduced into the culture medium within a bubbling chamber located upstream of the sample. The phantom was split into two compartments along the direction of media flow. Cells were immobilized by encapsulating them within solid alginate beads (Figure 52-b). One of the two compartments was filled with cells pre-incubated with the biosensor (the blue ones), and the other compartment was filled with the unlabeled cells (the red ones). Although the intracellular concentration of the bio-sensor is relatively low, the compartment bearing labeled cells could be clearly distinguished from the control compartment by Xe MRI after only 20 acquisitions (Figure 52-c).

III. Objectives

As an efficient method to overcome the lack of sensitivity of MRI, the technique using 129 Xe as a hyperpolarized species has been developing fast since a few years. Benefiting from the development of cryptophane-based biosensors functionalized with different targeted groups, this technique shows great interest for molecular imaging. After numerous studies of this kind of biosensors in water, buffer solutions, human plasma and lipid environment, the actual advancements in imaging internalized biosensors by NMR of clearly open new prospects for in vivo applications.

In this context, the objective of this thesis is to design new cryptophanes which can be used as molecular platforms for constructing new 129 Xe MRI biosensors usable for in vivo MRI molecular imaging. To meet this demand, two main difficulties should be resolved.

First of all, the high symmetry of cryptophanes is proved to be an important drawback to control the reactivity of a particular position with respect to other equivalent sites. The monofunctionalization step is always inevitable and leads to a complex statistical mixture in all kinds of existing synthetic pathways to biosensors. This strategy complicates the purification steps and jeopardizes the production of large quantities of cryptophane derivatives required for the arduous synthesis of bioprobes. For this reason, there is a systematic discussion about cryptophanes symmetry breaking and different attempts were made to synthesize monofunctionalized molecular cages.

Secondly, the synthesized biosensors still suffer from the high hydrophobicity of the cryptophane poly-aromatic core. Even though the grafting of various water-soluble linkers increases the biosensor overall solubility, the high hydrophobicity of the cage itself in aqueous milieu confers amphiphilicity to the molecule which is prone to form self-organizing systems, promoting a non-specific interaction with the lipid bilayers and the subsequent embedding of the probe inside the cell membranes. To avoid this problem which is detrimental for the application of these biosensors in vivo, a new cryptophane cage designed to be fully water-soluble in different pH environments must be developed. In this thesis, the polyethylene glycol (PEG) group was therefore used to address efficiently the issue of cryptophane water-solubility.

In this thesis, the main effort focuses on the hydrosolubilization and the desymmetrization of cryptophane [222]. This molecular cage was chosen thanks to its good affinity and selectivity for xenon, as well as the suitable exchange rate that it can offer. Synthesis of different water-soluble mono-functionalized cryptophane [222] have been achieved (Figure 53). All the strategies and synthetic pathways will be discussed in the next chapter.

B. CONCEPTION AND SYNTHESIS OF WATER-SOLUBLE AND MONO-FUNCTIONALIZABLE CRYPTOPHANES

I.Water-Soluble and Mono-Functionalizable Cryptophanes -Why and How

Background and objective

This thesis is part of a bigger project in our laboratory which aims at developing and promoting a new technique for imaging non-small cell lung cancer (NSCLC). In France 37 000 new cases of lung cancer are diagnosed each year with a high mortality rate (28 700 death per year). Eighty percent of all cases are NSCLC, a type of cancer that remains the leading cause of cancer-related mortality. Despite numerous clinical trials, no major outbreak has been made in the treatment of the large majority of NSCLC during the last decades. The detection and biochemical characterization of growing lung tumors by molecular imaging is a prerequisite to an effective treatment. Therefore, a fine detection and monitoring of the response of tumor to therapy through imaging techniques is critically important for the treatment of NSCLC.

To date, most of NSCLC cases are detected either fortuitously (in particular during chest radiograph) or on the basis of characteristic symptoms (coughing, hemoptysis, shortness of breath etc.). Both situations need a confirmation by invasive techniques such as bronchoscopy and biopsy. The systematic detection of NSCLC is difficult due the absence of circulating biomarkers and the detection of large proportion of false positive cases. Moreover, scintigraphy techniques (SPECT and PET) using short-period radio-isotopes, which use gamma and positron emitters respectively, can be used to confirm strongly suspected lung cancer cases and to follow up treatment efficacy but not to screen a large at-risk population.

As a consequence, NSCLC is frequently detected at late stages of development and is therefore difficult to cure. In this respect the development of an efficacious MRI technique for the detection and biological characterization of NSCLC is essential. In this project, we investigated the development of 129 109 for the epithelium growth factor receptor (EGFR), which is widely over-expressed on the surface of many cancer cells. It has been clinically approved for the therapeutic treatment of NSCLC. On top of its efficiency as anti-cancer agent, it exhibits a low toxicity at therapeutic concentrations (typical primary therapeutic dose of 400 mg/m 2 of body surface and no side-effect after injection of 1000 mg/m 2 of body surface in human). 110 The bioconjugation of diverse chemical entities onto this antibody is well documented 111,112 and bifunctional reagents are commercially available at a reasonable prize (1000 € / 500 mg).

2) A fluorescein moiety to allow the in vitro tracking of the biosensor by fluorescence confocal microscopy using the same probe, in order to make the proof of concept in cell cultures.

3) A cryptophane molecular cage encapsulating xenon which should be water-soluble, functionalizable and features an exchange rate adapted to the MRI experimental time scale. This part is also the focus of this thesis. However, though this cryptophane has enabled recent advances in the development of bioprobes for 129 Xe NMR, it displays several drawbacks. First of all, its solubility in water (only when pH is lower than 5) is relatively low. Secondly, its synthesis is rather long and difficult. Moreover, the presence of six identical possible active positions for desymmetrization undoubtedly leads to a very poor yield when reacting with the linker with a maximum of 13 % after optimization (Figure 56). It should be mentioned that cryptophane 48 and the linker carrying a fluorescein moiety are both high value-added and thus expensive products which are only available in limited amounts. In this context, it is necessary to develop a straightforward synthetic route leading to a water-soluble and mono-functionalizable cryptophane in good yield with the minimum waste of highly elaborated material.

In fact, this problem can be summarized by two interconnected issues: 1) how to break the symmetry of the system to make the cage mono-functionalizable and 2) how to efficiently solubilize the cryptophane moiety. In this thesis, different strategies to break the symmetry of cryptophane will be discussed in the latter parts. Regarding the problem of water-solubility, we focused our interest on the polyethylene glycol groups (PEG) which has been often used to augment the solubility of hydrophobic molecules in water.

Different strategies to synthesize asymmetric cryptophanes

The discussion of the strategy is based on the "template method" for synthesizing a 87 As the most widely-used method for synthesizing cryptophanes, the "template method" is selected thanks to its good yields and more noticeably, to the possibility of synthesizing cryptophanes with various symmetries. Compared with the "direct method" and the "coupling of CTVs", it is clearly obvious that the "template method" offers much more possibilities to break the symmetry of the system (Figure 57).

Polyethylene glycol -our choice for hydrosolubilization

The PEG group was chosen to be used as the water-soluble group for several reasons 87 :

1) The alternance of hydrophilic and hydrophobic groups on the PEG confers a very good solubility in both organic milieu and aqueous milieu, which facilitates not only the hydrosolubilty of the biosensor in vivo, but also the dissolution in organic solvents and enables a large variety of treatments during the different steps of the synthesis.

2) It is inert to many chemical reactions such as oxidation, reduction, acids and bases.

3) It is stable at high temperature.

4) It is biocompatible and used in many pharmaceutical and biomedical applications. 113,114 We have chosen the methoxy-polyethylene glycol 550 (mPEG 550), a polydisperse PEG with the average molar mass of 550, which is made of the concatenation of about eleven ethylene glycol units. This PEG is long enough to solubilize the cryptophane and not too long to cause a steric hindrance jeopardizing the subsequent functionalization and encapsulation.

According to the previous work done by our lab, the cryptophanes with three or six units of PEG moieties have satisfactory water-solubility (50 mg/mL) and possess very good abilities to encapsulate xenon gas (Figure 58). 87

II. Desymmetrization of Cryptophanes

The desymmetrisztion of a symmetric cryptophane was the more obvious and thus the first option attempted during my Ph.D. work. It does not require a complete revision of the synthetic pathways and only necessitates the addition of a selective desymmetrization step (Figure 60). Although it is already a well-developed synthesis, several points remain to be optimized.

First of all, the total yields (6 % for the cryptophane 23 and 2 % for the cryptophane 24)

are limited, mainly due to the poor yield (20 %) obtained for the functionalization step of CTV 20.

Secondly, the scale of this synthesis is limited to dozens of milligrams. These quantities are still far away from the need for the synthesis of reasonable amounts of biosensors (at least a dozen of miligrams of final compound for triplicate in vitro and in vivo experiments).

To date, mono-functionalization of the compounds 23 and 24 has not been achieved. The tri-propargyl cryptophane 24 leads to statistical mixtures after mono-functionalization and the presence of three PEG moieties is likely to make the purification even more difficult.

As reported for cryptophane 23, Delacour et al. have tried to carry out a total demethylation in order to make the cryptophane functionalizable. According to the LC-MS analysis of the crude product, the formation of the targeted cryptophane 49 was observed, together with partially demethylated cryptophanes 50 and 51, even when a large excess of PPh 2 Li was used (Figure 62). From her point of view, the small scale of this reaction made it difficult to secure a fine anhydrous environment which is necessary when the water-sensitive reagent PPh 2 Li was employed. Although the total demethylation seems to be difficult, the presence of the monodemethylated cryptophane 51 has demonstrated the validity of this approach.

Therefore our research plan was:

1) An optimization of the synthesis of cryptophane 23, especially a yield improvement for the functionalization step of CTV 20. Different reagents and reaction conditions will be tested to avoid the β-elimination of the leaving group X leading to the decrease of yield.

2) The scale-up of the synthesis for obtaining at least several hundred milligrams of cryptophane which can be used to synthesize biosensors.

3) To perform the desymmetrization, in particular a mono-demethylation of cryptophane 23 in order to obtain the water-soluble mono-functionalized cryptophane 51.

Optimization and scale-up

As shown in Figure 57, the first step does not necessitate to be optimized since the PEGylated vanillyl alcohol 18 is obtained at the 10 g scale in 97% yield.

a. Optimization of the cyclotrimerization and the demethylation

The conditions of the first cyclotrimerization step and the subsequent demethylation are also well-optimized. Cyclotrimerization was performed in the classical conditions (perchloric acid/methanol 1:2). The crude product was purified by reversed-phase chromatography to give the PEGylated CTV 19 in 50 % yields. Demethylation of 19 was done using a freshly prepared solution of LiPPh 2 and was purified by silica gel chromatography in 80 % yield.

For these two steps, the necessity to purify the product by silica-gel chromatography was a strong limitation in terms of yields and quantities. Therefore, we plan to skip the purification step after the cyclotrimerization reaction and to purify the product at the end of the demethylation step. Successive liquid-liquid extraction was also proposed as an alternative to chromatography.

Thus, after cyclotrimerization, 4 g of obtained crude product was directly engaged in the next step.

After the demethylation, tests of solubility were carried out with the crude product and different deuterated solvents in order to find the best solution for product purification (Table 4). In most cases, a homogeneous solution was obtained, suggesting that the expected product and the impurity were both very soluble in these solvents. Conversely, none of them was soluble in cyclohexane. The best result was obtained with toluene, in which the impurity is more soluble than the targeted CTV 20.

Solvent

b. Optimization of the alkylation of CTV

As discussed previously, the alkylation of CTV 20 is a key problem which negatively affects yields putatively due to:

1) The chelation of Cs + by the PEG oxy groups forming bulky chelate which might sterically hinder the alkylation of CTV. Different bases (like NaH) will be tested to solve this problem.

2) The β-elimination of the benzyl alcohol derivatives during the CTV grafting (Figure 63), which was already observed by LC-MS analysis of the crude product. 87 Different quantities of benzyl alcohol derivatives with different leaving groups were tested. 5, the less benzyl alcohol is used, the better yield is. This is probably caused by the side-reaction between the excess of 54 and targeted product 21 (Figure 66). With 4 equivalents of 52, the obtained yield (28 %) was already improved relative to the original yield (20 %). It should be noticed that the products in entry 1 and 2 were actually purified by reversedphase chromatography and the real yields were calculated whereas in other cases, yields were evaluated according to the HPLC chromatogram using yields in entries 1 and 2 as the references. Although all the factors in the condition of this reaction were investigated, the optimization was difficult due to limitations in our attempts for scaling up the reaction. In order to improve the efficiency of the purification in this step, the free alcohol group of derivative 39 was protected by tetrahydropyran group (THP). Thus, the corresponding functionalized CTV 55 was anticipated to be less polar and was expected to be purified by normal phase chromatography (Figure 67). In fact the THP motif was chosen among all the alcohol protecting groups because it can be easily cleaved concomitantly to cyclotrimerization in acidic conditions.

R

Figure 67: Alkylation of CTV with THP-protected benzyl alcohol

The protection of the alcohol group of compound 39 with THP was easily achieved in standard conditions. After several liquid/liquid extractions, the obtained product was already pure enough to be engaged in the next step without any additional purification. Several grams of 34 were isolated (yield 80 %).

The following functionalization of CTV 21 with protected benzyl alcohol derivative 34 was achieved in pretty good yield (70 %). As expected, the functionalized CTV 55 was purified by normal-phase chromatography. Moreover, thanks to the protection of the benzyl alcohol, NaH could be used in excess. It should also be mentioned that this alkylation of CTV 21 was scaled up to 500 mg without decreasing the yield.

As previously mentioned, the THP groups were removed concomitantly to the cyclotrimerization step (Figure 68). Since a low concentration (about 1 mg/mL) of reaction milieu is necessary to promote the preferential intramolecular cyclization rather than intermolecular reactions, the scale-up of this step was limited by the size of available flasks.

Even though, more than 500 mg PEGylated cryptophane 23 could be obtained in 80 % yield.

c. Conclusion of the optimization and scale-up

The total yield of the synthesis of PEGylated cryptophane 23 was improved from 5 % to 43 %, and the scale of the synthesis was raised from dozens of milligrams to several hundreds of milligrams.

Demethylation of symmetric cryptophane

After completing the synthesis of the symmetric PEGylated cryptophane 23, the next challenge was the mono-demethylation step necessary for cryptophane desymmetrization (Figure 69). In the literature, two methods for demethylation of cryptophanes have been reported. The use of a lithium diphenylphosphide (PPh 2 Li) solution was firstly reported for the synthesis of cryptophane[222] hexa-carboxylic acid. 63 In order to secure the efficient demethylation, this PPh 2 Li solution should be freshly prepared each time from lithium and cholorodiphenylphospine in a strictly anhydrous and anoxic environment and thus requires the use of a glove box (Figure 70). (3) As mentioned before, the diphenylphospine and diphenylphospine oxide generated in this reaction are very difficult to eliminate since they are soluble both in water and organic solvents, and thus contaminate all the fractions during the purification.

(4) On the safety point of view, the use of lithium is clearly not the perfect choice. It is difficult to manipulate due to its sensitivity for water and oxygen with an associated hazard related to explosion and inflammation.

The other demethylation procedure was achieved with trimethylsilyl iodide (TMSI), which was previously used in the synthesis of cryptophanol. 94 Compared with the above-mentioned method using LiPPh 2 , this method has several advantages in particular a simpler manipulation, treatment and purification. Moreover, TMSI is a commercially available product which is relatively stable and can be directly engaged without a bias regarding the exact quantity necessary to perform mono-demethylation.

However, the demethylation of non-aromatic methoxy groups with TMSI has been reported, 115,116 suggesting that the chemoselectivity might be insufficient for securing a mono-deprotection of the aromatic methoxy group (Figure 71). In contrast to the demethylation of cryptophane A in which the six methoxy groups are identical, the presence of methoxy groups at the end of the PEG chains in cryptophane 23 might lead to a mixture of two similar PEGylated cryptophanes which are presumably difficult to separate. Anticipating a sufficient stability of the PEG chains, we decided to use TMSI in order to precisely control the quantity of reagent.

a. Demethylation of the PEGylated cryptophane with TMSI

Cryptophane 23 was demethylated with 1.1 equivalents of iodotrimethylsilane (Figure 72).

Analysis of the crude product by LC-MS revealed that the product with a mass corresponding to cryptophane 51 was present in small quantity together with the starting cryptophane 23 which was still predominant. It should be noticed that the quantification of different products was difficult due to the polydispersity of the PEG and the predominance of the polarity of PEG moieties. A particular effort was done to determine the quantity of TMSI necessary for selective mono-demethylation of product 51 starting form cryptophane 23, while minimizing the formation of the undesired di-demethylated product 50 (Figure 73). This strategy was chosen because the starting material 23 could be recycled for a new run of deprotection whereas the di-demethylated product 50 is useless. The result of this optimization is shown in the Table 8. ----+ Table 8: Optimization of the quantity of TMSI. In this table, "-" means no formation of this product; "+" means observation of the formation of this product; "++" means it is the major product of this reaction.

The formation of di-demethylated product 50 was detected when using at least 2 equivalents of TMSI. For 3 equivalents of TMSI and more, the quantity of 51 was almost equal to that of 23 with an unneglectable proportion of tri-demethylated product 49. At 6 equivalents of TMSI, the mass of a tetra-demethylated product was detected by LC-MS analysis. This strongly suggests that at least one of the methoxy groups at the end of the PEG chains was cleaved. At 12 equivalents of TMSI, all the six methoxy groups (3 on the aromatic ring, 3 at the end of the PEG chain) were removed, as shown by the NMR spectrum of the crude product.

As one might dread, TMSI not only reacts with the methoxy groups on the aromatic rings but also with the ones at the end of PEG chains. Anticipating that the methoxy groups on the aromatic rings are more active than the PEG methoxy termini, the maximum quantity of TMSI was kept to 1.5 equivalents to favor the formation of product 51 without generating a mixture which was too complicated to purify.

Anyway, with 1.5 equivalents of TMSI, a mixture of starting cryptophane 23 and targeting crypotphane 51 was obtained. As mentioned before, these two products were very close in polarity but could be efficiently separated by HPLC in standard conditions. Surprisingly, two different adjacent fractions with the same mass corresponding to the targeted product 51 were obtained. Considering that demethylation of the methoxy groups at the end of the PEG chains can occur, the formation of mono-demethylated cryptophane 56 as the by product was anticipated (Figure 74). In order to conclude about the formation of compound 51, we treated the isolated product with MeI, considering that Cs 2 CO 3 is not strong enough to deprotonate the alcohol group at the end of the deprotected PEG chains but should abstract the proton of the phenol group.

