M Ali

Taher Mansour

thesis; and to Pr. Kamal Bouhadir for being the examiner. I am honored to have all of you as jury members.

I would also like to express my gratitude to my research supervisors; Dr. Ogaritte Yazbeck who always believed in my potentials, and tough me how to have the spirit of a researcher; Dr. Sylvie Robin who patiently followed my work on daily basis; Pr. David J. Aitken who provided top class scientific supervision, and Pr. Daoud Naoufal who was especially helpful in setting up a wok place for organic chemistry in his lab, here in Lebanon. You were all encouraging, supportive, and vital for the success of this research.

A special thanks goes to all the personnel at the Chimie Peptidomimétique Photochimie, Procédés Alternatifs (CP3A) group, from permanents to students, for their hospitality and scientific guidance, especially Ms. Florence Charnay-Pouget for her help with the HPLC, Mr. Jean-Pierre Baltaze for his help with the NMR experiments, Dr. Thomas Boddaert for his help with the photoreactor setup, Dr. Julien Buendia for his technical advices and Elodie Tran whose masters 1 work was integrated within this thesis. It has been a great pleasure, I learned enormously from each one of you. I would also like to show my appreciation to Dr, Ali Hachem and all his research group (LCMPN), especially my colleagues, Layal Haris, Tourin Bziea, Ranin Kawtharani, and Ali Solaiman.

I am also grateful to the CIOES for their financial support, and to my family, and friends, who stood by me throughout this doctoral research.

Chapter 1: Introduction 1.1 Photochirogenesis

The majority of natural and biologically relevant chiral molecules on Earth have a defined configuration which is linked to their molecular function. It is marvelous how living organisms have the ability to preferentially choose or selectively biosynthesize only a single enantiomer of a given molecule. 1 Chemists are envious of this and seek to imitate biological systems; thus, the asymmetric synthesis of organic compounds has become a popular playground for research. [START_REF] Avalos | [END_REF] Selective preparation of molecules in single enantiomer form also has industrial importance, due to the requirement of stereochemically pure molecules in the field of medicine and material science.

Over the last few decades, the asymmetric synthesis of organic compounds has been conventionally achieved by either catalytic 3 or enzymatic [START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] transformations, and a wide range of methods have been developed in order to gain stereochemical control over a variety of chemical transformations. Photochemistry has proven itself to be a versatile tool in organic synthesis, 5,6 and it is receiving even more attention today since it qualifies as a method for the preparation of new materials in an "environmentally friendly" context, referred to as green chemistry. 7 Nonetheless, asymmetric photochemical transformations are not widely studied. The term "photochirogenesis" is rather new. It was coined in 1996, and is conceptually synonymous to more traditional "asymmetric photochemistry" or "chiral photochemistry" with the emphasis on the creation of molecular chirality through the use of a photon. 8 Asymmetric induction from the excited electronic state is hard to achieve due to fundamental factors that make conventional approaches difficult to apply in photochemical reactions. [START_REF] Inoue | [END_REF] For instance, a chiral catalyst in thermal reactions often functions by decreasing the reaction activation barrier and channels the reaction profile towards an enantioselective pathway; whereas in a photochemical reaction, the irradiated molecules gain sufficient energy to undergo fast subsequent reactions, which do not require further catalysis. So, the biggest challenge in photochirogenesis relates to the inherent high-energy nature of the electronical excited state. Indeed, the much smaller activation parameters for excited-state (as opposed to ground-state) reactions leave little space for energetically differentiating the diastereomeric transition states leading to a pair of enantiomers. So how can chirality be controlled in the highly reactive, short lived excited state? Photochirogenesis research has made great progress both quantitatively and qualitatively due to new methodologies developed for controlling the processes involved. Essentially, in order to achieve stereochemical control over a photochemical reaction, one must make sure that the substrate molecule already resides in a chiral environment during the excitation step. 10 In this regard, photochirogenesis can be convenienttly split into two major categories.

The first includes reactions in which chirality is transferred to a reacting molecule due to the presence of a chiral substituent, covalently or ionically connected to the reactant. Chirality, with such an approach, can be induced through the intramolecular interaction between the chiral center, acting as a "chiral auxiliary", and the reacting moiety. The second category, supramolecular photochirogenesis, can be described as a host-guest interaction between the reactant, as the guest, and a host molecule that provides a chiral environment for the photochemical reaction to proceed in an asymmetric manner. Here, chirality is transferred from one molecule to another through intermolecular interactions between the substrate and a neighboring molecule bearing some chiral properties.

Non-supramolecular photochirogenesis

1.1.1.1 Photochirogenesis via a chiral photosensitizer Hammond and Cole,11 were the first to exploit chiral photosensitizers for asymmetric induction in photochemistry. They studied the geometrical photoisomerization of racemic 1,2-diphenylcyclopropane sensitized by a chiral (R)-N-acetylnaphthylethylamine (see Scheme 1). By monitoring the development of the optical activity during the course of the photoisomerization, they established a photostationary state at a cis/trans ratio of 1.03 with an enantiomeric excess of 7% of the chiral trans-isomer, thus showing the potential of using a chiral photosensitizer to induce chirality in a photoreaction.

Scheme 1: The asymmetric photoisomerization of 1,2-diphenylcyclopropane sensitized by a chiral naphthylethylamine derivative.

However, after 20 year of rather slow progress; the unprecedentedly high 53% ee achieved by the Inoue group, 12 in a systematic study of the enantiodifferentiating isomerization of cyclooctenes sensitized by chiral polyalkyl benzene(poly)carboxylates (see Scheme 2), triggered a boost of interest in photochirogenesis.

