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

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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 15N in the urea and 89Y in the chelates of yttrium (III) has all been succeeded using DNP. However, as discussed previously, only polarized 13C could be used for in vivo MRI applications, in particular thanks to its higher sensitivity.
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.
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.
The hyperpolarization of 129Xe 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).

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 129Xe 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.

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 R1 and R2.
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.

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 PPh2Li 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).

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).
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(C6H5)3]4 as a catalyst and Bu3SnH.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.

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Cryptophane-based biosensors for 129Xe MRI

The first cryptophane-based biosensor for 129Xe 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).
Although this biosensor gave unsuitable complex NMR signals due to the presence of four diastereoisomers (there is one stereocenter in the linker and the cryptophane is used as a racemate)92, it enables the detection of avidin when linked to the biosensor. Selective detection of the biotin/avidin complex is based on the changes in rotational and vibrational movements of the cryptophane causing deformation and distortion of the electron cloud of xenon when the bioprobe is bound to the large protein. In the NMR spectrum, this change was visualized by a shift from 72 ppm to 74 ppm (Figure 49). Afterward, this biosensor has been continuously optimized by changing the peptide linker between biotin and the cryptophane in order to improve the chemical shift difference.

Table of contents :

GENERAL INTRODUCTION
A. BIBLIOGRAPHIC STUDY
I. The Hyperpolarized 129Xe MRI and Its Encapsulating Cages
1. General presentation of MRI
a. Principles of NMR
b. Principles of MRI
c. Advantages and Disadvantages of MRI
2. How to improve the sensibility of MRI
a. The MRI contrast agents
b. Hyperpolarization
i. Parahydrogen
i. Hyperpolarized 3He
ii. Hyperpolarized 13C
3. Hyperpolarized 129Xe
a. General presentation of xenon
b. Hyperpolarization with optical pumping
c. Encapsulating structures of xenon
i. The selection criteria
ii. Cyclodextrins
iii. Calixarenes
iv. Hemicarcerands
v. Cucurbiturils
vi. Cryptophanes
II. Cryptophanes: Structure, Synthesis and Applications
1. Structures – stereochemistry, symmetry and conformers
a. Stereochemistry and symmetry
b. Conformers
2. Synthesis
a. The “direct method”
b. The “template method”
c. Coupling of CTVs
3. Strategies for the hydrosolubilization of cryptophanes
4. Strategies of mono-functionalization of cryptophanes
5. Cryptophane-based biosensors for 129Xe MRI
III. Objectives
B. CONCEPTION AND SYNTHESIS OF WATER-SOLUBLE AND MONO-FUNCTIONALIZABLE CRYPTOPHANES
I.Water-Soluble and Mono-Functionalizable Cryptophanes – Why and How
1. Background and objective
2. Different strategies to synthesize asymmetric cryptophanes
3. Polyethylene glycol – our choice for hydrosolubilization
II. Desymmetrization of Cryptophanes
1. Context
2. Optimization and scale-up
a. Optimization of the cyclotrimerization and the demethylation
b. Optimization of the alkylation of CTV
c. Conclusion of the optimization and scale-up
3. Demethylation of symmetric cryptophane
a. Demethylation of the PEGylated cryptophane with TMSI
b. Demethylation of the PEGylated cryptophane by LiPPh2
c. Scan of other reagents to perform the demethylation of cryptophane
4. Conclusion and perspective
III. Desymmetrization of CTVs
1. Cryptophanes based on the functionalization of CTVs with different benzyl alcohol derivatives
a. Conception and retrosynthetic analysis
b. Synthesis towards a mono-allyl PEGylated cryptophane
i. Synthesis of the allylic benzyl alcohol derivative 37
ii. Synthesis of the PEGylated benzyl alcohol derivative 58
iii. Synthesis towards the mono-allylated PEGylated cryptophane
iv. Functionalization of the mono-allylated PEGylated cryptophane
v. Attempts to synthesize PEGylated cryptophanol
vi. Conclusion
c. Synthesis towards a mono-benzylated PEGylated cryptophane
i. Synthesis to the mono-benzyl PEGylated cryptophane
ii. Attempts to synthesize the PEGylated cryptophanol
d. Synthesis towards a mono-acid PEGylated cryptophane
i. The synthesis of the benzyl alcohol derivative with methyl acetate
ii. Synthesis to the mono-acid PEGylated cryptophane
iii. Encapsulation of xenon inside cryptophane
iv. Hyperpolarized 129Xe NMR analysis of cryptophane
v. Conclusions and perspectives
2. Cryptophanes based on mono-halogenation of CTVs
a. Synthesis of mono-halogenated CTV
i. Synthesis of the symmetric CTV 97
ii. Mono-halogenation of CTV 97
b. Synthesis of a PEGylated mono-iodinated cryptophane
i. The analysis of cryptophane 106 by 129Xe NMR
ii. Study of anti-/syn- isomers
c. Synthesis towards a mono-ester PEGylated cryptophane
d. Conclusion and perspectives
3. Cryptophanes based on mono-protected CTVs
IV Synthesis of Asymmetric CTVs
1. Design and retrosynthetic analyse
2. Synthesis of the benzyl alcohol derivatives
3. Synthesis of asymmetric CTVs by cyclotrimerization with different monomers
4. Synthesis of a mono-acid PEGylated cryptophane
GENERAL CONCLUSIONS AND PERSPECTIVES
REFERENCES
EXPERIMENTAL PART

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