Consequently only the cryptophane 51 was expected to lead to compound 23 (Figure 76). Molecules like PEGylated cryptophanes are not prone to be ionized easily in relatively soft conditions used for electrospray MS due to its lack of ionizable chemical groups. To solve this problem, MALDI-TOF analysis was performed and revealed the presence of the mass of cryptophanes 51/56. Surprisingly, a mass corresponding to cryptophane 57 was also observed (Figure 77). As the long PEG chains predominantly determine the polarity of these molecules, it is difficult to separate them, even by HPLC. Consequently we turned back to the classical condition with LiPPh 2 which does not affect the methoxy group at the end of the PEG chains during demethylation of CTV 19.

b. Demethylation of the PEGylated cryptophane by LiPPh2

As mentioned before, it's difficult to ensure the quality of LiPPh 2 and thus to control an accurate quantity of LiPPh 2 necessary for a selective mono-demethylation. Therefore we needed to determine the precise concentration of the freshly-prepared LiPPh 2 solution using a titration test. After several attempts, the procedure using butanol and a solution of 1,10phenanthroline in xylenes as the indicator revealed to be the most reliable. 117 Optimization of the demethylation of compound 23 with LiPPh 2 was tricky. Progressive increase in LiPPh 2 from 1.1 equivalents to 6 equivalents had only a little effect, resulting in a mixture of cryoptophane 51 and majoritary 23. This situation hardly changed with 30 equivalents of LiPPh 2 after 48 h. On the contrary, if we directly added 12 equivalents of LiPPh 2 at the beginning of the reaction, the mono-demethylated cryptophane 51 was already the major product after 3 hours, together with starting material 23 as well as a little amount of cryptophane 50 (Figure 79). The possible explanation for this phenomenon is that the formed MePPh 2 and its oxide may hinder the further reaction when we re-added the LiPPh 2 solution.

c. Scan of other reagents to perform the demethylation of cryptophane

As mentioned before, the other reagents were tested simultaneously in order to achieve the demethylation of cryptophane 23.

In the literature, the most widely-used reagent for demethylation is boron tribromide. 118 However in our case, BBr 3 is clearly too reactive since it is expected to cleave not only the C-O bonds of the aryl methoxy groups, but also those located in the alkyldioxy bridges. The same problem can be observed with HBr and NaI, which has been proven to be an efficient method for the removal of methyl groups from aryl methyl ether in amino acids. 119 Recently, the 1-decanethiol (Et 2 NCH 2 CH 2 SH.HCl) has been shown to efficiently monodeprotect an aryl methyl ether group when used simultaneously with of a base like NaOtBu. 120 Unfortunately, this milder reagent seems to be not efficient enough to carry out the demethylation of cryptophane 23. Even with a large excess of the reagents, the only product recovered was the starting material. A similar lack of reactivity was also observed with PhSH-(Catalytic) KF in N-Methyl-2-pyrrolidone (NMP), which has been reported as an efficient method for selective cleavage of aryl alkyl ethers under non-hydrolytic and neutral conditions. 121 We have also tried to realize the demethylation with magnesium iodide (MgI 2 )

in ionic liquid like [bmim]BF 4 . 122 Once again, the starting material 23 was recovered after the reaction without trace of deprotected products.

Finally LiPPh 2 seems to give the best result for mono-deprotecting a methyloxy motif selectively despite several major drawbacks.

Conclusion and perspective

As the first attempted strategy to achieve the synthesis of a PEGylated monofunctionalizable cryptophane, this "desymmetrization of cryptophane" approach is based-on a two-step procedure including the synthesis of a symmetric PEGylated cryptophane, and the mono-deprotection of a single methoxy aryl group in order to break the symmetry.

The first step was achieved and optimized leading to the production of about 500 mg of the PEGylated symmetric cryptophane 23 synthesized in five steps with an overall yield of 43 %.

Desymmetrization of the cryptophane through mono-demethylation was attempted using several different methods. The best result was obtained with a solution of LiPPh 2 , which generated a mixture of the targeted water-soluble mono-functionalized cryptophane 51 as majoritary product which was recovered along with two cryptophanes 23 and 50. These last two cryptophanes are difficult to separate at the preparative scale by silica gel flashchromatography but could be eliminated by HPLC. As underlined previously, the separation of different PEGylated cryptophanes is very difficult due to the predominant effect of the long PEG chains on the polarity of these molecules. To achieve an efficient synthesis which can meet the demand for large quantities of products in order to construct biosensors, the introduction of the PEG group might be done after the desymmetrization of the system. This approach was developed in the following strategies.

III. Desymmetrization of CTVs

In this chapter, we will describe the synthesis of a symmetric non-PEGylated CTV which will be used as a template for the step-by-step construction of a desymmetrized cryptophane with PEGylated moieties. These motifs will be introduced lately in order to avoid recurrent problems during purifications for separating structurally similar products. We anticipated that the introduction of only two PEG units should be sufficient to ensure the hydrosolubility of the final molecule.

Three different strategies to break the symmetry of CTV have been envisioned:

1) The functionalization of CTVs firstly with benzyl alcohol derivative linkers of series A and then with series B (Figure 80)

2) The mono-halogenation of CTVs (Part III-2)

3) The selective deprotection of protected CTVs (Part III-3).

Cryptophanes based on the functionalization of CTVs with different benzyl alcohol derivatives

a. Conception and retrosynthetic analysis

This synthetic route is inspired by the classic synthesis of cryptophanol 31. 90 The key step to achieve the desymmetrization is the functionalization of CTV 25 with different benzyl alcohol derivative linkers, including a PEGylated benzyl alcohol derivative 58. Therefore the desymmetrization is achieved prior to the introduction of the PEG moieties.

The retrosynthesis is depicted in Figure 81: As we can see in the Figure 81, the asymmetric cryptophane is basically composed of three moieties: the CTV 25 and two different benzyl alcohol derivatives bearing either a PEG moiety (2 benzyl units) or a protected phenol moiety OR (1 benzyl unit) which can be used for mono-functionalization.

The synthesis of the CTV 25 is already well-described in the literature. Briefly, the phenol group of the commercially available vanillyl alcohol 16 was protected with an allyl group.

Cyclotrimerization of this allylic vanillyl alcohol 14 led to the formation of CTV 32. The allyl groups were subsequently deprotected with palladium acetate (Figure 82), the resulting CTV 25 could be obtained in three steps in 42 % overall yield (5 g scale).

Figure 82: Synthesis of CTV 25

The benzyl alcohol derivative linkers with various R groups can be synthesized from commercial 3,4-dihydroxybenzaldehyde 59. Their synthesis will be introduced latter.

b. Synthesis towards a mono-allyl PEGylated cryptophane

The allyl group is the most widely used phenol protecting group in the chemistry of cryptophanes and therefore was chosen since it is easily cleaved by using a pallado-catalyzed reaction.

i. Synthesis of the allylic benzyl alcohol derivative 37

A straightforward synthesis of the allylic benzyl alcohol derivative 37 is depicted in Figure 83. In the second step, the dibromoethane should be used in a large excess in order to minimize the formation of the by-product 66 (Figure 85). Reduction of the aldehyde motif was easily achieved by using sodium borohydride (NaBH 4 ) in standard conditions. Pure compound 37 was obtained after several extractions without any further purification. It should be mentioned that the alcohol group in molecule 37 was kept unprotected to facilitate the purification in the next step.

To conclude, the allylic benzyl alcohol derivative 37 is obtained in three steps in 42 % overall yield (2 g scale).

ii.

Synthesis of the PEGylated benzyl alcohol derivative 58

In order to circumvent a possible lack of selectivity for the PEGylation of diphenol 59 which could give two position isomers presumably too difficult to separate, we firstly protected the phenol group on position 4 of the aromatic ring with an allyl group (Figure 86).

This selective protection was achieved by reducing the quantity of NaH to 1.1 equivalents. An alternative would be also to recycle side-product 63 formed in the synthesis of 37. The remaining of this synthesis was quite similar to the previous synthesis of 37. However the alcohol group of derivative 70 was protected as a THP group in order to avoid the formation of a tri-alcohol which might be too polar for purification in the subsequent step.

To sum up, the PEGylated benzyl alcohol derivative 58 was synthesized in 6 steps in 34 % overall yield (3 g scale).

iii.

Synthesis towards the mono-allylated PEGylated cryptophane

The synthetic route to the mono-allylated PEGylated cryptophane 73 implies the connection of benzyl alcohol 37 and 58 to CTV 25 as depicted in Figure 87. This reaction was optimized by changing the quantity of 37 in order to suppress the formation of CTV 74. The best result was obtained with 0.5 equivalents of 37, which yielded 37 % of 71 and the reaction yield based on recycling was 45 % (Table 9). No significant improvement was observed when reducing the quantity of 37 and the production of the targeted product became insignificant.

CTV As reported in the case of cyclotrimerization, the classic acidic conditions with pure formic acid were replaced by milder conditions using a mixture 50/50 of formic acid and chloroform in order to prevent hydrolysis of O-allyl groups. 90 However a Claisen rearrangement involving the allyl moiety was still observed in several attempts of this reaction (Figure 89).

Figure 89: Possible Claisen rearrangement in the cyclotrimerization

After purification, cryptophane 73 was obtained in 60 % yield. Cyclotrimerization with scandium triflate Sc(OTf) 3 was also tested. This method was reported in the synthesis of calixarene leaving unreacted allyl groups. 124 According to the LC-MS of the crude product, the targeted cryptophane 73 was formed together with many impurities.

To conclude, the mono-allylated PEGylated cryptophane 73 was synthesized in six steps in 7 % overall yield (50 mg scale). This cryptophane was sufficiently soluble in water to be used for the synthesis of bioprobes (30 mg/mL). Considering that the synthesis of a water-soluble asymmetric cryptophane was already achieved, we investigated various possible chemical routes aiming at functionalizing the PEGylated cryptophane.

iv. Functionalization of the mono-allylated PEGylated cryptophane

We firstly planned to take full advantage of the O-allyl group already grafted onto cryptophane 73. Consequently we tested an olefin metathesis reaction with hex-1-ene and Grubbs catalyst II (Figure 90). Although the formation of targeted product 75 was observed in the LC-MS analysis, the purification revealed to be so complicated that even HPLC was not capable to purify it. Different catalysts were tried (Grubbs I, Grubbs II, Hoveyda-Grubbs II), but the result remained unchanged. Since the functionalization with the O-allyl group has been proven to be difficult, the deprotection of the allyl-protected phenol was therefore carried out on cryptophane 73 in order to release the free phenol group as a convenient access for the introduction of different functional groups.

v. Attempts to synthesize PEGylated cryptophanol

As mentioned in part B-I-1, the expected monofunctionalized cryptophane is supposed to be coupled with a peptide linker in order to approach the synthesis of biosensors. In this linker, a free amine group will be used for grafting onto the cryptophane through an amide coupling. Consequently the most straightforward strategy to access this biosensor was the conversion of cryptophane 73 into a mono-acid PEGylated cryptophane 76. From this standpoint, PEGylated cryptophanol 78 is a very important intermediate (Figure 91). To achieve this goal, a deprotection of the O-allyl group in cryptophane 73 was attempted with Pd(OAc) 2 (Figure 92). This method proved to be efficient in the previously mentioned synthesis of CTV 25 and PEGylated linker 37. According to the NMR spectrum of the obtained crude product, all the allylic protons disappeared. The formation of PEGylated cryptophanol 78 was observed by LC-MS, however this one could not be isolated from the mixture of several non-identifiable PEGylated impurities. Although there are reports about the sensitivity of free phenol groups to the condition used for cyclotrimerization 2 , the formation of PEGylated cryptophanol 78 was still observed by LC-MS analysis of the crude product after cyclotrimerization with a mixture of formic acid and chloroform 50/50. The apparent purity was improved compared to those observed in the deprotection of cryptophane 73, but the existence of a PEGylated impurity jeopardized the purification once more. Additionally, the percentage of cryptophanol 78 in the obtained fraction was even worse after purification suggesting that the PEGylated cryptophanol 78 might be unstable during silica gel flash-chromatography purification.

vi. Conclusion

Mono-allylated PEGylated cryptophane 73 was synthesized in six steps in 7 % overall yield (50 mg scale). This cryptophane is enough soluble in water for being used to synthesize bioprobes in aqueous media. However, neither the functionalization of the double bond in this cryptophane nor the deprotection of the O-allyl group was successful.

The apparent lack of suitable reactivity of cryptophane 73 combined with the previously observed problem of Claisen rearrangement as well as potential isomerization of the double bond led us to conclude that the allyl group might not be the best choice as the protecting group in this strategy.

c. Synthesis towards a mono-benzylated PEGylated cryptophane

In order to overcome the above-mentioned problems encountered with the allyl protecting group, we focused our attention on the benzyl group for suitable phenol protection. Benzyl was expected to be stable in all the anticipated synthetic conditions used and its cleavage was anticipated to be easily archieved by hydrogenolyse. Moreover its applications as a protecting group in CTVs as well as cryptophanes were also reported (Figure 94). 83,125 i.

Synthesis to the mono-benzyl PEGylated cryptophane

The synthesis of benzylic linker 82 is similar to the one of the allylic linker 37 and was achieved as follows: commercial product 3,4-dihydroxybenzaldehyde 59 was O-benzylated with benzyl bromide. The chemoselectivity of this step was ensured by using two equivalents of NaH as the base. Then the O-benzylated product 80 was reacted with a large excess of dibromoethane to yield 81 and its aldehyde function was subsequently reduced with NaBH 4 .

Thus, benzylic ether 82 was obtained in three steps in 20 % yield (0.5 g scale) without any optimizations (Figure 95). The mono-benzyl PEGylated cryptophane 85 was thus obtained in six steps in 9 % overall yield (50 mg scale). This cryptophane was sufficiently soluble in water to be used for the synthesis of bioprobes (30 mg/mL).

ii.

Attempts to synthesize the PEGylated cryptophanol 78

Cryptophane 85 is an asymmetric water-soluble cryptophane which is similar to the monoallyl PEGylated cryptophane 73. Unlike the O-allyl group, the lack of potentially reactive position in the O-benzyl group led us to deprotect the benzyl moiety to isolate the PEGylated cryptophanol 78 as starting material for the construction of biosensors.

The deprotection of benzyl group in cryptophane 85 was attempted by using 10% Pd/C in a methanolic solution of ammonium formate, as reported for the synthesis of a tri-benzylated cryptophane. 125 However, although the 1 H NMR spectrum of the debenzylated product strongly suggested that hydrogenolysis was completed, the expected cryptophanol 78 could not be identified by ES or MALDI-TOF mass spectrum analyses (Figure 97). Therefore, we tried to deprotect the benzyl group of compound 85 with H 2 , Pd/C and CH 3 COOH (Figure 99), which was also reported as a well-working method in the debenzylation of CTV. 83 Unfortunately we did not succeed either though the starting compound disappeared. In order to better understand the fate of this reaction, we removed the acetic acid to slow down the kinetics. A follow-up by LC-MS analysis showed that after 18 h, the reaction milieu contained a mixture of targeted cryptophanol 78 and starting material 85.

After 24 h, the quantity of both the targeted product and the starting material decreased, while a new unidentified product began to form. After 40 h, the reaction was completed leading to the exclusive formation of the unexpected product, the mass of which was identical to that of the unidentifiable product obtained previously by debenzylation with ammonium formate and Pd/C. In order to check this hypothesis, a conversion of the phenyl formate to phenol group was attempted with this unexpected product (Figure 100). 126 No matter how long the reaction was kept, the LC-MS analysis of the crude product showed that the starting compound was not consumed. Therefore we still failed to determine the structure of this isolated compound.

Combined with the previous results obtained with the mono-allylated PEGylated cryptophane 73, the hypothesis of a possible instability of PEGylated cryptophanol 78 might be evoked. This might be explained by a strong dissymmetry of the cryptophane structure induced by two long chains of PEG which could distort and then facilitate the breaking the structure of the cage. This hypothesis explained why cryptophanol 78 was observed but unexpectedly transformed into another product either during the reaction or the purification steps. However we could not produce any hypothetical structure accounting for this phenomenon.

d. Synthesis towards a mono-acid PEGylated cryptophane

According to the previous results discussed in parts III-1-b and III-1-c, it was necessary to develop an alternative strategy avoiding the use of the PEGylated cryptophanol 78 as an intermediate. Since a mono-acid PEGylated cryptophane was highly desirable to synthesize the biosensor, we decided to introduce a methyl acetate group on the tri-functionalized CTV 79 at an earlier stage of the synthesis (Figure 101). Surprisingly, the targeted product 86 was obtained along with by-product 87, suggesting that the central carbon atom of methyl bromoacetate was too electrophilic and thus reacted not only with the phenol group but also with the alcohol group, thus promoting a bis-alkylation (Figure 101). Once again, as the long chains of PEG were predominant for determining the polarity of these molecules, this by-product was impossible to be separated from compound

86.

Consequently we decided to introduce the methyl acetate group directly into the benzyl alcohol derivative linker.

i. The synthesis of the benzyl alcohol derivative with methyl acetate

The synthesis of methyl ester 90 is similar to that of allylic benzyl alcohol derivative 37, with the replacement of allyl bromide with bromomethyl acetate (Figure 102).

Figure 102: Synthesis of the benzyl alcohol derivative with methyl acetate

We had also vainly attempted other optimizations of the original reducing condition (NaBH 4 , MeOH, THF) such as replacing methanol with ethanol or carrying out the reaction without any alcohol.

Finally, the best result was obtained by simply reducing the time of the reaction from 30 min to 10 min. yielding 77% of product 90 though by-product 91 was also obtained along in small quantity (Figure 105). In conclusion, the benzyl alcohol derivative 90 was obtained in three steps in 27 % overall yield (2.5 g scale).

ii. Synthesis to the mono-acid PEGylated cryptophane 76

CTV 91 was obtained as previously described with CTV 25 and 0.5 equivalent of linker 90, yielding 25 % of the mono-functionalized compound based on recovered starting material.

This result was less attractive than that obtained with allyl or benzyl protecting groups, which was actually caused by the instability of the ester group. After purification, the hydrolyzed mono-functionalized CTV 93 was obtained in 29 % yield based on recycled starting material.

The same problem was also observed in the subsequent functionalization of the CTV with the PEGylated linker 58. Due to the presence of the grafted PEG groups on the benzyl moieties, it was complicated to separate the targeted product 92 from the hydrolyzed product 94.

Fortunately, as the ester group was expected to be converted into the corresponding acid at the end of the synthesis, we decided to work on the mixture, anticipating that the presence of the hydrolyzed product would not jeopardize the subsequent cyclotrimerization. It should be noticed that the 55 % yield for this step was calculated for both ester 92 and corresponding acid 94 (Figure 106). At the same time, the recovered mono-functionalized CTV 93 was also used to synthesize cryptophane 76 through the same chemical route as described above. Cryptophane 76 could be obtained in two steps in 24 % yield (Figure 111). This moderate yield probably results from the presence of the carboxylic acid group which complicates the purification process.