Scheme 2: The asymmetric photoisomerization of cyclooctene sensitized by chiral polyalkyl benzene(poly)carboxylates.

Most recently the Inoue group 13 exploited chiral methyl (R)- [10]paracyclophane-12carboxylate as a sensitizer, to achieve an enantiomeric excess of 87% in the photoisomerization of (Z,Z)-1,5-cyclooctadiene (see Scheme 3). In this system the decamethylene bridge provided a shield on one of the enantiotopic faces of the chiral cyclophanes sensitizer. This shielding was attributed by the authors to both enthalpically and entropically factors, which makes the approach, and subsequently the isomerization of the substrate more enantioselective.

Scheme 3: The asymmetric photoisomerization of cyclooctadiene sensitized by (R)- [10]paracyclophane-12-carboxylate derivative.

Aside from this quite unique example of enantioselective photosensitized isomerization of cyclooctadiene, it had pretty much been established that conventional photochirogenesis induced via chiral sensitizers alone are generally considered not very efficient. The exciplex between an excited chiral sensitizer and a ground-state substrate, despite being at lower energy than the exited state alone, still has large conformational freedom which minimizes the prospects for chiral induction. 14

Photochirogenesis via a chiral auxiliary

After the mid-1990's, with more understanding of chirality transfer, much greater stereoselectivity was reported in photochemical reactions. Griesbeck and coworkers studied the photodecarboxylation of N-phthaloylanthranilic acid derivatives (see Scheme 4). 15 Photodecarboxylation of the corresponding chiral potassium salt allowed access to the pentacyclic pyrrolo [1,4]benzodiazepine photo-adduct with a diastereoselectivity greater than 98% and an enantiomeric excess up to 86%.

Scheme 4: The asymmetric photodecarboxylation of N-phthaloylanthranilic acid derivatives.

At around the same period, the Mariano group studied the [2+2] photocycloaddition of chiral pyrolidino-cyclohexeniminium perchlorates (see Scheme 5). 16 The presence of the chiral pyrrolidine makes the photocycloaddition facially selective favoring the anti transition state that gives rise to an enantiomeric excess of up to 82%.

Scheme 5: The asymmetric photocycloaddition of chiral pyrrolidino-cyclohexeniminium perchlorate.

Later, much higher enantiomeric excess was found through photochirogenesis using chiral auxiliaries. Scheffer and Wang studied the photochemical synthesis of β-lactams via the asymmetric Norrish Type II photochemical reaction of salt crystals formed from ionic chiral auxiliaries and 4-(2-(diisopropylamino)-2-oxoacetyl)benzoate (see Scheme 6). 17 Irradiation of the chiral crystals in the solid state yielded the target photoadduct in ee up to 99%. The high enantioselectivity was attributed by the authors to the ability of the chiral salt substrate to crystallize into a single conformational enantiomer.

Scheme 6:The asymmetric Norrish Type II photochemical reaction of chiral salts of 4- (2-(diisopropylamino)-2-oxoacetyl)benzoate, forming a β-lactam derivative.

Supramolecular Photochirogenesis

Supramolecular photochirogenesis 18 is an evolving field of research implicating photochemistry, asymmetric synthesis, and supramolecular chemistry. Stereochemical control in supramolecular photoreactions is mostly attributed the non-covalent interactions between the host and the substrate; by confining a prochiral substrate in a chiral supramolecular environment prior to photo-excitation, there is more likelihood of effective chirality transfer in the excited state. The substrate is likely to have a preferred conformation within the host cavity; this is conveyed to the product chirality upon irradiation. Also, confinement extenuates the difference in free energy of activation between two diasteromeric transition states, thus facilitating their discrimination, and boosting enantioselectivity. Several macromolecules have been used as hosts in asymmetric photosynthesis, including zeolite, urea clatharates, cucurbiturils, octaacids, metal nanocages, chiral organic templates, and cyclodextrins (CDs). There are a number of excellent recent reviews on supramolecular photochirogenesis; 19 here we shall illustrate some of the more recent studies or examples with fairly high enantioselectivity.

Chiral templates

Chiral templates are among the best supramolecular hosts utilized in the field of asymmetric photochemistry. Several chiral templates have been used to induce high enantioselectivity.

Chiral templates possess a well-defined three-dimensional space to confine a guest substrate; they do not have a cavity, but achieve chirality transfer by directly interacting with substrates through strong and directional non-covalent interactions, such as hydrogenbonding and/or electrostatic interactions. In their interactions they are considered supramolecular hosts, even though they are more "molecular" due to their size and architectural simplicity. Several chiral templates have been used to induce high enantioselectivity.

The Lipkowska group utilized chiral diols as templates to perform enantioselective photocyclizations of 1-alkyl-2-pyridones in inclusion crystals, to give β-lactams in good yield with ee up to 99% (see Scheme 7). 20 The high enantioselectivity was attributed to the spatial situation of the pyridone derivative substrate relative to the template molecule in the solid crystal form, which sterically allows the formation of one enantiomer.

Scheme 7:

The asymmetric photocyclizations of an 1-alkyl-2-pyridone into a β-lactam derivative.

A similar approach was later studied by the Bach group who developed a lactam/xanthone based template for the intramolecular [2+2]-photocycloaddition of prochiral 4-(but-3enyloxy)quinolone (see Scheme 8 ). 21 The template allowed high directionality in the binding of its lactam segment to a photoreactive substrate, making only one enantiotopic face available for the [2+2]-cycloaddition reaction to proceed. This was validated by the high enantioselectivity of the reaction, with ee values up to 94%.

Supramolecular photochirogenesis with cyclodextrins

Cyclodextrins (CDs) are macrocyclic nano-buckets that are capable of forming stable inclusion complexes with many molecules. Due to this property, CDs have many applications in supramolecular chemistry, [START_REF] Szejtli | Cyclodextrin Technology[END_REF][START_REF] Szejtli | [END_REF] in the catalysis of organic reactions, 25 and in encapsulation of pharmaceuticals in order to serve as drug delivery agents. 26,27 CDs also play a prominent role in the field of supramolecular photochirogenesis, as we shall illustrate below.

Before discussing asymmetric photochemistry induced by cyclodextrins, one must appreciate the structural features of these molecules. Natural CDs are cyclic oligomers comprised of 6, 7, or 8 D-glucopyranose units (corresponding to α-, β-, and γ-CD respectively), that are linked through α- (1,4) glycosidic bonds. Due to the chair conformation of the glucopyranose units the CD is shaped like a truncated cone rather than a perfect cylinder (see Figure 1). 28 The internal cavity of the truncated cone is lined by the H3 and H5 protons and the lone pair of the glycosidic oxygen atoms, thus rendering the interior of the structure lipophilic; this allows CDs to form host-guest complexes with a variety of organic and inorganic molecules. On the other hand, the hydroxyls on C-2, C-3, and C-6 are directed towards the exterior, thus rendering the outer face hydrophilic. As a combined effect of this arrangement of atoms, CDs are soluble in polar (e.g. physiological) media and at the same time can carry lipophilic molecule in their cavity. [START_REF] Dodziuk | Cyclodextrins and Their Complexes[END_REF] Figure 1: Glucopyranose unit (left), the β-CD torus (middle), and the orientation of the CD protons relative to its cavity (right).

CDs are transparent in the UV-vis region of electromagnetic radiation and are inherently chiral and compatible with many electronically excited species, making them among the most frequently employed supramolecular hosts for mediating photochirogenic reactions. [START_REF] Mori | [END_REF] In the past 30 years, the CD cavity has provided a chiral environment for a wide range of photochemical reactions, including photolysis reactions, 31,32 unimolecular photoelectrocyclizations, intermolecular 33 and intramolecular photocycloadditions, 34,35 as well as photocyclodimerization reactions. 36,37,38 Since CDs can be chemically modified, 39 sensitizer-grafted CDs can be envisaged as supramolecular photosensitizing systems for better enantiodifferentiation.

In 1980, Takeshita and coworkers studied the intramolecular photochemical electrocyclization of tropolone and its methyl ether derivative in the presence of αor β-CDs, in water, methanol, acetone, or hexane (see Scheme 10). 40 The authors did not report ee values for the photoproducts; rather, they validated the occurrence of enantioselective transformations through optical rotation measurements. They also noted that the amplitude of the optical rotation was largest in photoadducts obtained from irradiation in aprotic solvents in the presence of β-CD compared to those obtained in the presence of α-CD.

Scheme 10: The intramolecular photochemical electrocyclization of tropolone and its methyl ether derivative in the presence of CDs.

Twenty years later, the Ramamurthy group conducted new studies on the photochemical electrocyclizations of tropolone alkyl ethers bearing different alkyl substituents, in the presence of α, β and γ-CDs, both in solution and in the solid state. 41 observed a near zero ee in solution and a moderate ee (20-30%) in solid state, suggesting that the rigidity of the medium is an important parameter for the chiral induction during this photoreaction.

Moreover, they suggested that a tight-binding complexation between the reactant and the CD host is a requirement for a good enantioselectivity.

Subsequently, the same group studied the photochemical electrocyclization of N-alkyl pyridines in the presence of β-CD (see Scheme 11). 42 Enantiomeric excesses 60% was obtained by irradiating a mechanically ground mixture of the β-CD host and the pyridone guest in solid state.

Scheme 11:

The photochemical electrocyclization of N-alkyl pyridones in the presence of β-CD.

Despite the higher ee, the authors were unable to exceed a conversion of 15% even when controlling the wavelength of the irradiation, an observation which was attributed to the photoreversibility of the reaction.

Chirally modified CDs were utilized as hosts for Norrish Type II / Yang cyclization reactions of chiral derivatives of N,N-diisopropyl-2-oxo-2-arylacetamides, in aqueous solutions as well as in sloid state (see Scheme 12). 43 Although an increase of the enantiomeric (up to 21% ee and 47% de) was noted when either the host or the guest were chirally modified; neither a reliable model that would help to predict the outcome of the chiral induction in a photochemical reaction, nor an understanding of the effect of the chiral center in the substrate emerged from this investigation. Inoue and coworkers investigated the photoisomerization of Z-cyclooctene in the presence of native β-CD 44 and functionalized α, β, and γ-CDs bearing a photosensitizer on the primary face. 45,37 They observed an improvement in the ee from near 0% with native β-CD to 24% with a sensitizer functionalized β-CD (see Scheme 13). The authors pointed out the importance of the "fit" between the CD cavity and the guest for the enantioselectivity. The same photoisomerization reaction was further investigated in aqueous methanol by Li and coworkers. 46 This group showed that, in the presence of a sensitizer functionalized β-CD, the ee and the photostationary E/Z ratio are dependent on the solvent composition. They reported ee values up to 47% through optimization of the methanol/H2O ratio and the temperature of the reaction mixture (See Scheme 13).