The NMR spectrum of the product also displayed the two unexpected signals in 1 H NMR which disappeared upon reflux in methanol. In conclusion, the PEGylated cryptophane carrying a carboxylic acid 76 was synthesized in seven steps in 4 % overall yield (50 mg scale). However, when including the recycling of mono-functionalized CTV 93, the overall yield is improved to 7 % at the hundred mg scale.

As anticipated, we observed that cryptophane 76 displayed a satisfactory hydrosolubility (30 mg/mL).

iii.

Encapsulation of xenon inside cryptophane 76

Cryptophane 76 was dissolved in water and then tested via hyperpolarized 129 Xe NMR.

The equipment for hyperpolarizing xenon has already been introduced in Part A-I-3-b and was used as described therein. Freshly hyperpolarized xenon is condensed in a tube submerged in liquid nitrogen. In the presence of an adapted magnetic field, the hyperpolarization of xenon in solid phase can be conserved for several hours. 130 The tube will be heated by putting into hot water which permits to turn xenon back to the gas phase.

Hyperpolarized xenon gas is introduced into the NMR tube through a three-way valve (Figure 112) Xenon gas contained inside the NMR tube is then frozen and remains in contact with the sample. In order to conclude about this point, a simple deprotection of THP of the trifunctionalized CTV 92 was carried out (Figure 113). The corresponding 1 H NMR spectrum was compared with every spectrum recorded after cyclotrimerization of 92. Detailed analysis showed that the cryptophane was effectively formed after cyclotrimerization, however we realized that the cage was reopened upon heating in refluxing methanol during our attempts to eliminate the two inexplicable signals in 1 H NMR. Then we focused our interest on the two unexpected peaks in 1 H NMR, which probably arises from a molecule who has got stuck in the cage and thus blocks the entrance of xenon.

This process is only possible during the cyclotrimerization because once the cage is formed, the molecules which can enter the cage should also be able to get out. During the cyclotrimerization step, only formic acid and chloroform are brought into contact with the product. Since formic acid has been largely used for the cyclotrimerization of cryptophane and even in the cyclotrimerization of PEGylated cryptophane 87 without being encapsulated, chloroform seems to be the only possibility. Consequently, the cyclotrimerization of molecule 94 was carried out in pure formic acid (Figure 114). The obtained cryptophane 76 was then tested by 1 H NMR, confirming the absence of the two unexpected peaks at around 5.1 and 8.1 ppm (Figure 115). The encapsulation of chloroform by cryptophane with different cavity sizes has been already reported. 131,132,133 Chloroform was currently used as a guest molecule in particular for the crystallization of cryptophane. 134,135 It is anticipated that the affinity between these two molecules is mainly caused by Van de Waals interactions. 136 However, with an affinity constant which is by far lower than that of xenon, chloroform should not hamper the encapsulation of the gas. For example, the affinity constant between chloroform and cryptophane-A (which has more or less the same cavity size than cryptophane 76) is 230 M - 1 , 137 while the affinity constant for xenon is 3900 M -1 .

Moreover, cyclotrimerization with a mixture of chloroform and formic acid has been widely used for synthesizing cryptophane carrying relatively sensitive substituent groups like allyl and no problem for xenon encapsulation has been reported. Therefore it seems highly probable that the PEG moieties have played a role in this unexpected phenomenon.

A possible explanation is that the presence of PEG has changed the cryptophane's affinity

iv. Hyperpolarized 129 Xe NMR analysis of cryptophane 76

As shown in Figure 116, there is more than one signal corresponding to encapsulated xenon, suggesting that this multi-signal results from the coexistence of several distinct complexes in the tested sample.

As this product seemed already very pure in LC-MS (from the complicated 1 H NMR, it was difficult to estimate the purity of product), a purification by HPLC was carried out.

Without discarding any obtained fraction, the original sample was separated into three adjacent fractions and each of them was retested by 129 Xe NMR (Figure 118). collected fractions gave the same 129 Xe NMR spectrum than that observed from the third fraction, thus suggesting either the presence of syn-/anti-isomers, the chelation of cations by the PEG chains, the formation of aggregates etc. This question is presently under investigation.

v.

Conclusions and perspectives

Cryptophane 76 was synthesized in seven steps in a non-optimized 1.4 % yield (if only the green fraction in Figure 115 was considered as the good product). This cryptophane displayed a good water-solubility (30 mg/mL) thanks to the presence of two PEG chains and is mono- Optimizations of yields and scale-up attempts are undergoing, leading us to explore the reasons accounting for the multi-signal problem in 129 Xe NMR. On the other hand, the coupling reaction between cryptophane 76 and a peptide linker is actually being investigated (Figure 119). The protected thiol group (underlined in red in Figure 119) in the targeted cryptophane-linker will be subsequently activated and used to bind the Cetuximab antibody through a classical thiol/maleimide addition, thus completing the construction of the biosensor for NSCLC.

functionalized
Figure 119: Perspectives of cryptophane 76 on its application for constructing biosensors An alternative to the desymmetrization of cryptophane by successive grafting of different benzyl moieties is the mono-halogenation of the CTVs followed by the subsequent conjugation of identical benzyl motif and cyclotrimerization. In the literature, halogenation reactions of phenoxy functions have been extensively described. 138 The presence of multiple chemically equivalent phenoxy functions in cryptophanes offers the possibility to achieve this kind of functionalization.

Cryptophanes based on mono-halogenation of CTVs

Several successful attempts of the halogenation of cryptophanes have been already achieved by our group. For the cryptophanes lacking of reactive positions (like cryptophane[111] 7), this is the only reported method to achieve its functionalization. 83,84,86 The halogen atoms in cryptophanes 9 and 96 can be used for halogen-metal exchange reactions 139 or palladium-catalyzed coupling reactions 140 (Figure 121). Thus, the water-soluble moieties 84,86 and different linkers 83,84 can be added in view of applications to the development of biosensors.

a. Synthesis of mono-halogenated CTV

i. Synthesis of the symmetric CTV 97

CTV 97 was chosen to be the platform for preliminary attempts of mono-halogenation because all the previous halogenation reactions were achieved on cryptophane[111] 7 which is obtained by coupling two units of CTV 97. 59,83 To the best of our knowledge, there are three existing methods for synthesizing CTV 97. 83,141,142 In our case, we chose the synthetic route developed by T. Traoré in our group thanks to its simplicity, inexpensiveness and the potentially easy scale-up (Figure 123). 143 Iodination took place regioselectively: the 1 H NMR spectrum of CTV 100 clearly showed that iodination occurred at the ortho position of a phenoxy ring (Figure 124). The two singlets in the aromatic area at 7.74 and 6.99 ppm represented the two protons in para position at the iodinated aromatic ring (Figure 125). Similarly, mono-bromination of CTV 97 was achieved using N-bromosuccinimide (Figure 126). Surprisingly, a mixture of two regioisomers was obtained. Both compounds were easily separated by chromatography on silica gel to afford compounds 102 and 103 in 81% overall yield. Di-brominated CTV 104 was also observed and recovered (22 %).

b. Synthesis of a PEGylated mono-iodinated cryptophane

The synthesis was performed according to the classic "template method" (Figure 127).

Deprotonation of CTV 100 with Cs 2 CO 3 , followed by treatment with the previously synthesized PEGylated linker 58 in DMF led to the tris-functionalized CTV 105 in 74% yield.

Subsequent cyclotrimerization of this preorganized precursor with formic acid gave cryptophane 106 in a satisfying 41% yield. To conclude, from CTV 97, the PEGylated monoiodinated cryptophane 106 could be obtained in three steps in 22 % overall yield (1.3 % for five steps when starting from the commercially available product (3-methoxyphenyl)methanol 98).

b

In order to decide on the purity of the product, HPLC repurification of the original sample of cryptophane gave two different fractions with identical LC-MS profiles and 1 H NMR spectra. Both of the two fractions (which were called fractions A and B) were also analyzed by MALDI-TOF MS analysis in order to decrease the potential risk that the PEGylated cryptophane was not well-ionized in electrospray. Surprisingly, these two fractions revealed to be fully similar (Figure 129). In both fractions, the minor mass distribution corresponded to If the spectra of these two fractions were overlapped with the one from the sample before purification, it was obvious that these two sharp peaks in fraction A and the "crude" sample were well overlapped.

On the other hand, the broad peak in fraction B was shifted about 5 ppm and had turned even more broad (Figure 130-c). The same test performed with the fraction A demonstrated also the existence of big particles, and their sizes were even larger than the ones presented in fraction B (Figure 132).

Figure 132: The results of DLS of fraction A

To confirm or invalidate this hypothesis, we performed a test of 129 Xe NMR in organic solvent in order to minimize the formation of aggregates. The two fractions were solubilized in tetrachloroethane since this bulky solvent is not expected to compete with xenon for encapsulation inside the molecular cage. However, except for the regular shifting caused by the change of solvent, there was no difference observed between the signals in water and in organic solvents (Figure 133). This result suggested that the multi-peak problem was caused neither by the formation of aggregations, nor the chelation between cations and PEG moieties. ii.

Study of anti-/syn-isomers

Another possible reason for the multi-peak problem in 129 Xe NMR was the formation of syn-/anti-isomers of cryptophanes (Figure 134). For cryptophane [222], the formation of synisomers is reported 141, 145 but very disfavored because cryptophanes with an even number of carbon atoms in the bridges preferentially adopts anti-conformation. In fact, in our previous research on the tri-PEGylated cryptophanes 23 and 24, the formation of syn-isomer was never observed. 87 However, in the case of cryptophane 106, the presence of iodine might weaken the preference for the anti-conformation versus the syn-conformation. In order to study the influence of iodine atom on the syn-/anti preference, we decided to synthesize a mono-iodinated cryptophane without PEG moieties and to separate the two possible isomers (Figure 135). The presence of two different products in this fraction was also proven by thin layer chromatography (TLC) (Figure 137). With dichloromethane/ethyl acetate = 9/1 as the elution solvent, there was just one spot under UV. For the same sample with chloroform/ethyl acetate = 9/1 as the eluting solvent, the original spot was divided into two spots. In order to separate these two products, a purification by preparative TLC was carried out using a mixture of chloroform/ethyl acetate = 9/1 as the eluent. The two products isolated, characterized with NMR and LC-MS, exhibit the same mass with different retention time in LC-MS and totally different spectrums in NMR. Therefore, this strongly suggests the formation of syn-isomer upon cyclotrimerization.

The two isomers of cryptophane 108 were also tested by 129 Xe NMR for comparison with the results obtained with PEGylated cryptophane 106. The 108-anti showed a nice single peak at about 74 ppm as expected (Figure 138). On the other hand, the encapsulation of xenon by 108-syn was difficult to observe after only one scan, but the subsequent selective impulsion at the relative region revealed its signal at about 78 ppm (Figure 139). Unfortunately, there was no reliable explanation accounting for the broad signal in fraction B of 106. However, fraction B was a separable minor fraction (less than 20 %) which is not a concern for the synthesis and applications of cryptophane 106.

c. Synthesis towards a mono-ester PEGylated cryptophane

In the study carried out by G. Milanole, the mono-iodinated CTV 100 was tested towards different reagents to take full advantage of the presence of a suitably positioned iodine which is anticipated to be used for grafting a variety of chemical functions. Among them, the organolithium reagents have drawn a particular attention since the CTV could be easily functionalized with an electron-withdrawing group such as an ester which was useful for the conjugation of a peptide linker.

Thus, a synthetic route leading to the mono-ester PEGylated cryptophane 109 was designed as depicted in figure 140: Surprisingly, the time needed for this reaction was much longer than expected. The reaction was monitored by LC-MS to track the evolution of different intermediates.

According to the result of the test (Table 10), the conversion was not totally completed after 96 h of reaction. During a long period of reaction, the major product was a mono-benzylated CTV. This very interesting observation suggests that it is possible to selectively deprotect a CTV and might be a simple and straightforward strategy for breaking the symmetry of the cryptophanes which will be discussed in part III-3.

CTV The rest of the synthesis was performed according to the classical "template method"

(Figure 144). The functionalization of CTV 110 with the PEGylated linker 58 was achieved in 51 % yield. The subsequent cyclotrimerisation was carried out in pure formic acid. The targeted cryptophane 109 was formed as the major product. Its purification by HPLC is presently under investigation.

d. Conclusion and perspectives

Based on the mono-iodination reaction of CTV 97, a PEGylated mono-functionalized cryptophane 106 was synthesized. This cryptophane could be obtained as a mixture of syn-/anti-isomers in three steps in 18 % overall yield (1.1 % for five steps when starting from the commercially available product 98).

In order to study the formation of syn-/anti-isomers, a non-PEGylated mono-iodinated cryptophane 108 was also synthesized in three steps from CTV 97 (non-optimized 23 % overall yield). To the best of our knowledge, this is the first report of the synthesis of a monohalogenated cryptophane [222]. The syn-/anti-isomers of cryptophane 108 were discriminated and separated by preparative TLC and their properties for encapsulating xenon were tested.

In order to hydrosolubilize cryptophane 108, a demethylation reaction needs to be carried out. The resulting three free phenol groups can be used to introduce PEG moieties. However, considering the stability of the iodine group in the conditions used for demethylation (possibly with LiPPh 2 ), the iodine should be firstly removed to introduce a more stable and easily functionalizable group through a Sonogashira coupling reaction (Figure 145). On the other hand, the synthesis of the PEGylated mono-ester cryptophane 109 was also attempted (Figure 144). The formation of 109 was well observed and its purification was under investigation. Pines. 92 The key step to achieve the desymmetrization is the selective deprotection of CTVs and then different benzyl alcohol derivative linkers, including the PEGylated linker 58, can be grafted.

Cryptophanes based on mono-protected CTVs

In 2004, the group of A. Pines has reported a synthesis of cryptophanol 31 based on a mono-protected CTV 33 which was prepared through a selective deprotection of the tri-allylic CTV 32 using Pd[P(C 6 H 5 ) 3 ] 4 as catalyst and Bu 3 SnH (Figure 147). The unquestionable advantage of this method is that the mono-protected CTV permits to use the benzyl alcohol derivative linkers in large excess in order to functionalize the desymmetrized CTV in better yields.

Taking advantage of our previous experience with the debenzylation of CTV 112, we investigated the synthesis of mono-benzylated CTV 115 in order to functionalize it with an excess of PEGylated linker 58 as a precursor of desymmetrized cryptophanes. According to this synthetic route, the obtained di-functionalized CTV 114 should be successively debenzylated, functionalized with an excess of another benzyl alcohol derivative linker carrying a R group. The resulting grafted CTV can be finally cyclized to yield the targeted PEGylated mono-functionalized cryptophane (Figure 148). Theoretically, the key step to implement this strategy is the synthesis of mono-protected CTV 115 from the corresponding tri-benzyl ether. For this purpose, treatment of CTV 97 with benzyl bromide, K 2 CO 3 and a catalytic amount of tetrabutylammonium iodide (TBAI) as reported for CTV 100 was attempted (Figure 149). Surprisingly, in the absence of iodine, the kinetics of the benzylation was much slower and, after one night of reaction at room temperature, the major product obtained was the mono-benzylated product 115 in 55 % yield after purification (non-optimized). Therefore, this lower reactivity of the CTV revealed to be extremely interesting for the one-step early desymmetrization of cryptophanes.

Figure 149:

The benzylation of CTV 97

The subsequent functionalization of CTV 115 with PEGylated linker 58 was easily achieved in classical conditions in 96 % yield. Unfortunately, the debenzylation reaction could not be achieved through hydrogenolysis with Pd/C or Pd(OH) 2 /C in the presence of acetic acid and therefore, we unfortunately failed to get product 116 (Figure 150). Another option for the cleavage could be proposed such as using ozone 149 or 2,3-dichloro-5,6dicyano-p-benzoquinone (DDQ) 150 to oxidize the benzyl ether to benzoate which allows for the subsequent hydrolysis of the resulting ester under basic conditions. There are also reports about using strong Lewis acid like boron trichloride (BCl 3 ) 151, 152 or using HBr in ionic liquid 153 to achieve this deprotection.

IV Synthesis of Asymmetric CTVs

Our previous attempts to break cryptophane symmetry suggest that the strategy is difficult to implement efficiently and is often marred by the uncontrollable formation of non-recyclable side-products and a large proportion of remaining starting material. Therefore, our experience suggests that the earlier the symmetry is broken, the more efficient the strategy is. For the "template method", the earliest possible point to break the symmetry of the system could be the first cyclotrimerization reaction leading to the formation of asymmetrical CTVs (Figure 151). Therefore we focused our interest on strategies based on the synthesis of an asymmetrized CTV scaffold and then using it as a universal precursor for the construction of unsymmetrical cryptophanes.

Design and retrosynthetic analyse

Symmetrical CTVs are usually formed by cyclotrimerization of three identical monomers.

In part B-III, we have overall discussed how to break the symmetry of symmetrical CTVs. An alternative to this strategy could be to cyclize a mixture of two different monomers in order to form directly an asymmetric CTV which avoids the further desymmetrization of the molecule.

Then the asymmetrical CTV is used as a scaffold for synthesizing mono-functionalized PEGylated cryptophanes through the standard "template method" procedure.

Ideally, the PEG groups should be born by the monomers in order to meet simultaneously the demand for hydrosolubility and for asymmetry. Therefore grafting a bis-PEG CTV with molecule 34 followed by a subsequent cyclotrimerization is expected to give a desymmetrized cryptophane (Figure 152). As a matter of fact, we investigated both strategies which were evaluated for the simple and straightforward synthesis of asymmetric CTVs.

Synthesis of the benzyl alcohol derivatives

The preparation of the methylated monomer 44 (Figure 154) has already been presented in the synthesis of CTV 25 (Figure 82) One step of allylation from vanillyl alcohol gave quantitatively monomer 44. Other monomers can all be obtained in two steps from the previously synthesized allyl ether 63 (Figure 155). The main difficulty for synthesizing monomer 121 is that the reaction time of the reduction of the aldehyde moiety needs to be controlled after 10 min in order to avoid the simultaneous reduction of the methyl ether. Anyway, the yield of this reduction step is lower than that of the similar reduction of molecules 67 and 118.

Synthesis of asymmetric CTVs by cyclotrimerization with different monomers

The first attempted cyclotrimerisation was performed with 2 equivalents of PEGylated monomer 117 and 1 equivalent of benzylated monomer 119, aiming to generate CTV 122 bearing two PEG groups and one benzyl group usable for the further functionalization of the molecule (Figure 156). Unfortunately, the crude product revealed to be a complex mixture, the LC-MS analysis of which could not permit to detect the formation of the desired product.

We anticipated that a polymerization occurred instead of the expected cyclotrimerization, leading to a mixture of unidentified oligomers. In this respect, cyclotrimerization of benzyl derivatives bearing side chains too different in size (-OPEG and -OBn) might favor the formation of bigger oligomers or polymers instead of the expected trimer. However it is difficult to draw a general tendency from this result since only one condition was tested for cyclotrimerization. The cleavage of the allyl ether was performed on the mixture of CTV 32 and 125 and, after liquid/liquid extraction, CTV 25 distributed into the organic phase whereas the targeted CTV 127 was essentially dissolved into the aqueous phase. CTV 127 was then purified by chromatography in order to eliminate a few oligomers, giving the pure asymmetric CTV 127 carrying one carboxylic group in 14 % overall yield (two steps without optimization).

The main advantage of this strategy was the easy elimination of CTV 25 (the main sideproduct) by simple liquid-liquid extraction. Moreover, the unexpected acidification permits to save one step for the whole synthesis. Nevertheless, to carry out this synthesis successfully, the ratio of the two monomers should be fixed to 6 : 1 or even more and optimization of the relatively poor yield seems illusive.

In order to solve this problem, an alternative strategy was investigated. This strategy implies the saponification of the mixture of CTV 32, 125 and 126 with NaOH which do not affect CTV 32 (Figure 160). Therefore it is possible to discriminate between CTV 129 (mono-acid) and CTV 130 (di-acid) which display significantly different polarities. Thus, the asymmetric CTV 129 was purified to yield 14 % for two steps without any optimization.

Although this yield was relatively low, it might be optimized by increasinging the quantity of monomer 121 used for the cyclotrimerization in order to favor the formation of CTV 125 versus CTV 32. Tuning the 44:121 ratio (Figure 157) for this purpose is currently under investigation. Asymmetric CTVs 124 and 127 are very interesting intermediates which can be directly used for the synthesis of water-soluble mono-functionalized cryptophanes.

Synthesis of a mono-acid PEGylated cryptophane

As mentioned above, the crude product of CTV 127 can be directly engaged in the functionalization by O-alkylation with the PEGylated linker 58. Synthesis of the functionalized CTV 131 was performed in 49 % overall yield (two steps together) (Figure 161). Then CTV 131 was cyclized in pure formic acid (Figure 162) to give cryptophane 132 which was isolated in 65 % yield after purification. This CTV showed to be water-soluble with three PEG chains and is mono-functionalized with one free carboxylic acid. As a conclusion, the PEGylated mono-functionalized cryptophane 132 was performed in seven or eight steps (depending on the strategy for obtaining CTV 125) from the commercial 3,4-dihydroxybenzaldehyde 59. This synthetic route enables to isolate compound 132 at a scale of 30 mg in a non-optimized 1.5 % overall yield. There is no doubt that the yields can be improved in particular by augmenting the proportion of monomer 121 used in the cyclotrimerization step (Figure 157). Before this research is undertaken, we are presently investigating the xenon-encapsulation properties of cryptophane 132 in order to evaluate its response in xenon NMR.

The objective of this Ph.D. work was to design and synthesize new cryptophanes usable for the construction of 129 Xe NMR biosensors. These cryptophanes should satisfy three criteria: a satisfactory affinity and exchange rate for xenon gas compatible with in vivo 129 Xe MRI experiments, a good solubility in water, and the capacity to be specifically bio-conjugated in order to be inserted inside a bio-probe. We selected cryptophane [2,2,2] as the molecular cage since it displays both a good affinity and an exchange rate which enables xenon renewal with a sufficient turnover. The intrinsic poor hydrosolubility of cryptophanes was overcome by the grafting polyethylene glycol (PEG) moieties although the presence of bulky and polydisperse moieties raises specific synthetic issues. Different strategies were envisioned to synthesize PEGylated mono-functionalized cryptophanes and their development was the core of this PhD work.

After an overall analysis of the classic "template method" of synthesizing cryptophane [222], five possible strategies to achieve the mono-functionalization of the molecular cage were investigated. The resulting conclusions and corresponding perspectives will be summarized sequentially.

1) Mono-deprotection of a PEGylated C 3 symmetric cryptophane

In this strategy, based on a previous synthesis developed by L. Delacour, a PEGylated symmetric cryptophane 23 was synthesized in 5 steps. After several trials for optimizing and scaling up the synthesis, we improved the overall yield from 5 % to 43 %, and the scale of the synthesis was raised from dozens of milligrams to several hundreds of milligrams.

148

The mono-deprotection was tricky due to the presence of the PEG moieties which predominantly influence the polarity of products and thus complicate the purification steps.

After we have tested numerous deprotection conditions, the majority formation of the target molecule 51 was observed however its purification remains to be improved.

2) Functionalization of CTVs with different benzylic alcohol linkers

Following this strategy, 3 different PEGylated asymmetric cryptophanes were synthesized at the scale of 50 mg. Cryptophane 73 was synthesized in six steps in 7 % overall yield, cryptophane 85 was prepared in six steps in 9 % overall yield; cryptophane 76 was obtained in seven steps in 1.4 % yield (non-optimized). All of these 3 cryptophanes bearing two PEG moieties display sufficient solubility (30 mg/mL).

Cryptophane 73 and 85 need one more deprotection step to be used for grafting.

Unfortunately, the corresponding deprotections of allyl or benzyl groups could not be achieved successfully despite numerous attempts, suggesting that the PEGylated cryptophanol 78 might be unstable.

For this synthesis, optimizations need to be carried out in order to improve the overall yield as well as the production scale. Now the efforts are focused on the hydrosolubilization of this compound by introducing PEG moieties.

Applying the same strategy to the CTV, the tri-functionalized CTV 113 was synthesized in 5 steps in 21 % overall yield. The cyclotrimerization step leading to the corresponding cryptophane 109 as the major product was carried out however the purification stage revealed to be challenging once again.

In contrast to the previous startegies, the symmetry of the cryptophane motif can be broken at the early stage, either by mono-depotecting a symmetric CTV (strategy 4) or by directly constructing an asymmetric CTV with different monomers (strategy 5).

4) Selective deprotection of CTVs

The selective mono-deprotection of CTVs was an alternative to the previouslty exposed strategy. The PEGylated mono-protected CTV 114, which is an important and interesting intermediate in the synthesis of water-soluble mono-functionalized cryptophane, was obtained in two steps in 53 % non-optimized yield. However removal of the benzyl ether is difficult and therefore currently locks this synthetic pathway. Additional deprotection conditions have to be scanned in order to achieve the construction of the corresponding cryptophanes. Purification by chromatography on silica gel (dichloromethane / methanol 100 : 0  90 : 10) yielded quantitatively the compound 17 (6.4 g).

 Experimental protocol for the synthesis of methoxy-polyethylene glycol 550 (20g scale)

Methoxy-polyethyene glycol 550 (20 g, 39 mmol) and tosyl chloride (74 g, 387 mmol) were dissolved in 400 mL of anhydrous dichloromethane. Then triethylamine (37.8 mL, 271.1 mmol) was added dropwise under nitrogen atmosphere. The reaction was heated to reflux of dichloromethane overnight. The solvent was evaporated and the residue was taken up in diethyl ether and washed twice with water. The aqueous phase was then recovered and extracted with dichloromethane. Finally the organic phase was dried over MgSO 4 and the solvent was evaporated. The compound 17 was obtained in 60 % yield (15.7 g).

Yellow oil

Compound 106

In a 50 mL round-bottom flask equipped with a septum and a magnetic stirrer, 105 (40.0 mg, 0.015 mmol) was dissolved in formic acid (20 mL). The reaction mixture was slowly stirred at 60 °C for 3 hours. Subsequently, the colorless solution was allowed to cool down to room temperature and concentrated in vacuo. The brownish crude oil was purified by preparative HPLC (water / acetonitrile 100 : 0  0 : 100) to afford expected product 20 (14.5 mg, 41%).

Yellow oil Abstract: Among all the imaging techniques, magnetic resonance imaging (MRI) offers several advantages owing to its low invasiveness, its harmlessness and its spatial indepth resolution but suffers from poor sensitivity. To address this issue, different strategies were proposed, including the utilization of hyperpolarizable species such as 129 Xe.

Xenon is an inert gas with a polarizable electronic cloud which leads to an extreme sensitivity to its chemical environment. Its capacity of being hyperpolarized makes it possible to obtain a significant gain of sensitivity. Nevertheless, xenon has no specificity to any biological target therefore it needs to be encapsulated and vectorized. Different molecular cages were proposed and we are particularly interested in cryptophane.

In this context, the objective of this thesis is to design new cryptophanes which can be used as molecular platforms to construct novel 129 Xe MRI biosensors usable for in vivo imaging. To meet this demand, these cryptophanes should be mono-functionalizable and enough soluble in water.

In this thesis, the polyethylene glycol (PEG) group is used to improve the poor solubility of the hydrophobic molecular cage. And there is a systematic discussion of how to break the symmetry of cryptophanes and different strategies were attempted to synthesize monofunctionalized cryptophanes.

As a result, several PEGylated monofunctionalized cryptophanes were obtained and their properties for encapsulating xenon were tested.

L

  'Imagerie par Résonance Magnétique (IRM) est une technique d'imagerie médicale couramment utilisée dans le milieu hospitalier pour diagnostiquer des maladies sur des organes tels que le cerveau et les poumons. Elle est basée sur le principe de la Résonance Magnétique Nucléaire (RMN) et elle repose en général sur la détection des protons ( 1 H) présents dans le corps humain. Cette technique d'imagerie se caractérise par une haute résolution spatiale/temporelle et est devenue un outil incontournable pour l'observation de tissus en profondeur. Cependant, le principal inconvénient de l'IRM est sa faible sensibilité. L'utilisation d'agents de contraste est souvent nécessaire afin d'améliorer la qualité de l'image et donc de faciliter le diagnostic. Les plus utilisés actuellement sont les complexes de gadolinium et les nanoparticules d'oxyde de fer super paramagnétiques. Malgré l'utilisation d'agents de contraste, l'IRM 1 H n'est pas encore suffisamment sensible pour pouvoir faire de l'imagerie à l'échelle moléculaire. Cependant, l'observation des protons atteint rapidement ses limites. L'utilisation d'autres espèces hyperpolarisables telles que l'hélium ( 3 He), le carbone ( 13 C) ou le xénon ( 129 Xe) peut permettre l'accès à de meilleures sensibilités. Au cours de ma thèse, nous nous sommes intéressés à l'IRM du xénon (IRM 129 Xe). Le xénon est un gaz de spin 1/2, inerte, non toxique et déjà connu pour ses propriétés anesthésiantes et pour faire de l'imagerie des poumons humains par IRM 129 Xe. Il est très sensible à son environnement et possède donc une large gamme de déplacements chimiques. D'autre part, grâce à une technique d'hyperpolarisation du xénon, il est actuellement possible d'obtenir un gain en sensibilité considérable de l'ordre 10 4 à 10 5 . Cependant, n'étant spécifique d'aucun récepteur biologique, le xénon nécessite d'être vectorisé. Pour ce faire, des auteurs ont proposé son encapsulation dans une cage moléculaire capable de reconnaître la cible biologique à imager. Les meilleurs candidats pour une encapsulation optimale du xénon sont aujourd'hui les cryptophanes, qui possèdent les meilleures propriétés d'encapsulation du xénon, en termes de constante d'affinité, de vitesse d'échange entrée/sortie du xénon dans la cage et de relaxation du xénon dans le cryptophane.

(

  Figure II).

Figure II :

 II Figure II: La voie synthétique générale de « méthode de template » avec des 5 points possibles pour casser la symétrie des cryptophanes

c. 3 . 4 . 1 . 2 . 3 . 3 . 2 . 3 . 1 . 2 .TBAI

 3412332312 ABBREVIATION LIST

Figure 1 1 :

 1 Figure 1 1 : Two-dimensional views of the common landmark (the skin) for (a) PET with attenuation correction. (b) PET without attenuation correction. (c) CT. (d) MRI : T1. (e) MRI :T2. (f) MRI : fluid-attenuated inversion recovery (FLAIR)

Figure 2 :

 2 Figure 2: General structure of cryptophanes

Figure 3 :

 3 Figure 3: interaction of proton spins with a magnetic field in NMR

Figure 4 :

 4 Figure 4: Return of the magnetization to equilibrium

  field B 0 Switch of magnetization M z probe another zone of the object (Figure5). The magnetic signal which is emitted by nuclei after the resonance is recorded and then a data processing treatment allows reconstructing a three-dimensional image shown in successive cuts.

Figure 5 4 :

 4 Figure 5 4 : Principle of magnetic resonance imaging in NMR spectrum (left) and in 1D MRI (right)

Figure 6 :

 6 Figure 6: Commercial gadolinium-based contrast agents 9

Figure 7 :

 7 Figure 7: Magnetization of the super-paramagnetic iron oxide in the presence of a magnetic field

Figure 8 :

 8 Figure 8: Distribution of spins before and after hyperpolarization

  achieved by parahydrogen-induced polarization technique (PHIP) which will be presented in part A-I-3-b.

Figure 9 :

 9 Figure 9: Change in metabolic pattern visualized by 13 C-MR spectra originating from the area containing vena cave (A), skeletal muscle (B), and P22 tumor tissue (C). Whereas alanine is most prominent in skeletal muscle, the tumor shows a typical lack of alanine signal and has a high lactate signal 21

Figure 10 :

 10 Figure 10: Chemical shifts of xenon in different environments

Figure 11 :

 11 Figure 11: Equipment for hyperpolarizing xenon gas 46

Figure 12 :

 12 Figure 12: Electronic polarization of rubidium

Figure 13 :

 13 Figure 13: Transfer of polarization between rubidium and xenon

Figure 14 48 :

 48 Figure 1448 : Spectrums of 129 Xe NMR for a same sample (a) hyperpolarized (one scan)(b) non-hyperpolarized (15 h of acquisition)

Figure 15 :

 15 Figure 15: Concept of bio-sensors in 129 Xe MRI i.The selection criteria

3 .

 3 A NMR chemical shift for the encapsulated xenon which is significantly different from the free xenon. 4. A minimization of the relaxation of xenon inside the cage 5. A sufficient solubility of the molecular cage in aqueous milieu and an easy functionalization to allow in vivo applications.

Figure 16 :

 16 Figure 16: Structure of α-cyclodextrin iii.Calixarenes

54

 54

Figure 17 :

 17 Figure 17: (a) calix[4]arene with para-tert-butyl substituents. (b) Structure of STCAS iv. Hemicarcerands

Figure 18 :

 18 Figure 18: Structure of the hemicarcerand which can be used to encapsulate xenon v. Cucurbiturils

57

 57

Figure 19 :

 19 Figure 19: (a) General structure of cucurbit[n]urils (b) Structure of CB[5]. (c) Structure of CB*[6]

Figure 20 :

 20 Figure 20: General structure of cryptophanes

  via inhalation (Figure 21, unpublished results). The signal of xenon encapsulated in hydrophilic cryptophane which has crossed the lung epithelium unambiguously shows up at 60 ppm. A continuous exchange between the large reservoir of hyperpolarized xenon constituted by the lung (Xe gas ) and caged xenon has been highlighted, affording a further gain in sensitivity to the method.

Figure 21 : 1 .

 211 Figure 21: Hyperpolarized 129 Xe MRI of rat lungs and associated localized 129 Xe spectroscopy (Dr. P. Berthault, unpublished results)

Figure 22 :

 22 Figure 22: (a) General structure of CTVs. (b) Cryptophane A also called cryptophane[222](OMe) 6

Figure 23 :

 23 Figure 23: Two CTV enantiomers

Figure 24 :

 24 Figure 24: Configurations syn and anti of cryptophanes Different bridging chains (Z and Z') and different substituent groups (R 1 and R 2 ) also induce different symmetries.

Figure 25 :

 25 Figure 25: Different conformation of CTVs and their possible interconversions

Figure 26 :

 26 Figure 26: Different conformations of cryptophanes

Figure 28 :

 28 Figure 28: General synthesis of cryptophanes by the direct method

Figure 31 :

 31 Figure 31: Probable intermediates for cryptophane syn and anti in the template method

Figure 32 :

 32 Figure 32: Synthesis of the cryptophane[111] by the coupling of CTVs

Figure 33 :

 33 Figure 33: Synthesis of cryptophane[112] and [122] by coupling of CTVs

Figure 34 :C 5

 345 Figure 34: Hydrosolubilization of cryptophane A by introducing 6 carboxylic acid groups With an excellent affinity for xenon, cryptophane[111] 7 is also a potential candidate for the development of in vivo biosensors. The first synthesis of its water-soluble derivative was achieved by the η 6 -coordination of the arene rings by cationic, electron-withdrawing [(η 5 -C 5 Me 5 )Ru II ] + moieties (hereafter [Cp*Ru] + )78 , giving rise to the air stable chloride salt

Figure 35 :

 35 Figure 35: Hydrosolubilization of cryptophane[111] by cordination with 6[Cp*Ru] +

Figure 37 :

 37 Figure 37: Hydrosolubization of cryptophane[222] by introducing dendrons

Figure 38 :

 38 Figure 38: Hydrosolubilization of cryptophane[111] by introducing a trisulfonated linker

Figure 40 :

 40 Figure 40: Synthesis of the water-soluble PEGylated cryptophanes

Figure 41 :

 41 Figure 41: Cycloaddition with one of three propargyl groups on the cryptophane

Figure 42 :

 42 Figure 42: Peptide coupling with one of six carboxylic acid groups on the cryptophane

Figure 43 :

 43 Figure 43: Synthesis of cryptophanol by the group of T. Brotin and J.P. Dutasta

Figure 44 :

 44 Figure 44: Synthesis of cryptophanol by the group of A. Pines

Figure 45 : 25 .

 4525 Figure 45: Mono-deprotection of cryptophane A

Figure 46 :

 46 Figure 46: Synthesis of mono-functionalized cryptophane[223]

Figure 47 :

 47 Figure 47: Mono-functionalization of cryptophane[111]

Figure 48 :Figure 49 :Figure 50 :

 484950 Figure 48: Structure and conception of the cryptophane-based biotin/avidin bio-sensor

Figure 51 :

 51 Figure 51: a) structure of the pentafluorophenol/transferrin biosensor. b) Internalization of the biosensor. c) Fluorescence (up) and bright field (down) images of cells incubated with biosensor, with (left) and without (right) pronase treatment. d) 129Xe NMR spectrum of cells incubating biosensor (left) and corresponding supernatant (right)

Figure 52 :

 52 Figure 52: a) structure of the biosensor with a fluorescein moiety. b) Conception of the experiment. c) Images of the hyperpolarized Xe MRI

Figure 53 :

 53 Figure 53: Different targeting water-soluble mono-functionalized cryptophane[222]

  Xe MRI-based biosensors for the diagnosis of NSCLC and follow up of treatment efficacy in an orthotopic tumor mice model.The designed biosensors are composed of three conjugated parts which are assembled by a peptide linker (Figure54).