(dimethylamino)ethylamino)-6 A -deoxy-γ-CD (DMAEA-γ-CD) 3 (see Scheme 15). 36,48 In the case of native γ-CD, the head to tail enantiomer b was the major compound, obtained with an ee of 32% at 25 °C. This was improved to an ee of 41% when the reaction temperature was lowered to 0 °C. On the other hand, when DMAEA-γ-CD was used as the chiral host, ionic interactions between the carboxylate part of the ACs and the ammonium part the DMAEA-γ-CD led to substrate reorientation inside the CD cavity such that the head to head enantiomer c became the major compound, still with 41% ee.

Scheme 15: The chiral photo-cyclodimerization of 2-anthracenecarboxylate inside γ-CDs.

Later, the same group devised a novel, "dual chiral, dual-supramolecular" system in which they anchored two AC acid molecules to the primary face of an α-CD in order to pre-orient the two photosubstrates before adding the γ-CD as the chiral confining host. This duel system allowed selective access to the major cyclodimer c in 98% yield and unprecedented 99% ee (see Scheme 16). 49 Scheme 16: The dual-supramolecular system for a chiral photocyclodimerization of 2anthracenecarboxylate.

Continuing their research, Inoue's group recently published the cyclodimerization of ACs encapsulated by two 6 A ,6 C -dideoxy-6 A, 6 C-triethylammonium derivatives of β-CD (see Scheme 17). 50 In this 2:2 AC/β-CD complex, two ACs moieties are partially stacked to each other exclusively in a head-to-tail fashion which allowed access to the non-conventional slipped cyclodimer with an ee of 71% under optimized conditions.

Scheme 17: Formation of the non-conventional slipped cyclodimer as a product of the photocyclodimerization of the 2:2 AC/β-CD complex.

Study of CD complexes 1.2.1 Complex stoichiometry

When studying a molecular inclusion complex, a key matter is to determine its stoichiometry (see Figure 2). For CD complexes several host:guest ratios have been reported, among which the most commonly observed is the 1:1 complex, wherein one CD molecule includes only one guest inside its cavity. 51 Nevertheless, other ratios are known, e.g. The most common binding stoichiometries between CDs and guest molecules. [START_REF] Linde | Changes to Taste Perception in the Food Industry[END_REF] Three-component CD complexes are also known. These ternary complexes are usually composed of the CD, a guest, and an alcohol that serves to optimize the fit of the guest in the CD cavity. 51 Interestingly, the stoichiometry in a CD complex can be temperature dependent. For instance, the 1:1 stoichiometry of a β-CD/1-adamantanecarboxylic acid complex at 25 o C changes to a 1:2 stoichiometry when the temperature is lowered to 0 o C. 56

Determination of a CD complex stoichiometry

One of the frequently applied procedures to determine the stoichiometry of a molecular complex is the Job's method of continuous variation. [START_REF] Dodziuk | Cyclodextrins and Their Complexes[END_REF] In a typical experiment, a physical property that is sensitive to complex formation is recorded while the relative proportions of the host and the guest are varied in a solution in which the total solute concentration is kept constant. 57 The most common variable used for a Job's plot is UV-vis absorbance, but any property that correlates linearly with the concentration of complex formation, including kinetics, conductivity, NMR spectroscopy etc., can be used. 58 The collected data are usually plotted in the form of the molar fraction () of one species (host or guest) multiplied by the induced change in the observed property (P), against that molar fraction (), where  =

[𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝒂] [𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝒂]+[𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝒃]
(see Figure 3).

The position of the maximum in a Job's plot identifies the stoichiometric ratio of the formed complex. Although the data obtained is not convenient for estimating the binding constant, 57 the shape of the Job's plot curve provides qualitative insight into the magnitude of the binding constant. Strong binding (Kb >> 1) affords a more angular plot, whereas a lower bonding constant affords a gentle curvature. 58 Figure 3: A general form of a Job's plot for a 1:1 complex showing the difference in curvature shape when Kb << 1 and , Kb>> 1.

1D NMR studies of CD complexes

The simplest NMR experiment for studying an inclusion complex is to monitor the changes in the proton chemical shifts between the free host and guest species and the putative complex. Thakkar and Demarco described an NMR method for examining the mode of interaction of CDs with a variety of aromatic substrates. 59,60 Their method is based upon the rationale that when an aromatic guest is incorporated inside the cavity of the CD molecule, protons located within the cavity (H3, H5 and, possibly, H6), (see Figure 4), undergo a noticeable shift in their 1 H NMR signals due to the anisotropy of the aromatic moiety. On the other hand, protons which are oriented toward the outside of the CD cavity (H1, H2, and H4), are relatively unaffected. These induced variations in the chemical shifts could provide evidence for the formation of an inclusion complex in solution. 61,62 Figure 4: Orientation of CD protons with respect to the cavity.

Apart from qualitative information, NMR titration experiments can lead to valuable information not just for how the host and guest(s) interact, but can also be used to quantitate how strong this interaction is. Indeed, values exceeding 10 5 for the equilibrium binding constant Kb can be estimated using modern NMR instruments from the observed induced chemical shifts (Δδ). 63 The induced chemical shift is defined as the difference in the chemical shift of a proton belonging to one molecule, in the presence and absence of another molecule. 64 In our case, the induced shifts were calculated by the following equation: Δδ = δ(free) -δ(complexed). In this convention, a positive sign of Δδ designates an upfield shift in the presence of another molecule.