Figure 54 :

 54 Figure 54: Conception of the cryptophane-based biosensor for lung cancer

  Cryptophane[222] hexa-carboxylic acid 48 was chosen as the molecular cage for constructing the biosensor. This compound was kindly provided by Dr. Thierry Brotin (ENS Lyon). With a well-developed synthetic route, cryptophane 48 can be obtained in several hundred milligrams in 1.4 %overall yield (Figure55).101

Figure 55 :

 55 Figure 55: Synthesis of the cryptophane[222] hexa-acid carboxylic 48

Figure 56 :

 56 Figure 56: The step of mono-functionalization of cryptophane in the synthesis of the biosensor

Figure 57 :

 57 Figure 57: General pathway to synthesize a cryptophane[222] using the "template method"with the possible points to break its symmetry

Figure 58 :

 58 Figure 58: 129 Xe NMR spectrum of a) tri-PEGylated cryptophane. b) hexa-PEGylated cryptophane

Figure 59 :

 59 Figure 59: Activation of mPEG-550

Figure 60 :

 60 Figure 60: General description of the strategy "desymmetrization of a symmetric cryptophane". In this figure, R 2 represents the functionalizable group, R 3 represents the solubilizing group

Figure 61 :

 61 Figure 61: Synthesis of the PEGylated cryptophanes by L. Delacour

Figure 62 :

 62 Figure 62: Attempts of Delacour to achieve the demethylation of the PEGylated cryptophane 87

Table 4 :

 4 Result of the test of solubilityThe crude product was solubilized in water and washed several times with toluene until the elimination of the impurity from the organic phase. Then the aqueous solution was extracted with dichloromethane, affording 3 g of very pure CTV 20. This improvement enabled to increase the scale of purification by 15 times, leading to an optimization of the yield (2 steps) from 40 % to 85 %.

Figure 63 :

 63 Figure 63: β-elimination of the benzyl alcohol derivatives

Figure 64 :

 64 Figure 64: Synthesis of the benzyl alcohol derivative with different leaving groups

Figure 65 :

 65 Figure 65: General image of the alkylation reaction of CTV

Figure 66 :

 66 Figure 66: Possible side-reaction during the functionalization of CTV 20

  should be mentioned that the quantity of sodium hydride must be minimized(3.5 equivalents) because it can also react with the alcohol group of compound 39. The purity of the sodium hydride suspension (60 % or 95 %) and the quantity of compound 39 were also important factors: the best result was obtained with NaH (95%) as the base and 5 equivalents of 39 (57 % yield, entry 8).

  This product was purified by reversed-phase chromatography (water / acetonitrile = 100 : 0  0 : 100) because the CTV triol 21 is highly polar.Therefore CTV 20 was functionalized with 5 equivalents of 39 in the presence of 3.5 equivalents of NaH 95 % as the base, to yield 200 mg of functionalized CTV 21 (yield 58 %).

Figure 68 :

 68 Figure 68: Cyclotrimerization of the THP-protected cryptophane precursor 55

Figure 69 :

 69 Figure 69: Mono-demethylation of the cryptophane 23

Figure 70 :

 70 Figure 70: Synthesis of the PPh 2 Li solutionThis method, most widely used in demethylation of cryptophanes, has been proven to be efficient with the demethylation of PEGylated CTV 19 but has also several drawbacks:

Figure 71 :

 71 Figure 71: Examples of using TMSI in the dealkylation of aliphatic alkyl ethers 107, 108

Figure 72 :

 72 Figure 72: Mono-demethylation of PEGylated cryptophane 23 with TMSI

Figure 73 :

 73 Figure 73: Structures of di-demethylated cryptophane 50 and tri-demethylated cryptophane

Figure 74 :

 74 Figure 74: Possible by-product 56 in the demethylation with TMSI

Figure 75 :

 75 Figure 75: Iron(III) chloride -pyridine test for water-Insoluble phenols

Figure 76 :

 76 Figure 76: Methylation of the cryptophane 51

Figure 77 :Figure 78 :

 7778 Figure 77: Possible by-product cryptophane 57 in the demethylation with TMSI.This unexpected iodination can be explained by the particular mechanism of the demethylation reaction with TMSI (Figure78). As a matter of fact, TMSI can cleave the C-O bond at both the two sides of the oxygen. Although cleavage of the methyl is preferential, the competitive formation of iodide by-products like cryptophane 57 has been already reported.116

Figure 79 : 19 .

 7919 Figure 79: Mono-demethylation of PEGylated cryptophane with LiPPh 2

Figure 80 :

 80 Figure 80: General description of the strategy "functionalization of CTVs with the different benzyl alcohol derivatives". In this figure, R 2 represents a functionalizable group and R 3 represents the solubilizing group

Figure 81 :

 81 Figure 81: Retrosynthetic analysis of the mono-functionalized PEGylated cryptophane based on the mono-functinalization of CTV 25

Figure 83 :

 83 Figure 83: Synthesis of the benzyl alcohol derivative 37

Figure 84 :

 84 Figure 84: Explanation of the formation of different products in a) using 1 equivalent of base. b) using 2 equivalents of base

Figure 85 :

 85 Figure 85: The possible by-product in the reaction between molecule 60 and dibromoethane.

Figure 86 :

 86 Figure 86: Synthesis of the PEGylated benzyl alcohol derivative 58.

Figure 87 : 37 .

 8737 Figure 87: Synthesis towards the cryptophane 73

Figure 88 :

 88 Figure 88: Mono-functionalization of CTV 25 with benzyl alcohol derivative 37.

Figure 90 :

 90 Figure 90: Attempts of metathesis reaction with cryptophane 73.

Figure 91 :

 91 Figure 91: Retrosynthetic analysis of PEGylated mono-acid cryptophane 76.

Figure 92 :

 92 Figure 92: Attempt to synthesize PEGylated cryptophanol 78.

Figure 93 :

 93 Figure 93: Deprotection of CTV 72 and its following cyclotrimerization

Figure 94 :

 94 Figure 94: Utilization of benzyl as a protecting group in the chemistry of a) CTV 83 . b) cryptophane 125

Figure 95 :

 95 Figure 95: Synthesis of benzyl alcohol derivative 82.

Figure 96 :

 96 Figure 96: Synthesis towards cryptophane 85

Figure 97 :

 97 Figure 97: Attempts to hydrogenolyse the benzyl ether of cryptophane 85 with Pd/C and ammonium formate

Figure 98 :

 98 Figure 98: Attempts to hydrogenolyse the benzyl group of the cryptophane precursor 84 with Pd/C and ammonium formate

Figure 99 :

 99 Figure 99: Attempts to debenzylate cryptophane 85 with Pd/C and H 2 1 H NMR analysis of this unexpected product ensured the total cleavage of benzyl group without giving more meaningful information due to the complexity and asymmetry of this molecule. The LC-MS and MALDI-TOF analyses showed that the mass of this product is M unexpected = M compound 78 + 6/50/94… (because of the polydispersity of PEG). The only plausible explanation for M unexpected is the mass of cryptophane 86 (Figure 100) plus a sodium cation.

Figure 100 :

 100 Figure 100: Attempts to prove the formation of cryptophane 86

Figure 101 :

 101 Figure 101: Attempt to synthesize the precursor of the mono-ester PEGylated cryptophane 86.

Figure 105 :

 105 Figure 105: Optimization of the reduction by reducing the time of reaction.

Figure 106 :

 106 Figure 106: Functionalization of CTV 25 with the benzyl alcohol derivatives carrying methyl acetate groups and PEG groups

Figure 107 :

 107 Figure 107: Cyclotrimerization to form the PEGylated cryptophanes carrying methyl acetate and carboxylic acid groups

Figure 108 :

 108 Figure 108: Saponification of the cryptophane 77

Figure 109 :

 109 Figure 109: The two unexpected peaks in NMR spectrum after cyclotrimerization

Figure 110 :

 110 Figure 110: The disappearance of the two unexpected peaks by heating the cryptophane in MeOH

Figure 111 :

 111 Figure 111: Synthesis of cryptophane 76 from mono-functionalized CTV 93.

Figure 112 :

 112 Figure 112: Introduction of hyperpolarized xenon into the NMR tube

Figure 113 :

 113 Figure 113: Deprotection of THP group in molecule 92

Figure 114 :

 114 Figure 114: The cyclotrimerization of 94 with pure formic acid

Figure 116 :

 116 Figure 116: 129 Xe NMR spectrum of cryptophane 76 in water

  for chloroform, thus challenging the xenon encapsulation process. However the same sample of cryptophane 76 used for xenon NMR was previously dissolved in chloroform for proton NMR analysis without hampering the encapsulation of xenon in the latter experiments.Another more plausible hypothesis might account for this phenomenon: when the cage is closed, an ethylene glycol motif might penetrate inside the cavity of cryptophane and then might block a molecule of chloroform inside the cage. Therefore the PEG moiety hampers the release of chloroform out of the cage, thus blocking the entrance of xenon molecule independently of the intrinsic affinity of xenon for cryptophanes (Figure117).

Figure 117 :

 117 Figure 117: The simple conceptual drawing to explain the hypothesis that a part of the PEG chain, stuck inside the cryptophane cage, retained a molecule of chloroform inside the cryptophane. This hypothesis might also account for the presence of the two unexpected signals in 1 H NMR. The peak around 5.1 ppm corresponds to protons of a partially encapsulated ethylene glycol motif and the other at around 8.1 ppm might be the signal of the encapsulated chloroform. Both signals are shifted for about 1 ppm related to their theoretical value because of interactions between the protons and the surrounding structure of cryptophane. Moreover,as the guest molecule inside the cage is so cumbersome, the cage itself has become more vulnerable and less stable than the free ones. This might explain propensity of the cage to

Figure 118 :

 118 Figure 118: The 129 Xe NMR result for the three fractions after HPLC. Each spectum represents one of the fractions

Figure 120 :

 120 Figure 120: General description of the strategy "synthesis of PEGylated mono-functionalized cryptophane based on mono-halogenated CTVs". In this figure, R 2 represents a functionalizable group and R 3 represents the solubilizing group

Figure 121 : 96 .

 12196 Figure 121: Structure of cryptophane[111] 7 and its halogenated product cryptophane 9 and 96.

Figure 122 :

 122 Figure 122: Retrosynthetic analysis of the PEGylated mono-halogenated cryptophanes.

Figure 123 :

 123 Figure 123: Scalable synthesis of CTV 97 developed by T. Traoré

Figure 124 :

 124 Figure 124: Mono-iodation of CTV 97.

Figure 125 :

 125 Figure 125: The aromatic area of the 1 H NMR spectrum of the mono-iodinated CTV 100.

Figure 126 :

 126 Figure 126: Mono-bromination of CTV 97.

Figure 127 :

 127 Figure 127: Synthesis of cryptophane 106 from CTV 100. i. The analysis of cryptophane 106 by 129 Xe NMR The obtained cryptophane 106 was dissolved in water and then tested by 129 Xe NMR (Figure 128). Beside the large signal at about 197 ppm which corresponds to free xenon in water (Figure 128a), the signal related to the cryptophane-encapsulated xenon corresponds to 2 sharp signals at 70 and 56 ppm and a broad peak around 66 ppm (Figure 128-b).

Figure 128 :

 128 Figure 128: The 129 Xe NMR spectrum of a) the full range. b) the zoomed-in region between 50 ppm and 80 ppm.

  a

  the correct mass of cryptophane 106 (M = 2383 +/-44) and the major distribution corresponded the mass of cryptophane 106 plus a sodium cation (M + [Na + ] = 2406 +/-44).

Figure 129 :

 129 Figure 129: Result of MALDI-TOFMS analysis of fractions A and B. The samples were solubilized in a mixture 50/50 of water and methanol with 0.1 % of TFA.

Figure 130 :

 130 Figure 130: 129 Xe NMR spectra of a) fraction A. b) fraction B. c) spectra overlapped with the one obtained from the sample before purification of HPLC, in which the red curve represents the fraction A, the blue curve represents the fraction B, and the green curve represents the "crude" product

Figure 131 :

 131 Figure 131: The results of DLS of fraction B

Figure 133 :

 133 Figure 133: 129 Xe NMR results of fraction A (up) and fraction B (down) in tetrachloroethane

Figure 134 :

 134 Figure 134: The possible anti-/syn-isomers of cryptophane 106

Figure 135 :

 135 Figure 135: Synthesis of cryptophane 108 from mono-iodinated CTV 100.

Figure 136 :

 136 Figure 136: LC-MS chromatogram of two peaks (t=10.70 and 11.01) showing the same mass of cryptophane 108

Figure 137 :

 137 Figure 137: TLC result of the previously discussed fraction. On the left, the sample was eluted with chloroform/ethyl acetate = 9/1, and on the right with dichloromethane/ethyl acetate = 9/1

Figure 138 :Figure 139 :

 138139 Figure 138: The 129 Xe NMR spectrum of the anti-isomer of cryptophane 108

Figure 140 :

 140 Figure 140: Retrosynthetic analysis of cryptophane 109

Figure 141 :

 141 Figure 141: Protection of the phenol groups in CTV 100

Figure 142 :

 142 Figure 142: Halogen-metal exchange reaction of CTV 111

Figure 143 :

 143 Figure 143: Removal of the benzyl groups in CTV 122

Figure 144 :

 144 Figure 144: Synthesis to the mono-ester PEGylated cryptophane 109

Figure 145 :

 145 Figure 145: Perspective on the hydrosolubilization of cryptophane 108

Figure 146 :

 146 Figure 146: General description of the strategy "synthesis of PEGylated mono-functionalized cryptophane based on mono-protected CTVs". In this figure, R 2 represents a functionalizable group and R 3 represents the solubilizing group

92

 92

Figure 147 :

 147 Figure 147: Selective deprotection of the allyl groups in CTV 32.

Figure 148 :

 148 Figure 148: Retrosynthetic analysis of the strategy based on the use of the mono-protected CTV 115.

Figure 150 :

 150 Figure 150: Functionalization of CTV 115 and subsequent debenzylation

Figure 151 :

 151 Figure 151: General description of the strategy "synthesis of PEGylated mono-functionalized cryptophane based on the synthesis of asymmetric CTVs". In this figure, R 2 represents a functionalizable group and R 3 represents the solubilizing group

Figure 152 :

 152 Figure 152: One possible retrosynthetic analysis of the mono-functionalized PEGylated cryptophanes based on the asymmetric CTV

Figure 153 :

 153 Figure 153: The other possible retrosynthetic analysis of the mono-functionalized PEGylated cryptophanes based on the asymmetric CTV.

Figure 154 :

 154 Figure 154: Synthesis of methylated monomer 44

Figure 155 :

 155 Figure 155: Synthesis of different substituted monomers

Figure 156 :

 156 Figure 156: Cyclotrimerization between monomer 117 and 119

Figure 157 :

 157 Figure 157: Cyclotrimerization between monomer 44 and 119 and the subsequent deprotection

Figure 158 :

 158 Figure 158: Hetero-cyclotrimerisation of monomers 44 and 121

Figure 159 :

 159 Figure 159: Deallylation of the CTV 32, 125 and 126

Figure 159 :

 159 Figure 159: Saponification of the mixture of CTV 125 and 126.

Figure 160 :

 160 Figure 160: Cleavage of the O-allyl groups in the CTV 129.

Figure 161 :

 161 Figure 161: Deprotection of CTV 129 and the subsequent functionalization with 58.

Figure 162 :

 162 Figure 162: Cyclotrimerisation of functionalized CTV 131.

5 ) 17 

 517 Synthesis of asymmetric CTVs through the cyclotrimerizations with different monomersBy implementing this strategy, two asymmetric CTVs were synthesized in two steps (25% overall yield for CTV 124, 14 % yield for CTV 127, non-optimized). CTV 127 was then engaged in the synthesis of the PEGylated mono-functionalized cryptophane 132. This synthetic route enabled to isolate compound 132 in 7 steps in 1.5 % overall yield which has to be optimized. We are presently investigating the xenon-encapsulation properties of this cryptophane and preliminary results suggest that its capacity to encapsulate and exchange xenon complies with the specifications determined by the development of bio-probes usable for xenon MRI. This work demonstrates that the solubilization of cryptophane by grafting polyethylene glycol moieties which is a prerequisite to the development of bio-probes recurrently complicates the purification of intermediates and final compounds which only slightly differ in polarity. Therefore, desymmetrization at the late stage of the synthesis is challenging.Moreover, as a bulky group, PEG moities can trap water or ions, which may jeopardize further chemical modification of the cryptophanes. Presently, the most promising synthetic routes imply the early desymmetrization of the CTV unit and the subsequent introduction of three benzyl-PEG adducts which are finally cyclotrimerized. After optimization and scale-up of the synthesis, cryptophanes 79 and 132 will be conjugated to a lysine-containing peptide bearing a fluoresceine moiety in order to produce a dual imaging (fluorescence/ 129 Xe MRI) bio-probe in sufficient quantities for in vivo experiments on NSCLC mice models.Compound Experimental protocol for the synthesis of methoxy-polyethylene glycol 550 (5g scale) Methoxy-polyethyene glycol 550 (5 g, 9.7 mmol) and tosyl chloride (18.5 g, 96.8 mmol) were dissolved in 100 mL of anhydrous DCM. Then triethylamine (9.5 mL, 70 mmol) was added dropwise under nitrogen atmosphere. The reaction was heated to reflux of dichloromethane overnight. The solvent were evaporated and then an extraction water / dichloromethane was carried out. The organic phase was dried over MgSO 4 and dichloromethane was evaporated.