Determination of the binding constant Kb

The equilibrium constant (K) for a complex formed between a host (H) and a guest (G) can be formulated from the general equilibrium reaction:

When the stoichiometry of a β-CD/guest complex is 1:1, the desired equilibrium (in this case binding) constant Kb is given by:

𝐾 𝑏 = [𝐻𝐺] [𝐻][𝐺] (1)
Where [G] is the concentration of free guest, [H] is the concentration of free host, i.e. β-CD, and [HG] the concentration of the formed host/guest complex.

Because the complexation phenomenon is a dynamic equilibrium, the individual concentration of each species is not readily measurable. Instead of measuring separate concentrations, a well-known approach to determine the binding constant is to generate the binding curve then to deduce Kb via a regression analysis based on the Benesi-Hildebrand equation. 65,[START_REF] Conners | Binding constants[END_REF] A plot of 1/Δδ vs [G] establishes a straight line whose slop is directly related to the value of Kb, for a 1:1 -Guest:Host complex (see Figure 5). (For more detailes see ndex -A)

2D NMR in the characterization of CD host/guest complexes

In order to investigate the geometry of a complex and to propose a mode of complexation between CD and a guest, 2D ROESY NMR experiments are very useful. At the level of NMR spectroscopy, nuclear spins can interact via both through-bond phenomena (spin-spin coupling) and through-space phenomena (dipolar coupling). The through-bond interaction is commonly observed as a split in the NMR signal and characterized by the coupling constant J. On the other hand, the through-space dipolar interactions do not split the NMR signal since the effect is completely averaged by the normal tumbling of molecules in isotropic solutions. Nevertheless, these dipolar interactions manifest themselves indirectly, generating the Nuclear Overhauser Effect (NOE). [START_REF] Dodziuk | Cyclodextrins and Their Complexes[END_REF] NOE magnitudes depend, among others, on the effective correlation times τeff characterizing overall molecular tumbling and intramolecular motions. CD complexes with molecular masses around 1-2 kDa display τeff of a fraction of a nanosecond and are placed near the transition between positive and negative NOEs. Fortunately NOE in the rotating frame (ROE) is always positive, (see Figure 6), thus becoming a method of choice in the studies of medium-sized molecules and complexes. [START_REF] Dodziuk | [END_REF] Consequently the (ROESY) pulse sequence is typically the NOE-based experiment of choice for CD complexes. 68 In Rotating-frame Overhauser Effect Spectroscopy (ROESY) experiment, the spatial proximity between atoms of the host and guest molecules is obtained by observing the intermolecular dipolar cross-correlations. The intensities of the cross peaks are proportional to 1/r 6 , where r is the mean distance between the protons in dipolar interaction. 69 A NOE cross-peak between a proton of a guest molecule and a proton of the CD moiety will be observed in a ROESY spectrum if these two protons are closer than 4 Å through space.

Therefore, if the guest molecule is included in the CD cavity, NOE correlations between the protons of the guest and the protons (H3, H5, or H6) that appear in the CD inner cavity will be observed. 62 Although 1D NMR titration experiments could provide evidence on the formation of an inclusion complex between two molecules, 2D NOESY or ROESY experiments are essential to understand the dynamics and the overall geometry of the inclusion complex. 64 Several research groups have relied on interpretation of 2D as well as the 1D (selective)

ROSEY spectra to extract valuable information on the mode of complexation of various organic guest molecules inside CD cavities.

For instance, Forgo and D'Souza used 1D ROE experiments to study the host-guest interactions of β-CD with p-nitrophenol (see Figure 7). 70 The selective excitation of H3' and between β-CD and vanillylmandelic acid (VMA), homovanillic acid (HVA) and vanillin (VA). The ROE correlations extracted from a 2D ROESY experiment allowed the authors to distinguish between partial and deep inclusion loci of the guest inside the CD, as well as on the face from which the guest enters the cavity (see Figure 8).

γ-Amino acids

Amino acids are elementary building blocks of the biological system. From a structural point of view, amino acids can be classified as α, β, γ, etc., depending on the relative position of the amino group (-NH2) with respect to the carbocyclic acid (-COOH) on the common carbon chain (see Figure 9). While β-and γ-amino acids are non-proteinogenic, they have a great importance in the field of biology and in medicinal chemistry. 72,73 1.

GABA and constrained analogs

Structurally, -amino acids are sub-classified according to the position of the substituents they bear. For example, a γ-amino acid which is monosubstituted at the C-2 position is labelled a γ 2 -amino acid, while a a γ-amino acid which is disubstituted at its C-2 and C-3 positions is considered a γ 2,3 -amino acid (see Figure 10). In the human body, -amino acids operate mainly as neurotransmitters. In particular, the parent compound γ-aminobutyric acid (GABA) serves as the principle inhibitory neurotransmitter of the central nervous system. 74 GABA also has an influence on different biological events during the nervous system development. 75 Abnormal levels of GABA in the human brain have been correlated with serious neurological disorders such as epilepsy, anxiety, 76 schizophrenia, 77 and Alzheimer's disease. 78 Due to their potential significance in human biology and their potential applications in pharmacology, 79 GABA analogs have gained considerable attention over the past decades and a large number of strategies have been developed for their stereoselective synthesis. In order to acquire analogs that mimic a particular bioactive conformation of GABA, the synthesis of conformationally restricted GABA analogs has been of recent interest. 81 Various types of GABA analogs, both acyclic, 82 and cyclic, 81 have been reported in the literature. Figure 11 shows some of these analogs that act as agonists for specific GABA receptors. For example, trans-4-aminocrotonic acid (TACA) was more potent than GABA as a ligand for the ionotropic receptors GABAA and GABAC, but less potent for the metabotropic receptor GABAB. 83,84 . The cis-isomer (CACA) showed preferred binding to the GABAC receptor over GABAA, whereas 4-aminotetrolic acid (ATA) had the opposite selectivity profile. 82 cis-2-(Aminomethyl)cyclopropanecarboxylic acid ((+)-CAMP) was found to be a selective agonist of GABAC. 85 Gabapentin (GBP, Neurontin®) is a