Compound 97 13 C

 9713 Boron tribromide (200 mL of a 1 N solution of BBr 3 in CH 2 Cl 2 ) was added dropwise to a suspension of 2,7,12-Trimethoxy-10,15-dihydro-5H-tribenzo[a,d,g]cyclononene 99 (10 g, 28 mmol) in dry CH 2 Cl 2 (40 mL) at 0 °C. The solution was warmed to room temperature and stirred overnight. Then it was poured onto an ice and H 2 O slurry. The aqueous solution was neutralized to pH 6 and filtered. The solid residue was washed with 120 mL of hot water and dried to provide a slightly colored powder. The powder was poured into 30 mL of acetonitrile and sonicated for 20 min. The precipitate was filtered, washed with 45 mL of acetonitrile and dried to furnish 8.8 g (100 % yield) of pure 97. White powder 1 H NMR (400 MHz, acetone-d6, δ in ppm): 3.54 (d, J = 13.4 Hz, 3H), 4.77 (d, J = 13.4 Hz, 3H), 6.54 (dd, J1 = 8.0 Hz, J2 = 2.6 Hz, 3H), 6.87 (d, J = 2.6 Hz, 3H), 7.21 (d, J = 8.0 Hz, 3H), 7.99 (s, 3H) NMR (100 MHz, acetone-d6, δ in ppm): 30.79, 113.55, 116.16, 130.58, 130.92, 141.56, 155.78 HRMS-ESI (m/z): 317.1190, [M-H] -

Compound 121 Compound 120 ( 1 . 13 C 13 C 76 - 6 . 97 (m, 6H) 13 C

 121113137669713 95 g, 7.8 mmol) was dissolved in THF(25 mL). The solution was cooled to 0 °C with an ice bath and then NaBH 4 (296 mg, 7.8 mmol) and methanol (3.2 mL, 78 mmol) were added. The reaction was kept at 0 °C with stirring for 10 minutes and then it was quenched with a solution of HCl 1N and extracted with dichloromethane. The organic phase was dried over MgSO 4 and concentrated under vacuum. A purification by chromatography on silica gel (cyclohexane / ethyl acetate 50 : 50  0 : 100) afforded the product 121 in 61 % yield (1.20 g). White solid 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 3.80 (s, 3H), 4.59 (s, 2H), 4.61 (d, J = 5.4 Hz, 2H), 4.71 (s, 2H), 5.28 (dd, J1 = 10.5 Hz, J2 = 1.3 Hz, 1H), 5.41 (dd, J1 = 17.3 Hz, J2 = 1.3 Hz, 1H), 6.00 -6.14 (m, 1H), 7.85 -7.97 (m, 3H) NMR (100 MHz, CDCl3 , δ in ppm): 52.19, 65.01, 66.61, 69.99, 114.21, 117.87, 121.20, 133.15, 133.95, 147.73, 148.22, 169.52 MS (ESI-TOF) m/z: 252.8, [M+H-H 2 O] +Compound 127To a solution of alcohol 44 (1.23 g, 6.35 mmol) and alcohol 121 (200 mg, 0.794 mmol) in methanol (10 mL), cooled in an ice bath and magnetically stirred, was added dropwise 5 mL of 70 % perchloric acid, and the resulting pink solution was stirred under nitrogen at room temperature for 18 h. The formed precipitate was dissolved by adding dichloromethane and the organic phase was thoroughly washed with water until neutral. The dichloromethane solution was dried over magnesium sulfate and evaporated under vacuum, affording a crystalline residue (1.47 g). This residue was dissolved with THF (50 mL) and then palladium acetate (30 mg, 0.13 mmol), triphenylphosphine (5 mg, 0.39 mmol), diethylamine (10 mL), and H 2 O (10 mL) were added. The resulting solution was stirred at 80 °C under a nitrogen atmosphere for 3 h. The dark solution was then stripped off under reduced pressure and extracted with water / ethyl acetate. The aqueous layer was evaporated under reduced pressure to leave a dark residue. A purification by reversed-phase chromatography (water / acetonitrile 100 : 0  0 : 100) afforded the product 127 in 14 % overall yield for two steps (50 mg).Pale yellow solid1 H NMR (400 MHz, methanol-D 4 , δ in ppm): 3.28 -3.39 (m, 3H), 3.74 (s, 6H), 4.32 -4.65 (m, 5H), 6.69 -6.93 (m, 6H) NMR (100 MHz, CDCl 3 , δ in ppm): Not enough sample kept to achieve a satisfying 13 C NMR spectrum MS (ESI-TOF) m/z: 470.1, [M+H+H 2 O] + Compound 129 To a solution of alcohol 44 (308 mg, 1.59 mmol) and alcohol 121 (200 mg, 0.794 mmol) in methanol (3 mL), cooled in an ice bath and magnetically stirred, was added dropwise 1.5 mL of 70 % perchloric acid, and the resulting pink solution was stirred under nitrogen at room temperature for 18 h. The formed precipitate was dissolved by adding dichloromethane and the organic phase was thoroughly washed with water until neutral. The dichloromethane solution was dried over magnesium sulfate and evaporated under vacuum, affording a crystalline residue (444 mg). To this residue, ethanol (1.5 mL) and a solution of 2N NaOH (1.5 mL) were added. The resulting yellow suspension was heated to reflux under a nitrogen atmosphere overnight. The solvent was stripped off under reduced pressure and the residue was extracted with 1N HCl / ethyl acetate. The organic layer was dried over MgSO 4 and concentrated under vacuum. A purification by chromatography on silica gel (dichloromethane / methanol 100 : 0  90 : 10) afforded the product 129 in 14 % overall yield for two steps (63 mg). Pale yellow solid 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 3.44 -3.55 (m, 3H), 3.83 (s, 6H), 4.51 -4.65 (m, 8H), 4.67 -4.78 (m, 3H), 5.21 -5.31 (m, 3H), 5.33 -5.45 (m, 3H), 5.98 -6.12 (m, 3H), 6.NMR (100 MHz, CDCl 3 , δ in ppm): Not enough sample kept to achieve a satisfying 13 C NMR spectrum MS (ESI-TOF) m/z: 573.3, [M+H] + Titre : Conception et synthèse de nouveaux cryptophanes pour des applicationss en l'imagerie moléculaire par RMN du xénon Mots clés : cryptophane, hydrosolubilisation et mono-functionalisation Résumé : Entre tous les techniques de l'imagerie, imagerie par résonance magnétique (IRM) offre plusieurs avantages en raison de sa faible invasivité, son innocuité et sa résolution spatiale en profondeur, mais souffre d'une mauvaise sensibilité. Pour résoudre ce problème, différentes stratégies ont été proposées, y compris l'utilisation de l'espèce hyperpolarisée comme 129Xe. Le xénon est un gaz inerte avec un nuage d'électrons polarisable qui est très sensible à son environnement chimique. Sa capacité d'être hyperpolarisée permet d'obtenir un gain significatif de la sensibilité. Néanmoins, le xénon n'a aucune spécificité à une cible biologique, par conséquent, il a besoin d'être encapsulé et vectorisée. Des cages moléculaires différentes ont été proposées et nous sommes particulièrement intéressés à cryptophane qui est l'un des meilleurs candidats pour l'encapsulation du xénon. Dans ce contexte, l'objectif de cette thèse est de concevoir des nouveaux cryptophanes qui peuvent être utilisés comme les plates-formes moléculaires pour construire des nouvelles biosondes de 129 Xe IRM utilisables pour l'imagerie in vivo. Pour cette raison, ces cryptophanes devraient être hydrosoluble et monofonctionnalisable. Dans cette thèse, le polyéthylène glycol (PEG) est utilisé pour améliorer la faible solubilité de la cage moléculaire hydrophobe. Il y a également une discussion systématique des façons de casser la symétrie des cryptophanes et les stratégies différentes ont été tentées pour synthétiser cryptophanes mono-fonctionnalisé. En conséquence, plusieurs cryptophanes PEGylés et mono-fonctionnalisés ont été obtenus et leurs propriétés d'encapsulation du xénon ont été testées. Title : Conception and synthesis of the new cryptophane for the applications in xenon NMR molecular imaging Keywords : cryptophane, hydrosolubilization and mono-functionalization

Table

Table 1 :

 1 ). Comparison

		PET	CT	Optical	ultrasound	MRI
				imaging
	Spatial	1-10 mm	100 m	25-50 m	30 m	10-100 m
	resolution
	Temporal	60-1000 s	10-100 ms	1-200 ms	1-100 ms	10-100 ms
	resolution
	Sensitivity	pM	M	fM-nM	M-mM	M-mM
	Cost	+++	++	+	+	+++
	Ionizing	Yes	Yes	No	No	No
	radiation
	Other limit		Limited	Limited in	Limited in	No analysis
			contrast for	depth	depth	of solid
			soft tissues			tissues

of common medical imaging techniques Among all the techniques, MRI offers several advantages owing to its low invasiveness, its harmlessness and its spatial/time in-depth resolution but suffers from its expensive cost and poor sensitivity. The improvement of the sensitivity of MRI is always one of the main issues of research in this field.

Table 2 :

 2 Volume of cavity and affinity constant to xenon of different cryptophanes

Table 2

 2

	also suggests that for cryptophanes with equivalent sizes, water-soluble molecules
	always display a better affinity for xenon than organosoluble ones. This is due to the
	hydrophobicity of the xenon gas which prefers the hydrophobic environment of the
	cryptophane cavity to the aqueous milieu.
	The exchange rate is a very important factor for encaging hyperpolarized 129 Xe NMR. It
	directly affects the Xe NMR signal and is directly correlated to the width of peak at mid-
	height of the encapsulated xenon: wider is the peak, higher is the exchange rate.
	As discussed previously, the exchange rate should be slow enough for visualizing the
	encapsulated xenon by NMR, suggesting that the difference in chemical shifts between free
	and encapsulated xenon should be large enough. Until now, this difference is larger than 112
	ppm for all the reported cryptophanes (Table 3).
	Cyptophanes Organosolubles [111] [lmn]	R1, R2 H, H	δ encapsulated Xe (ppm) 31	[δ free Xe -δ encapsulated Xe ] (ppm) 194	Ref . 59
	[222]	OMe, OMe	68	156	3
	[223]	OMe, OMe	60	164	62
	[233]	OMe, OMe	47	177	62
	[333]	OMe, OMe	30	186	62
	Water-solubles [111]	H + 6[Cp*Ru] +	308	112	58
	[222]	OCH 2 COOH, OCH 2 COOH	64	133	63
	[223]	OCH 2 COOH, OCH 2 COOH	52	145	63
	[233]	OCH 2 COOH, OCH 2 COOH	42	155	63
	[333]	OCH 2 COOH, OCH 2 COOH	35	162	63

Table 3 :

 3 Chemical shifts of encapsulated xenon in different cryptophanes

	Concomitantly, the exchange rate should also be fast enough to enable the permanent
	refilling of the molecular cage with freshly hyperpolarized xenon. Generally, the exchange
	rate increases with the cage size. For example, cryptophane[111] has an exchange rate of 2.4

Table 5 :

 5 ). The influence of equivalent of benzyl alcohol on the reaction As shown in Table

		R	Base	Eq. of 54 Eq. of base	Yield
	1	-OTs	Cs 2 CO 3	9	6	7 %
	2	-OTs	Cs 2 CO 3	4	6	28 %

Table 6 :

 6 The influence of the leaving group on the reaction For results reported in table 6, the quantity of benzyl alcohol derivative was fixed to 4 equivalents, while using Cs 2 CO 3 as the base, and different leaving groups were tested. The best result was obtained with the benzyl bromide 39 (36 % yield, entry 4).

			Base	Eq. of 54	Eq. of base	yield
	2	-OTs	Cs 2 CO 3	4	6	28 %
	3	-OMs	Cs 2 CO 3	4	6	trace
	4	-Br	Cs 2 CO 3	4	6	36 %
		R	Base	Eq. of 54	Eq. of base	yield
	4	-Br	Cs 2 CO 3	4	6	36 %
	5	-Br	K 2 CO 3	4	6	22 %
	6	-Br	NaH (60%)	9	3,5	24 %
	7	-Br	NaH (95%)	9	3,5	31 %
	8	-Br	NaH (95%)	5	3,5	57 %
	9	-Br	NaH (95%)	4	3,5	33 %

Table 7 :

 7 Influence of the base on the reaction yieldResults reported in table 7 highlight the influence of different bases on the reaction. It

Table 9 :

 9 Optimization of the mono-functinalization of CTV 25 with compound 37

		25	Mono 71	Di 74	Yield based on recycling
	1 eq. of 37	31 %	30 %	16 %	39 %
	0.7 eq. of 37	32 %	35 %	11 %	43 %
	0.5 eq. of 37	38 %	37 %	8 %	45 %
	During

the next step we grafted the PEGylated linker 58 onto CTV 71. The purification step was facilitated by the presence of THP groups which avoided the formation of a too polar product. Normal-phase chromatography yielded 60 % of expected tri-functionalized CTV 72.

Table 10 :

 10 Evolution of different intermediates during the debenzylation. Percentages were calculated according to the chromatograms of LC-MS

		112	Di-benzylated CTV	Mono-benzylated CTV	CTV 110
	0 h	100 %	0 %	0 %	0 %
	24 h	75 %	4 %	17 %	4 %
	48 h	31 %	9 %	43 %	17 %
	72 h	2 %	15 %	16 %	67 %
	96 h	0 %	0 %	0 %	100 %

Table 11

 11

		).
	Ratio 44 : 121	2 : 1	3 : 1	4 : 1	5 : 1	6 : 1	8 : 1
	CTV 32	50 %	57 %	62 %	66 %	68 %	71 %
	CTV 125	35 %	32 %	30 %	30 %	32 %	27 %
	CTV 126	15 %	11 %	8 %	4 %	2 %	2 %

Table 11 :

 11 Results of cyclotrimerization with different ratios of monomer 44:121 engaged. All the percentages were calculated according to their LC-MS chromatograms

  The reaction was heated to reflux of acetone, under inert atmosphere overnight.Acetone was evaporated and then an extraction using water / dichloromethane was carried out.The organic phase was washed twice with an aqueous solution of potassium carbonate (pH = 11) and twice with water. It was finally dried over MgSO 4 and dichloromethane was evaporated. Compound 18 was obtained without purification in 98 % yield (6.0 g). CDCl 3 , δ in ppm): 55.84, 58.95, 65.08, 68.64, 69.61, 70.50, 70.77, 71.86, To a solution of 18 (4.05 g, 6.15 mmol) in 40 mL of MeOH was slowly added 20 mL of perchloric acid 70 % at 0 °C under nitrogen atmosphere. The reaction mixture was then warmed to room temperature and stirred for 24 hours. The mixture was diluted in dichloromethane and neutralized with a solution of NaOH 1N at 0 °C. The recovered organic phase was washed successively with water, brine and was finally dried over MgSO 4 and dichloromethane was evaporated. The obtained residue was directly engaged in the next step.NaH 95 % (9 mg, 0.35 mmol) was added to a solution of 20 (183 mg, 0.1 mmol,) in 20 mL of anhydrous DMF. After one hour of stirring at 70 °C, 39 (131 mg, 0.5 mmol) was added in the solution and this mixture was heated at 70 °C overnight. DMF was evaporated and then an extraction water / dichloromethane, followed by washing with brine was carried out. The organic solution was finally dried over MgSO 4 and the dichloromethane was evaporated. A overnight at room temperature under argon. The solvent was stripped off to leave a residue, which was extracted with diethyl ether. The combined organic layers were washed twice with brine, dried over MgSO 4 and evaporated to give an oily residue. stirred at 80 °C under an argon atmosphere for about three hours. The dark solution was then stripped off under reduced pressure and the product was extracted with ethyl acetate.The organic layer was washed with water followed by filtration with filter paper to remove insoluble material. The combined organic layers were washed three times with brine and then dried over MgSO 4 . Evaporation of the solvent under reduced pressure left a yellow solid, which was filtered and washed three times with diethyl ether to yield 25 (1.15 g, 67 %). The reaction was quenched with water and a solution of HCl 1N was added until reaching pH 2. An extraction water / EtOAc was carried out and the organic phase was dried over MgSO 4 and concentrated under vacuum. A purification by chromatography on silica gel (cyclohexane / ethyl acetate 90 : 10  70 : 30) afforded the product 60 in 60 % yield (3.87 g).The reaction was kept at 0 °C with stirring for 30 minutes and then it was quenched with a solution of HCl 1N and extracted with dichloromethane. The organic phase was dried over MgSO 4 and concentrated under vacuum. Product 37 was obtained quantitatively (2 g). The reaction was kept at room temperature with stirring for 18 hours. After, the reaction was quenched with water and a solution of HCl 1N was added until reaching pH 2. An extraction water / ethyl acetate was carried out and the organic phase was dried over MgSO 4 and concentrated under vacuum. A purification by chromatography on silica gel (cyclohexane / ethyl acetate 90 : 10  70 : 30) afforded the product 63 in 57 % yield (3.68 g).Compound 37 (0.35 g, 1.22 mmol) was added in one portion to a stirred solution of 25 (1 g, 2.45 mmol) and cesium carbonate (1.6 g, 4.90 mmol) in dry DMF (63 mL). The solution was heated for 18 h at 80 °C under nitrogen. After cooling, the dark mixture was poured into water and the product was extracted with ethyl acetate. The combined organic layers were washed several times with brine and then dried over MgSO 4 . The solvent was stripped off by rotary stirred at 80 °C under an argon atmosphere for 3 h. The dark solution was then stripped off under reduced pressure and the product was extracted with ethyl acetate. The organic layer was washed with water followed by filtration with filter paper to remove insoluble dark material. The combined organic layers were washed three times with brine and then dried over Mg 2 SO 4 . A purification by chromatography on silica gel (dichloromethane / methanol 100 : 0  90 : 10) afforded product 68 in 63 % yield (25 mg). H NMR (400 MHz, CDCl 3 , δ in ppm): 1.46 -1.66 (m, 8H), 1.67 -1.77 (m, 2H), 1.78 -1.90 (m, 2H), 3.37 (s, 6H), 3.48 -3.70 (m, 85H), 3.72 (s, 1H), 3.74 (s, 1H), 3.75 (s, 1H), 3.77 -3.83 (m, 4H), 3.86 -3.94 (m, 2H), 4.16 (t, J = 5.0 Hz, 4H), 4.21 -4.37 (m, 12H), 4.40 (d, J = 11.7 Hz, 2H), 4.55 (s, 2H), 4.63 -4.79 (m, 7H), 6.75 -6.98 (m, 15H) 13 C NMR (100 MHz, CDCl3 , δ in ppm): 19.32, 25.33, 30.47, 36.31, 56.07, 58.89, 60.86, 62.09, 64.47, 66.49, 67.86, 68.45, 68.66, 69.54, 70.41, 70.61, 71.79, 97.45, 113.68, 113.74, 113.85, 114.50, 114.59, 114.82, 116.29, 121.10, 131.70, 131.82, 132.76, 132.85, 134.87, 135.13, 146.69, 147.76, 147.82, 148.03, 148.19, 148.38, 148.47, 148.54, 148.60, 148.71, To a suspension of NaH 95 % (0.36 g, 14.5 mmol) in anhydrous DMF (3 mL), it was slowly added at 0 °C a solution of 3,4-dihydroxybenzaldehyde (1 g, 7.24 mmol) in anhydrous DMF (3 mL). After 30 min of stirring, a solution of benzyl bromide (0.9 mL, 7.24 mmol) in anhydrous DMF (3 mL) was added. The reaction was kept at room temperature with stirring for 18 h. The reaction was quenched with water and a solution of HCl 1N was added until reaching pH 2. An extraction water / ethyl acetate was carried out and the organic phase was dried over MgSO 4 and concentrated under vacuum. A purification by chromatography on silica gel (cyclohexane / ethyl acetate 90 : 10  70 : 30) afforded the product 80 in 39 % yield (0.64 g). CDCl 3 , δ in ppm): 70.05, 112.56, 114.01, 126.52, 127.29, 128.08, 128.64, 130.75, 137.63, 149.02, 155.24, 190.72 MS (ESI-TOF) m/z: 229.2, [M+H] +Compound 81 (0.43 g, 1.27 mmol) was dissolved in THF (5 mL). The solution was cooled to 0 °C with an ice bath and then NaBH 4 (58 mg, 1.52 mmol) and methanol (0.5 mL) were added. The reaction was kept at 0 °C with stirring for 30 minutes and then quenched with a solution of HCl 1N and extracted with dichloromethane. The organic phase was dried over MgSO 4 and concentrated under vacuum. Product 82 was obtained in 99 % yield (0.43 g). H NMR (400 MHz, CDCl 3 , δ in ppm): 3.64 (t, J = 6.4 Hz, 2H), 4.33 (t, J = 6.4 Hz, 2H), 4.57 (s, 2H), 5.13 (s, 2H), 6.85 -6.95 (m, 2H), 7.00 (d, J = 0.9 Hz, 1H), 7.29 -7.51 (m, 5H) 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 29.30, 64.93, 69.58, 71.18, 114.02, 115.70, 112.10, 127.31, 127.84, 128.47, 135.23, 136.91, 147.62, 149.14 MS (ESI-TOF) m/z: 337.3, [M+H] + Compound 82 (195 mg, 0.52 mmol) was added in one portion to a stirred solution of 25 (423 mg, 1.03 mmol) and cesium carbonate (674 mg, 2.07 mmol) in dry DMF(26 mL). The solution was then heated for 18 h at 80 °C under argon. After cooling, the dark mixture was poured into water and the product was extracted with ethyl acetate. The combined organic layers were washed several times with brine and then dried over MgSO 4 . The solvent was stripped off by rotary evaporation and the residue was purified by chromatography on silica gel (cyclohexane / ethyl acetate 50 : 50  0 : 100). 118 mg of expected product 83 was thus obtained (43 % yield based on recovered starting material). CDCl 3 , δ in ppm):36.21, 36.26, 36.36, 55.99, 56.02, 56.13, 65.23, 67.66, 68.14, 71.17, 110.92, 112.15, 113.68, 113.73, 115.48, 115.52, 116.73, 119.39, 127.06, 127.59, 128.88, 131.15, 131.22, 131.92, 132.20, 132.48, 133.05, 134.31, 136.72, 144.06, 144.09, 145.21, 146.62, 147.55, 148.48, 149.62 MS (ESI-TOF) m/z: 664.8, [M+H] +Compound 58 (0.36 g, 0.43 mmol) was added to a stirred solution of 83 (115 mg, 0.17 mmol) and cesium carbonate (0.23 g, 0.69 mmol) in dry DMF (7 mL). The solution was then heated for 18 h at 80 °C under nitrogen. The solvent was stripped off and the residue was extracted with dichloromethane / water. The combined organic layers were washed with brine, dried over Mg 2 SO 4 and evaporated to leave an oily residue. A purification by chromatography on silica gel (dichloromethane / methanol 100 : 0  90 : 10) afforded product 84 in 76 % yield (279 mg). CDCl 3 , δ in ppm):19.32, 25.33, 30.47, 36.31, 56.07, 58.89, 60.86, 62.09, 64.47, 66.49, 67.86, 68.45, 68.66, 69.54, 70.61, 71. 16, 71.79, 97.45, 113.68, 113.74, 113.85, 114.50, 114.59, 114.82, 116.29, 121.10, 127.13, 127.58, 128.93, 131.70, 131.82, 132.76, 132.85, 134.87, 135.13, 136.66, 146.69, 147.76, 147.82, 148.03, 148.19, 148.38, 148.47, 148.54, 148.60, 148.71, 148.80, 148.90 MS (ESI-TOF) m/z: distribution centered at 971.0 ± 22, [(M+2H-2THPOH-H 2 O)/2] + , irregular mass caused by an ionisation problem To a suspension of NaH 95 % (2.2 g, 90.6 mmol) in anhydrous DMF (17 mL), it was slowly added at 0 °C a solution of 3,4-dihydroxybenzaldehyde (5 g, 36.2 mmol) in anhydrous DMF (17 mL). After 30 min of stirring, a solution of methyl bromoacetate (3.4 mL, 36.2 mmol) in anhydrous DMF (17 mL) was added. The reaction was kept at room temperature with stirring for 18 h. The reaction was quenched with water and a solution of HCl 1N was added until reaching pH 2. An extraction water / ethyl acetate was carried out and the organic phase was dried over MgSO 4 and concentrated under vacuum. A purification by chromatography on silica gel (cyclohexane / ethyl acetate 90 : 10  70 : 30) afforded the product 88 in 67 % yield (5.1 g). H NMR (400 MHz, CDCl 3 , δ in ppm): 3.78 (s, 3H), 4.71 (s, 2H), 7.04 (d, J = 8.2 Hz, 1H), 7.38 (d, J = 1.7 Hz, 1H), 7.46 (dd, J1 = 8.2 Hz, J2 = 1.7 Hz, 1H), 9.76 (s, 1H) 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 52.60, 67.49, 113.90, 116.09, 128.65, 129.58, 146.22, 153.17, 170.08, 190.47 MS (ESI-TOF) m/z: 210.9, [M+H] + Compound 89 (3.1 g, 9.8 mmol) was dissolved in THF(33 mL). The solution was cooled to 0 °C with an ice bath and then NaBH 4 (0.37 g, 9.8 mmol) and methanol (4 mL) were added.The reaction was kept at 0 °C with stirring for 30 minutes and then it was quenched with a solution of HCl 1N and extracted with dichloromethane. The organic phase was dried over MgSO 4 and concentrated under vacuum. A purification by chromatography on silica gel (cyclohexane / ethyl acetate 50 : 50  100 : 0) afforded the product 90 in 77 % yield (2.4 g). H NMR (400 MHz, CDCl 3 , δ in ppm): 3.62 (t, J = 6.4 Hz, 2H), 3.75 (s, 3H), 4.30 (t, J = 6.4 Hz, 2H), 4.52 (s, 2H), 4.65 (s, 2H), 6.82 -6.94 (m, 3H 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 29.15, 52.07,64.33, 66.56, 69.44, 114.60, 115.54, 121.14, 135.28, 147.44, 147.93, 169.43