GABAergic agonist that has important anticonvulsant, 86 and analgesic 87 properties; on the other hand, Gabapentin lactam (GBP-L) is neuroprotective in retinal ischemia. 88 Figure 11: The parent γ-amino acid GABA and some of its bioactive constrained analogs.

GABA analogs in the field of foldamers

Aside from their biological importance as discrete molecules, -amino acids are also becoming increasingly popular as building blocks for peptidomimetics, in the form of γoligopeptides or of mixed α,or β,hybrid peptides, because of the distinct, well-defined 3D folding patterns that these compounds can adopt (as will be discussed in more detail in Chapter 4). 89,90 Here again, -amino acids with cyclic constraints are of particular interest since their rigid backbones provide the severe conformational restrictions needed to impose well-defined low-energy conformations in α,-and β,-hybrid peptides. 91,92 Figure 12 shows examples of three, 93 four, 94 five, 95 and six 96 membered ring cyclic constrained -amino acids which have been employed as building blocks for oligo-peptides that adopt well-defined 3D structures. 90

Literature synthesis of cis-and trans-2,3 CB-GABA

In this section we will focus our attention on two cyclobutane constrained -amino acids: 2-(aminomethyl)cyclobutanecarboxylic acid ( 2,3 CB-GABA) and 2-(2-aminocyclobutyl)acetic acid ( 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA) (see Figure 13).

Figure 13:The constrained -amino acids of interest: [START_REF] Avalos | [END_REF]3 CB-GABA and 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA

The synthesis of cis- More recently, the Aitken group established a synthetic route that allowed access to both enantiomers of cis-and both enantiomers of trans-2,3 CB-GABA. 99,100 The synthesis began with via a the photochemical [2+2]-cycloaddition reaction of (R)-1-(1-phenylethyl)-2,5dihydro-1H-pyrrole with ethylene, which provided the bicyclic photoadduct with an exclusive cis geometry and as a mixture of two diastereoisomers which were separated by chromatography. Each of the two compounds obtained was transformed in three steps into N-Boc-cis-2,3 CB-GABA in single enantiomer form. In turn, each cis derivative was subjected to an epimerization protocol to furnish the corresponding trans compound, in enantiomerically pure form (see Scheme 20).

Scheme 20: The Aitken group's access to all four stereoisomers of In the literature, access to cis-and trans- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA, each in racemic form, was described in 1982 by Kennewell and co-workers through a lengthy procedure starting from cis and trans-cyclobutane-1,2-dicarboxylic acids, respectively. 101 More recently, in order to improve the availability of these conformationally restricted cyclobutane analogues of GABA, our groups jointly reported two complementary synthetic routes that provided practical access to both enantiomers of cis- The first route involved a homologation strategy that started with the photochemical [2+2]cycloaddition reaction of Boc-uracil with ethylene, which provided a single bicyclic photoadduct with an exclusive cis geometry. Basic hydrolysis of this photoadduct led to the known N-Boc derivative of ()-cis-2-aminocyclobutanecarboxylic acid. 103 This cyclic βamino acid derivative was then homologated in five steps to provide the N-Boc derivative of ()-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA. The second route started with the known photochemical 4πelectrocyclization of azepinone (see Scheme 21). 102 The photoadduct was then transformed in three steps into the same N-Boc derivative of () cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA that was obtained from the first route. The second route employed fewer steps and had a better overall yield than the first one.

The chiral resolution of ()-N-Boc-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA was then described, using (4S,5R)-4methyl-5-phenyloxazolidin-2-one as a chiral auxiliary (See Scheme 21). 102 This endeavor required considerable effort; it was first necessary to identify the satisfactory chiral auxiliary, which then had to be prepared synthetically. The resolution protocol itself required three steps: coupling of the racemic -amino acids derivative with the oxazolidinone, tedious chromatographic separation of the two diastereoisomeric adducts, then cleavage of the chiral oxazolidinone from each, to provide both enantiomers of N-Boccis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA.

Aims of this project

In this work we sought to explore alternative synthetic strategies which might provide more convenient and/or efficient access to cis-2,3 CB-GABA and cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA in enantiomerically pure form. It was our intention to use these molecules as building blocks to prepare new mixed peptide sequences and to study these peptides for their foldamer behavior.

We indicated above that photochemical strategies, used in conjunction with covalently attached chiral motifs, can provide access to enantiopure cis-2,3 CB-GABA and cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA. In these preparations, chiral information is employed either in an "early-stage" role, such as in the synthesis presented in Scheme 20, or in a "late-stage" role, such as in the resolution protocol in Scheme 21. However, in each case the chiral fragment is (temporarily) attached covalently to the requisite -amino acid fragment, which implicates the formation of diastereoisomer mixtures at some point in the synthesis and the consequent requirement of diastereoisomeric separation by chromatography.