	Compound 19 Compound 20 Ethane-1,2-diyl bis(4-methylbenzenesulfonate) Compound 52 Ethane-1,2-diyl dimethanesulfonate Compound 34 Compound 14 Compound 32 Compound 25 Compound 60 Compound 61 Compound 37 Compound 63. Compound 70 Compound 71 Compound 72 Compound 79 Compound 80 Compound 82 Compound 83 Compound 84 Compound 88 Compound 90
	Over 30 min, small portions of TsCl (95 g, 0.5 mol) were slowly added to a solution of A solution of MsCl (2.55 mL, 33 mmol) in anhydrous dichloromethane (5 mL) was drop by
	ethylene glycol (11.2 mL, 0.2 mol) in pyridine (325 mL) under nitrogen at 0 °C. After it was drop added to the mixture of ethylene glycol (0.84 mL, 15 mmol) and trimethylamine (5.44 Vanillyl alcohol (2.6 g, 16.8 mmol) was dissolved in 20 mL of acetone. Compound 17 (7.5 g, continuously stirred at room temperature under nitrogen overnight, the solution was poured mL, 39 mmol) dissolved in 30 mL of dichloromethane under nitrogen at 0 °C. Then it was To a suspension of NaH 95 % (1.93 g, 76.3 mmol) in anhydrous DMF (17 mL), it was slowly A solution of 32 (4.4 g, 8.2 mmol), palladium acetate (122 mg, 0.56 mmol), 11.2 mmol) and potassium carbonate (2.6 g, 19 mmol) were then added in the reaction milieu with stirring. Pale yellow oil 110.95, 113.80, 119.28, 134.38, 147.73, 149.70 HRMS-ESI (m/z) : distribution centered at 675.3597 ± 44, (M+Na) + Transparent oil 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 3.38 (s, 9H), 3.50 (d, J = 13.7 Hz, 3H), 3.54 -3.70 (m, 120H), 3.81 (s, 9 H), 3.80 -3.90 (m, 6H), 4.11 -4.20 (m, 6H), 4.71 (d, J = 13,7 Hz, 3H), 6.81 (s, 3H), 6.89 (s, 3H) 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 36.44, 56.26, 58.99, 68.83, 69.61, 70.51, 70.72, To a solution of 19 (3.6 g, 1.8 mmol) in 27 mL of anhydrous THF was added slowly 12.3 mL of a freshly-prepared solution of 1M PPh 2 Li (12.3 mmol) under inert atmosphere. The reaction was stirred at room temperature for 3 hours. Then it was quenched with water and neutralized with a solution of HCl 1N. The organic phase was extracted with dichloromethane, then washed with a solution of saturated NaCl. The organic solution was finally dried over MgSO Transparent oil 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 3.36 (s, 9H), 3.44 (d, J = 13.7 Hz, 3H), 3.54 -3.75 (m, 126H), 4.08 -4.29 (m, 6H), 4.61 (d, J = 13.7 Hz, 3H), 6.84 (1s, 3H), 6.85 (1s, 3H) 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 36.13, 58.98, 69.48, 70.37, 70.49, 71.86, 116.51, 117.64, 130.87, 134.41, 144.50, 146.14 MS (MALDI-TOF) m/z: distribution centered at 1707.9 ± 44 ([M+Na] + ) onto crushed ice. The white precipitate formed was filtered and washed with cold water and methanol. Then it was dried for 48 h in vacuum to yield 55 g of Ethane-1,2-diyl bis(4-methylbenzenesulfonate) 75% yield (55.6 g). White powder 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 7.71 (d, 4H, J = 6.6 Hz), 7.32 (d, 4H, J = 8.0 Hz), 4.17 (s, 4H), 2.43 (s, 6H) Vanillyl alcohol (1 g, 6.5 mmol) was dissolved in DMF (30 mL) then K 2 CO 3 (4.5 g, 30 mmol) and ethylene ditosylate (12 g, 32.5 mmol) were added into the solution. The reaction was stirred at 60 °C for 16 hours. After, the mixture was poured into water (200 mL) and extracted with ethyl acetate (3 x 300 mL). The organic phases were collected and concentrated to about 200 mL and washed successively with a solution of NaOH 1N (2 x 200 mL), water (200 mL) and brine (2 x 200 mL). The organic phase was dried and the solvent was evaporated under vacuum. Compound 39 was obtained in 63 % yield (1.4 g). Brownish oil = 8.7 Hz, 2H) 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 21.62, 55.86, 67.02, 68.05, 71.71, 111.88, 114.65, 120.36, 127.96, 129.81, 132.37, 132.72, 144.87, 146.98, 149.84 MS (ESI-TOF) m/z: 335.0, [M+H-H 2 O] + stirred at room temperature under nitrogen for 2 hours. A solution of 5 % HCl was added until the reaction milieu turned to a limpid solution. The organic phase was collected and washed successively with a saturated solution of NaHCO 3 , water and brine. Then it was dried over MgSO 4 and the solvent was evaporated. After a recrystallization in methanol, the targeting product was obtained in 54 % yield (1.8 g). White powder Pale yellow oil 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 3.36 (s, 9H), 3.47 (d, J = 13.7 Hz, 3H), 3.24 -3.68 (m, 126H), 3.77 (s, 9H), 3.95 -4.05 (m, 6H), 4.32 (sl, 12H), 4.58 (s, 6H), 4.67 (d, J = 13.7 Hz, 3H), 6.82 -6.95 (m, 15H) 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 36.31, 55.74, 58.99, 64.84, 67.96, 68.57, 69.19, 69.62, 70.48, 71.86, 110.98, 113.92, 116.73, 117.57, 119.28, 132.73, 133.10, 134.97, 147.36, 147.51, A solution of PPTS (0.33 g, 1.3 mmol) in dichloromethane (5 mL) was added in one portion to a mixture of 39 (3.4 g, 13 mmol) and dihydropyran (1.64 g, 20 mmol) in THF (40 mL). The solution was stirred Chromatography on silica gel (diethyl ether / pentane 40 / 60) yielded compound 34 (3.6 g, 80 %) Colorless oil 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 6.91 -6.87 (m, 3H), 4.70 (d, J = 11.8 Hz, 1H), 4.65 (m, 1H), 4.42 (d, J = 11.8 Hz, 1H), 4.30 (t, J = 7.0 Hz, 2H), 3.90 (m, 1H), 3.86 (s, 3H), 3.62 (t, J = 7. 0 Hz , 2H), 3.55 (m, 1H), 1.90 -1.40 (m, 6 H) 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 149.75, 146.81, 132.34, 120.45, 114.60, 112.09, 97.61, 69.25, 68.63, 62.26, 55.94, 30.56, 28.84, 25.41, 19.44 MS (ESI-TOF) m/z: 345.6, [M+H] + A mixture of vanillyl alcohol (10 g, 0.065 mol), allyl bromide (6.3 mL, 0.073 mol), and potassium carbonate (9 g, 0.065 mol) in 15 mL of acetone was refluxed for 4 h with magnetic stirring. Then, after most of the solvent was stripped off, water was added and the organic material was extracted with dichloromethane, yielding 12.5 g (99%) of crude 14. This product was recrystallized from 25 mL of diisopropyl ether, affording 10 g (80%) of pure 14. White powder 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 3.89 (s, 3H), 4.59-4.65 (m, 4H), 5.28 (dd, J1 = 10.5 Hz, J2 = 1.2 Hz, 1H) 5.8-6.3 (dd, J1 = 17.3 Hz, J2 = 1.4 Hz, 1H), 6.02-6.14 (m, 1H) 6.86 (s, 2H) and 6.94 (s, 1H) 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 55.6, 64.6, 147.1, 113.2, 118.9 MS (ESI-TOF) m/z: 195.1, [M+H] + To a solution of phenolprotected vanillyl alcohol 14 (10 g, 52 mmol) in methanol (60 mL), cooled in an ice bath and magnetically stirred, was added dropwise 30 mL of 70 % perchloric acid, and the resulting pink solution was stirred under nitrogen at room temperature for 18 h. The formed precipitate was dissolved by adding dichloromethane and the organic phase was thoroughly washed with water until neutral pH. The dichloromethane solution was dried over magnesium sulfate and evaporated under vacuum, affording a crystalline residue, which was washed by 20 mL of diethyl ether and finally isolated by suction filtration, yield 4.8 g of CTV 32 (53%) 1.4 Hz, J2 = 17.3 Hz, 3H), 6.00-6.12 (m, 3H), 6.79 (s, 3H), 6.85 (s, 3H) 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 36.3, 55.9, 70.0, 113.5, 115.5, 117.2, 131.6, 132.2, 133.6, 146.6, 148.1 MS (ESI-TOF) m/z: 529.1, [M+H] + triphenylphosphine (0.42 g, 1.6 mmol), diethylamine (40 mL), THF (100 mL), and H 2 O (20 added at 0 °C a solution of 3,4-dihydroxybenzaldehyde (5 g, 36.2 mmol) in anhydrous DMF (17 mL). After 30 min of stirring, a solution of allyl bromide (3.1 mL, 36.2 mmol) in anhydrous DMF (17 mL) was added. The reaction was kept at room temperature with stirring Compound 60 (1.7 g, 9.7 mmol) was dissolved in DMF (15 mL) and then K 2 CO 3 (1.6 g, 11.7 mmol) and 1,2-dibromoethane (3.3 mL, 39 mmol) were added into the solution. The reaction was heated at 80 °C with stirring for 16 hours. After cooling, diethyl ether was added and it Compound 68 (3.25 g, 4.37 mmol) was dissolved in THF (15 mL). The solution was cooled to Compound 61 (2 g, 8.5 mmol) was dissolved in THF (19 mL). The solution was cooled to 0 °C with an ice bath and then NaBH 4 (0.3 g, 8.5 mmol) and methanol (2.3 mL) were added. To a suspension of NaH 95 % (1.05 g, 43.8 mmol) in anhydrous DMF (17 mL), it was slowly added at 0 °C a solution of 3,4-dihydroxybenzaldehyde (5 g, 36.2 mmol) in anhydrous DMF 0 °C with an ice bath and then NaBH 4 (165 mg, 4.37 mmol) and methanol (1.8 mL, 43.7 mmol) were added. The reaction was kept at 0 °C with stirring for 30 minutes and then it was mL) was White solid 1 H NMR (400 MHz, acetone-D 6 , δ in ppm): 6.99 (s, 3H), 6.92 (s, 3H), 4.73 (d, J = 13.5 Hz, 3H), 3.79 (s, 3 H), 3.43 (d, J = 13.5 Hz, 3H) 13 C NMR (100 MHz, acetone-D 6 , δ in ppm): 145.2, 144.1, 132.4, 131.2, 115.4, 112.2, 55.0, 36.3 MS (ESI-TOF) m/z: 426.2, [M+H+H 2 O] + was washed with a solution of HCl 1N. The organic phase was dried over MgSO 4 and (17 mL). After 30 min of stirring, a solution of allyl bromide (3.1 mL, 36.2 mmol) in quenched with a solution of HCl 1N and extracted with dichloromethane. The organic phase Compound 58 (0.42 g, 0.51 mmol) was added to a stirred solution of 71 (78 mg, 0.13 mmol) A solution of 72 (40 mg, 0.019 mmol), palladium acetate (0.4 mg, 1.8 µmol), for 18 hours. White Solid 7.05 (d, J = 7.8 Hz, 1H), 7.42 (s, 1H), 7.44 (d, J = 7.8 Hz, 1H), 9.82 (s, 1H) 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 69.89, 110.16, 114.55, 119.06, 127.45, 129.76, 131.93, 146.00, 151.78, 190.78 MS (ESI-TOF) m/z: 178.9, [M+H] + concentrated under vacuum. A purification by chromatography on silica gel (cyclohexane / ethyl acetate 80 : 20  50 : 50) afforded product 61 in 70 % yield (1.9 g). White Solid 9.85 (s, 1H) 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 28.26, 68.81, 69.81, 112.16, 112.99, 118.10, 126.34, 130.80, 132.50, 148.95, 153.31, 190.71 MS (ESI-TOF) m/z: 285.1, [M+H] + White solid 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 29.09, 65.05, 69.59, 69.96, 113.59, 115.69, 117.65, 119.89, 133.17, 135.22, 147.45, 149.07 MS (ESI-TOF) m/z: 287.1, [M+H] + was dried over MgSO 4 and concentrated under vacuum. Product 70 was obtained and cesium carbonate (0.17 g, 0.51 mmol) in dry DMF (6 mL). The solution was heated for triphenylphosphine (0.6 mg, 2.3 µmol), diethylamine (0.2 mL), THF (0.3 mL), and H 2 O (0.1 anhydrous DMF (17 mL) was added. White Solid 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 4.70 (d, J = 5.4 Hz, 2H), 5.37 (d, J = 10.3 Hz, 1H), 5.43 (dd, J = 17.2 Hz, 1H), 5.84 (s, 1H), 6.01 -6.12 (m, 1H), 6.96 (d, J = 8.3 Hz, 1H), 7.40 (dd, J1 = 8.3 Hz, J2 = 1.7 Hz, 1H), 7.45 (d, J = 1.7 Hz, 1H), 9.84 (s, 1H) 13 131.76, 146.22, 150.69, 190.98 5.35 (dd, J1 = 17.3 Hz, J2= 1.3 Hz, 1H), 5.93 -6.08 (m, 1H), 6.77 -7.00 (m, 15H) -7.00 (m, 9H) C NMR (100 MHz, CDCl 3 , δ in ppm): 69.91, 111.37, 114.34, 119.24, 124.29, 130.71, quantitatively (3.26 g). Transparent oil 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 29.42, 59.01, 64.84, 68.92, 69.75, 69.84, 70.52, 70.66, 70.80, 70.89, 113.95, 116.17, 120.01, 135.73, 147.44, 149.41 MS (ESI-TOF) m/z: distribution centered at 879.7 ± 44, [M+H] + White solid 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 3.43 -3.55 (m, 3H), 3.72 (s, 3H), 3.79 -3.85 (m, 6H), 4.25 -4.42 (m, 4H), 4.54 (d, J = 5.1 Hz, 2H), 4.59 (s, 2H), 4.65 -4.79 (m, 3H), 5.21 (dd, J1 = 10.5 Hz, J2= 1.2 Hz, 1H), 5.36 (dd, J1 = 17.2 Hz, J2= 1.2 Hz, 1H), 5.94 -6.08 (m, 1H), 6.74 18 h at 80 °C under nitrogen. The solvent was stripped off and the residue was extracted with dichloromethane / water. The combined organic layers were washed with brine, dried over Mg 2 SO Yellow oil (d, J = 5.4 Hz, 2H), 4.57 (s, 2H), 4.63 -4.78 (m, 7H), 5.20 (dd, J1 = 10.5 Hz, J2= 1.3 Hz, 1H), 2H), 6.74 -7.02 (m, 15H), 7.20 -7.32 (m, 3H), 7.38 (d, J = 7.0 Hz, 2H) 9H), 7.20 -7.33 (m, 3H), 7.39 (d, J = 7.0 Hz, 2H) 3.95 (m, 2H), 4.17 (t, J = 5.0 Hz, 4H), 4.24 -4.37 (m, 12H), 4.40 (d, J = 11.6 Hz, 2H), 4.54 2H), 4.17 (t, J = 5.2 Hz, 4H), 4.23 -4.46 (m, 14H), 4.56 (s, 2H), 4.63 -4.78 (m, 7H), 5.08 (s, 3H), 4.27 -4.41 (m, 4H), 4.57 (s, 2H), 4.70 (d, J = 13.7 Hz, 3H), 5.09 (s, 2H), 6.70 -7.00 (m, (m, 2H), 3.37 (s, 6H), 3.48 -3.70 (m, 88H), 3.71 -3.76 (m, 6H) 3.77 -3.85 (m, 4H), 3.86 -(m, 2H), 3.37 (s, 6H), 3.48 -3.72 (m, 91H), 3.74 (s, 3H), 3.77 -3.86 (m, 4H), 3.86 -3.96 (m, 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 3.42 -3.56 (m, 3H), 3.67 (s, 3H), 3.79 (s, 3H), 3.84 (s, 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 1.46 -1.66 (m, 8H), 1.68 -1.77 (m, 2H), 1.78 -1.90 White Solid White solid White solid Yellow oil 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 1.46 -1.66 (m, 8H), 1.67 -1.77 (m, 2H), 1.78 -1.90 White Solid mL) was Yellow oil White solid
	1 H NMR (400 MHz, CDCl 3 , δ in ppm): 2.44 (s, 3H), 3.37 (s, 3H), 3.54 -3.69 (m, 42H), 4.15 (t, J = 4.7 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 7.78 (d, J = 8.3 Hz, 2H) 71.88, 113.87, 116.08, 131.77, 132.61, 146.89, 148,37 MS (MALDI-TOF) m/z: distribution centered at 1705.9 ± 44 ([M+Na] + ) 147.79, 149.62 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 36.21, 36.26, 36.36, 55.99, 56.02, 56.13, 65.23, 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 19.32, 25.33, 30.47, 36.31, 56.07, 58.89, 60.86, MS (ESI-TOF) m/z: 178.9, [M+H] + 67.66, 68.14, 70.49, 110.92, 112.15, 113.68, 113.73, 115.48, 115.52, 116.73, 118.17, 119.39, 62.09, 64.47, 66.49, 67.86, 68.45, 68.66, 69.54, 70.41, 70.61, 71.79, 97.45, 113.68, 113.74, MS (MALDI-TOF) m/z: distribution centered at 2293.2 ± 44 ([M+Na] + ) 131.15, 131.22, 131.92, 132.20, 132.48, 133.05, 133.49, 134.31, 144.06, 144.09, 145.21, 113.85, 114.50, 114.59, 114.82, 116.29, 118.17, 121.10, 131.70, 131.82, 132.76, 132.85,
	13 146.62, 147.55, 148.48, 149.62 133.49, 134.87, 135.13, 146.69, 147.76, 147.82, 148.03, 148.19, 148.38, 148.47, 148.54, C NMR (100 MHz, CDCl 3 , δ in ppm): 21.55, 58.91, 68.57, 69.18, 70.45, 70.63, 71.83, 127.87, 129.74, 132.93, 144.70 MS (ESI-TOF) m/z: 597.4, [M+H-H 2 O] + ; 1193.9, [2M+1] + 148.60, 148.71, 148.80, 148.90 148.80, 148.90
	MS (ESI-TOF) m/z: distribution centered at 715.5 ± 44, [M+H] + MS (ESI-TOF) m/z: distribution centered at 968.1 ± 22, [(M+2H-2THPOH-H 2 O)/2] + , MS (ESI-TOF) m/z: distribution centered at 1850.4 ± 44, [M+H-2THPOH-H 2 O] + , irregular
	irregular mass caused by an ionisation problem mass caused by an ionisation problem
	Compound 18