We considered that the enantioselective synthesis of cis-2,3 CB-GABA and cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA derivatives warranted study. The clear advantage of this approach would be that the covalent attachment (and subsequent removal) of chiral auxiliaries would not be required, nor would chromatographic separation of diastereoisomeric pairs be necessary. Towards this objective, as a contribution to the growing area of photochirogenesis, we decided to investigate the use of β-CD as a readily-available, chiral supramolecular host for pertinent enantioselective photochemical transformations (see Scheme 22). The intramolecular photochemical [2+2]-cycloaddition of an N-allyl N-aryl acrylamide in the presence of β-CD might provide access to a bicyclic adduct in enantioenriched form, leading to enantioenriched cis-2,3 CB-GABA. The photochemical 4π-electrocyclization of azepinone in the presence of β-CD might provide access to the known bicyclic adduct in enantioenriched form, leading to enantioenriched cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA.

Scheme 22: The proposed photochirogenic reactions in the presence of β-CD, leading towards cis-2,3 CB-GABA (top) and cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA (bottom) in enantioenriched form.

We proposed to use the enantiomerically-pure CB-GABA derivatives obtained above to prepare γ/α-hybrid peptides and study their conformational behavior in solution. In this chapter we set out to establish a new synthetic access to cis-2,3 CB-GABA derivatives, based on the anticipated enantioselective intramolecular photochemical [2+2]cycloaddition of an N-allylacrylamide derivative hosted inside the chiral cavity of β-CD.

The selected N-allylacrylamide derivative was N-allyl-N-(4-methoxyphenyl)acrylamide, 1

(see Scheme 23). The methoxyphenyl group was chosen in order to favor the inclusion of compound 1 inside the hydrophobic (and asymmetric) cavity of the β-CD.

Scheme 23: The proposed enantioselective intramolecular [2+2]-photocycloaddition of compound 1 in the presence of β-CD.

methoxyphenyl group could be easily removed. 106 Compound 2 should therefore be transformed in only a few steps into the known N-Boc protected derivative of cis-2,3 CB-GABA (see Scheme 24).

Scheme 24: Transformation of the anticipated photoadduct 2 into a cis [START_REF] Avalos | [END_REF]3 CB-GABA derivative.

Our first objective in this chapter was to synthesize N-allyl-N- (4methoxyphenyl)acrylamide. Thereafter, a study of its host-guest interactions with β-CD was envisaged, followed by an assessment of its photochemical reactivity: both alone and in the presence of β-CD. Finally, the transformation of the anticipated cycloadduct to provide enantioenriched cis-2,3 CB-GABA would be carried out.

Results and discussion

Synthesis of N-allyl-N-(4-methoxyphenyl)acrylamide 1

A short synthesis of N-allyl-N-(4-methoxyphenyl)acrylamide 1 has been described, requiring as a starting material the unstable, not readily-available, toxic reagent acryloyl chloride. 104 We preferred to circumvent use of this reagent, and applied instead a variation of the literature procedures described by Bonete, 107 and Wang, 108 following the retrosynthetic approach depicted in Scheme 25.

Scheme 25: Retrosynthetic analysis for the preparation of N-allyl-N-(4methoxyphenyl)acrylamide 1.

The first step was the Michael addition of benzenesulfinate anion to acrylic acid, performed by stirring the reagents together in water overnight (see Scheme 26). In the literature procedure, 90% ethanol had been used as the solvent, however we encountered solubility problems in various ethanol/water mixtures. The use of water as the solvent greatly simplified the reaction. After the reaction time, the suspension was acidified with 2 M HCl and the desired product, 3-(phenylsulfonyl)propanoic acid 3, was extracted using diethyl ether. The organic layer was concentrated to give compound 3 as a white solid with a yield of 75%. The product 3 was used in the next step without further purification. Making use of the phenylsulfonyl group as leaving group, the third step involved a one pot elimination in the presence of potassium tert-butoxide (Scheme 28), followed by addition of allyl bromide in a nucleophilic substitution to give crude compound 1, N-allyl-N- (4methoxyphenyl)acrylamide. The final product was purified by silica gel chromatography, and isolated with an overall yield of 60%. It is important to note that compound 1 showed a tendency to polymerize into an insoluble hard solid, so it was important for it not to be in contact with traces of acids, nor be heated above 25 o C while evaporating the solvent after chromatography. hours then spread on a glass plate and left to evaporate to give a white clear film (see Figure 14).

Evidence of complexation by powder X-ray diffraction

In order to confirm the formation of a complex between 1 and β-CD, a powder X-ray diffraction analysis was done. Three samples were compared: β-CD alone, compound 1 alone, and the white film (β-CD/1 complex), as shown in Figure 17. The powder X-ray diffraction data obtained for the β-CD/1 complex film, were different from the data obtained for each component alone. This provides support for the argument that we obtained a genuine complex, rather than a simple physical mixture of the two discrete substances. 110

Determination of the complex stoichiometry

Job's method of continuous variation was used to investigate the stoichiometry of the β-CD/1 complex. Two separate 10 mL (0.9 mmol) mother solutions were prepared in water, one of β-CD and the other of 1. Specific volumes of each solution were taken and mixed in different proportions to prepare eleven sample solutions (each 2 ml) covering the rage of

mole ratios 0 <  = [𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑 𝟏] [𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑 𝟏]+[𝛽-𝐶𝐷] < 1
The UV absorbance for each of the eleven sample solutions was recorded at λ = 228 nm, and the difference in absorbance (ΔA) between compound 1 alone and in the presence of β-CD multiplied by the molar fraction () was plotted against molar fraction (), as shown in Figure 16. The maximum of the curve at  = 0.5 validates a 1:1 stoichiometry in the complex formed upon mixing β-CD with compound 1 in aqueous solution. The rounded shape of the curvature also provides an indication that the equilibrium constant of the complexation reaction is of low magnitude. 58

1D NMR studies of the β-CD/1 complex

To gain insight into the nature of the interactions between β-CD and compound 1, an NMR titration experiment was done. Six different NMR tubes containing a solution of β-CD in D2O at 7 mM were prepared. Each tube was prepared with a different equivalence of compound 1 to β-CD ranging from 0 equivalents in tube 1, to 2 equivalents in tube 6.