1

H NMR (400 MHz, CDCl 3 , δ in ppm): 3.37 (s, 3H), 3.54 -3.72 (m, 40H), 3.86 (s, 3H), 3.88 (t, J = 5.2 Hz, 2H), 4.17 (t, J = 5.2 Hz, 2H), 4.61 (s, 2H), 6.84 -6.92 (m, 3H) 13 C NMR (100 MHz, To realize the characterization of 132, a purification by reversed-phase chromatography (water / acetonitrile 80 : 20  20 : 80) allowed the obtention of compound 19 in 50% yield (1.95 g). 4 , filtered and concentrated in vacuo. A purification with silica gel chromatography (dichloromethane / methanol 99 : 1  90 : 10) allowed the production of compound 20 in 80 % yield (2.7 g).

13 C NMR (100 MHz, CDCl 3 , δ in ppm): δ 145.25, 132.23, 129.92, 127.91, 66.67, 21.65 MS (ESI-TOF) m/z: 370.9, [M+H] + 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 2.44 (s, 3H), 3.84 (s, 3H), 4.21 (t, J = 4.6 Hz, 2H), 4.36 (t, J = 4.6 Hz, 2H), 4.62 (s, 2H), 6.78 -6.92 (m, 3H), 7.33 (d, J = 8.7 Hz, 2H), 7.82 (d, J 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 3.09 (s, 6H), 4.74 (s, 6H) 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 33.75, 66.69 MS (ESI-TOF) m/z: 219.0, [M+H] + Compound 21 purification on reversed-phase column (water / acetonitrile 70 : 30  45 : 55) allowed the production of compound 21 in 58 % yield (139 mg).

1 H NMR (400 MHz, CDCl 3 , δ in ppm): 3.51 (d, J = 13.6 Hz, 3H), 3.84 (s, 9H), 4.53 -4.65 (m, 6H), 4.74 (d, J = 13.6 Hz, 3H), 5.25 (dd, J1 = 1.4 Hz, J2 = 10.4 Hz, 3H), 5.37 (dd, J1 = 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 4.68 (d, J = 5.5 Hz, 2H), 5.38 (dd, J1 = 10.5 Hz, J2 = 1.2 Hz, 1H), 5.46 (dd, J1 = 17.3 Hz, J2 = 1.2 Hz, 1H), 6.08 -6.18 (m, 1H), 6.26 (s, 1H), 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 3.68 (t, J = 6.6 Hz, 2H), 4,40 (t, J = 6.6 Hz, 2H), 4.65 (d, J = 5.5 Hz, 2H), 5.30 (dd, J1 = 10.5 Hz, J2= 1.2 Hz, 1H), 5.44 (dd, J1 = 17.3 Hz, J2 = 1.2 Hz, 1H), 6.07 -6.18 (m, 1H), 6.99 (d, J = 7.8 Hz, 1H), 7.44 (s, 1H), 7.46 (d, J = 7.8 Hz, 1H), 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 3.65 (t, J = 6.6 Hz, 2H), 4.33 (t, J = 6.6 Hz, 2H), 4.55 -4.65 (m, 4H), 5.30 (dd, J1= 10.5 Hz, J2= 1.2 Hz, 1H), 5.44 (dd, J1 = 17.3 Hz, J2 = 1.2 Hz, 1H), 6.07 -6.18 (m, 1H), 6.89 -6.96 (m, 3H) 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 3.37 (s, 3H), 3.50 -3.76 (m, 42H), 3.86 (t, J = 4.9 Hz, 2H), 4.19 (t, J = 4.9 Hz, 2H), 4.31 (t, J = 6.6 Hz, 2H), 4.59 (d, J = 5.4 Hz, 2H), 6.84 -6.92 (m, 2H), 7.01 (d, J = 1.1 Hz, 1H) evaporation and the residue was purified by chromatography on silica gel (cyclohexane / ethyl acetate 50 : 50  0 : 100). 277 mg of expected product 31 was obtained (30 % yield based on recovered starting material). 4 and evaporated to leave an oily residue. A purification by chromatography on silica gel (dichloromethane / methanol 100 : 0  90 : 10) afforded product 72 in 60 % yield (164 mg).

1 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 5.15 (s, 2H), 6.51 (s, 1H), 7.06 (d, J = 8.1 Hz, 1H), 7.33 -7.46 (m, 6H), 7.51 (d, J = 1.4 Hz, 1H), 9.80 (s, 1H) 13 C NMR (100 MHz,

1 13 C NMR (100 MHz, 13 C NMR (100 MHz, 1 1 MS (ESI-TOF) m/z: 318.9, [M+H] +

  H NMR (400 MHz, CDCl 3 , δ in ppm): 3.37 (s, 9H), 3.41 -3.97 (m, 141H), 4.03 -4.44 (m, In a 100 mL round-bottom flask equipped with a septum and a magnetic stirrer under nitrogen, 100 (280 mg, 0.63 mmol), potassium carbonate (1.74 g, 12.6 mmol) and tetrabutylammonium iodide (69.8 mg, 0.189 mmol) were dissolved in DMF (55 mL). Subsequently, benzyl bromide (1.48 mL, 12.6 mmol) was added and the reaction mixture was stirred overnight at room temperature. The pale yellow suspension was diluted with ethyl acetate (70 mL) and water (30 mL). The aqueous layer was separated and extracted with ethyl acetate (3 x 20 mL).The combined organic layers were washed with brine, dried over MgSO 4 and concentrated in vacuo. The resulting yellow crude oil was purified by column chromatography on silica gel (cyclohexane / ethyl acetate) to afford product 111 (414.2 mg, 92%). MHz, acetone-d6, δ in ppm): 157.6, 157.5, 155.8, 141.3, 140.8, 140.5, 140.3, 137.1, 137.0, 136.7, 133.6, 131.8, 131.1, 130.6, 128.6, 128.5, 128.0, 127.8, 127.7, 127.5, 127.3, 126.8, 116.4, 116.2, 114.3, 113.2, 113.1, 84.5, 71.0, 70.1, 69.9, 36.4, 36.1 HRMS-ESI (m/z): 715.1705, [M+H] + Compound 118 (0.29 g, 1.08 mmol) was dissolved in THF (4 mL). The solution was cooled to 0 °C with an ice bath and then NaBH 4 (49 mg, 1.30 mmol) and methanol (0.44 mL, 10.82 mmol) were added. The reaction was kept at 0 °C with stirring for 30 minutes and then it was quenched with a solution of HCl 1N and extracted with dichloromethane. The organic phase was dried over MgSO 4 and concentrated under vacuum. Product 119 was obtained in 96 % yield (0.28 g). H NMR (400 MHz, CDCl 3 , δ in ppm): 4.58 (s, 2H), 4.62 (d, J = 5.5 Hz, 2H), 5.16 (s, 2H), 5.27 (dd, J1 = 10.5 Hz, J2 = 1.3 Hz, 1H), 5.41 (dd, J1 = 17.3 Hz, J2 = 1.3 Hz, 1H), 6.00 -6.15 (m, 1H), 6.89 (s, 2H), 6.98 (s, 1H), 7.27 -7.51 (m, 5H) 13 C NMR (100 MHz, CDCl 3 , δ in ppm): 64.85, 70.49, 71.25, 114.14, 117.44, 118.26, 121.37, 112.56, 127.08, 127.56, 128.88, 133.62, 136.55, 146.69, 149.86 MS (ESI-TOF) m/z: 271.3, [M+H] +

	Compound 119
	White solid
	White solid
	12H), 4.47 -4.64 (m, 3H), 6.39 -7.21 (m, 12H) 1 H NMR (400 MHz, CDCl 3 , δ in ppm): 7.76 (s, 1H), 7.50-7.33 (m, 15H), 7.21 (d, J = 8.6 Hz,
	1H), 7.00 -6.97 (m, 2H), 6.95 (d, J = 2.6 Hz, 1H), 6.78 (s, 1H), 6.74 (dd, J1 = 2.6 Hz, J2 =
	8.6 Hz, 1H), 6.64 (dd, J1 = 2.6 Hz, J2 = 8.6 Hz, 1H), 5.22 -5.02 (m, 6H), 4.69 (m, 3H), 3.58
	(m, 3H)

1 13 C NMR (100 MHz, CDCl 3 , δ in ppm): Not enough products to achieve a satisfying resolution MS (ESI-TOF) m/z: distribution centered at 1347.6± 22, [(M+2H)/2] + Compound 111 13 C NMR (100 1

A. BIBLIOGRAPHIC STUDY

The development of the cryptophane-based hyperpolarized 129 Xe biosensors has highlighted the necessity to develop cryptophane chemistry for easy desymmetrization and water-solubilization of the cryptophane moiety. All the results encourage the researchers to continue the development of a cryptophane-based molecular imaging in vivo.

Selective O-alkylation of the phenol in position 3 of compound 59 with methyl bromoacetate and subsequent O-alkylation of position 4 with dibromoethane afforded compound 89 in 35% overall yield. Unfortunately, the reduction of the aldehyde with sodium borohydride as previously described clearly gave a mixture of two products which could be purified by chromatography to yield 59 % of the targeted product 90 and 28 % of the byproduct 90' containing an alcohol instead of the methyl ester (Figure 103). In order to decrease the proportion of the bis-reduction reaction, we attempted to lower the reactivity of NaBH 4 by using a mixture of dichloromethane, ethanol and acetic acid (35:5:1) as a solvent (Figure 104), leading to the formation of sodium borohydride acetate NaBH m (OAc) n (m + n = 4). 127,128 This compound is a reducing agent which is much less drastic than NaBH 4 and selectively reduces carbonyl compounds. 129 In these conditions, no reduction of the ester group was observed but the yield was not enough satisfying. In fact, after purification by silica gel chromatography, only 25 % of targeted product 90 was obtained and 47 % of starting material 89 was recovered. Even though the obtained starting material could be reused in the next cycle of reaction, the yield based on recycling was only 37 %, which was much worse than the one obtained with the original conditions (NaBH 4 , MeOH, THF). Moreover, prolongation of reaction time or reduction of the quantity of acetic acid did not efficiently improve the yield.

FRACTION A FRACTION B

With a carboxylic acid group, cryptophane 76 can be directly bio-conjugated for the construction of bio-probes. Its capacity to encapsulate and exchange xenon was studied but the analysis was marred by a multi-signal problem which suggests the formation of aggregates, the encapsulation of cations or a putative syn-/anti-isomerism. Therefore the yield and production scale of this compound still needs being optimized by avoiding the issues mentioned above.

3) Mono-halogenation of CTVs

A PEGylated mono-iodinated cryptophane 106 was synthesized in three steps in 18 % overall yield. This cryptophane has shown satisfying solubility in water (30 mg/mL) as well as good properties for xenon encapsulation. Unfortunately, it was obtained as a mixture of syn-/anti-isomers which are impossible to be separated.

Alternatively, a non-PEGylated mono-iodinated cryptophane 108 was also synthesized in three steps in 23 % overall yield (non-optimized). To the best of our knowledge, this is the first report regarding the synthesis of a mono-halogenated cryptophane [222]. The syn-and anti-isomers of cryptophane 108 were separately obtained and their properties for encapsulating xenon were evaluated.

Solvents and reagents

Anhydrous solvents are obtained by distillation under nitrogen atmosphere in the presence of drying agents: calcium hydride for dichloromethane and sodium (and benzophenone) for tetrahydrofuran.

The commercial reagents are directly used without purification.

Purification and analysis

Purifications by chromatography on silica gel column are carried out either by gravity or under pressure of nitrogen or air using Merck silica (particle size 40-63 μm). The eluent mixture is specified for each purification.

The automatic purifications by CombiFlash® were performed on a RediSep apparatus (Serlabo Technologies, Teledyn ISCO) using cartridges "pre-packaged" with SiO 2 for normal phase and C 18 for reversed phase.

Analytical thin layer chromatography (TLC) was performed on silica plates Merck 60 F254 on glass substrate.

Products are revealed either by ultraviolet illumination at 254 nm, or by reaction in the presence of iodine vapor, an aqueous solution of potassium permanganate and potassium carbonate or a phosphomolybdic acid solution of 5% by weight in ethanol.

Nuclear magnetic resonance spectra (NMR) 1 H, 13 Compound 39 103 Vanillyl alcohol (10 g, 74.9 mmol) was dissolved in DMF (120 mL) then K 2 CO 3 (45 g, 0.3 mol) and 1,2-dibromoethane (50 mL, 0.7 mol) were added into the solution. The reaction was heated with stirring to 60 °C for 16 hours. After, the mixture was poured into water (800 mL)

and extracted with ethyl acetate (3 x 400 mL). The organic phases were collected and concentrated to about 500 mL and washed successively with a solution of NaOH 1N (2 x 400 mL), water (500 mL) and brine (2 x 500 mL). The organic phase was dried and the solvent was evaporated under vacuum. Compound 39 was obtained in 60 % yield (11.7 g).

White solid

Compound 108 -syn

In a 250 mL round-bottom flask equipped with a septum and a magnetic stirrer, 107 (105 mg, 0.085 mmol) was dissolved in formic acid (100 mL). The reaction mixture was slowly stirred at 55 °C for 5 hours and kept stirring in the room temperature for 18 h. Subsequently, the colorless solution was cooled to room temperature and concentrated in vacuo. The crude product was purified by chromatography on silica gel (cyclohexane / chloroform 100 : 0  50 : 50). Then the fraction containing targeted product was purified by preparative TLC (chloroform / ethyl acetate 90 : 10) and the expected compound 108 -syn was obtained in 2 % yield (2 mg).