Figure 17 shows the 1 H NMR spectra for each tube.

Protons H3 and H5 of the β-CD cavity showed a gradual up-field shift with as the amount of compound 1 increased with respect to β-CD. At a 1:1 ratio, the change in the chemical shift reached 0.15 ppm for H3 and 0.2 ppm for H5. On the contrary, H1, H2, H4 and H6 of β-CD, located outside the cavity, did not show any significant shift change throughout the titration.

These observations can be interpreted as showing that compound 1 is being hosted inside the hydrophobic cavity of β-CD.

The induced shift Δδ, defined as the difference in the chemical shift of a proton belonging to one molecule, in the presence and absence of another molecule, 64 were calculated by the following equation: Δδ = δ(free) -δ(complexed) and are shown in Table 1. In this convention the positive sign of Δδ designates an upfield shift. Δδ (ppm) 0-100% 0.000 0.000 -0.15 0.000 -0.2 0.000

Table 1: The change in chemical shift (Δδ) of the β-CD protons between 0% and 100% of compound 1.

On the other hand, all protons of compound 1 show a down-field shift except for Hd and HAr2 which show an up field shift with each increase in the percentage of compound 1 to β-CD. It's also noteworthy that the multiplicity of Hc changes from a doublet in the free compound 1, to a doublet of doublets in the presence of β-CD (see figure 18, and table 2). Δδ (ppm) 0-100% 0.047 0.031 0.097 -0.009 0.081 0.175 not clear 0.041 -0.040

Table 2: The change in chemical shift (Δδ) of N-allyl-N- (4-methoxyphenyl)acrylamide (1) protons between 0% and 100% of compound 1 relative to β-CD.

The 1 H NMR titration experiment showed that all the protons of compound 1 and only the inside protons of the β-CD cavity are affected during the course of the titration, suggesting that compound 1 may be bound, totally or partially, inside the hydrophobic cavity of β-CD.

By identifying the affected protons, one can have a partial idea about the geometry of the inclusion complex but a ROESY experiment would be needed to more understand the orientation of a guest inside the CD cavity.

Determination of the binding constant Kb

The NMR titration experiment data allowed to establish a linear plot of 1/ΔδH3 vs. 1/ [compound 1],

(see Figure 19). The straight line obtained, has a slope of 1/(Kb × Δδsat) and an intercept at 1/Δδsat, from which the binding constant Kb for the β-CD/ 1 complex was estimated to be 14.7.

The photochemical reactivity of compound 1 2.2.3.1 Photoreactivity of compound 1 in solution, without β-CD

Water was the solvent of choice for the photochemical transformation of 1, taking account of our intention to perform subsequent reactions in the presence of β-CD. However, when a 20 mM solution of 1 in water was introduced into a Pyrex tube then irradiated in a Luzchem (LCZ-4V) reactor fitted with 14 UV-B (303 nm) lamps, no conversion was observed after 15 h of irradiation. The same lack of reactivity was observed when ether was used as the solvent in place of water.

When the reaction was carried out in the same conditions using acetone as the solvent, complete conversion of 1 was achieved after 9 h irradiation. The solvent was evaporated and the crude residue was purified by silica gel column chromatography to give 1-allyl-6- In compound 1 the resonance of the nitrogen lone pair gives the C-N bond of the acrylamide a partial double bond character which establishes a 6π-electron system with the methoxyphenyl ring. Upon irradiation, compound 1 moves into a singlet exited state, at this stage there is a competition between a 6π-electron conrotatory ring closure, and an evolution of the exited compound 1 into a triplet state. Due to an inefficient inter system crossing only the latter is observed, favoring the formation of compound 5 over the anticipated compound 2.

There are literature precedents for photochemical 6π-photocyclization reactions of N-aryl α,β-unsaturated carboxamides. they have been used to make quinolone or isoquinoline alkanoids derivatives. Enantiodifferentiating 6π-photocyclization of N-phenylcyclohex-1enecarboxamide was mediated by chiral template (see Scheme 30). 111

Scheme 30: Chiral template mediated 6π-photocyclization of N-phenylcyclohex-1enecarboxamide.

Analogously, non-aromatic reductive 6π-photocyclization was used as a key step in a the total synthesis of (+)-coniceine as its HBr salt (see Scheme 31). 112

Scheme 31: The 6π-photocyclization as a key step in the preparation of (+)-coniceine HBr salt.

Interestingly, the photosensitized photochemical reaction of an N-allyl-Nbenzylacrylamide derivative, which cannot participate in a 6π-photocyclization, proceeds smoothly according to a [2+2]-cycloaddition to give a racemic mixture of the photoadduct.

(see Scheme 32). 113

Conclusion

Following our goal of developing an enantioselective route to cis-2,3 CB-GABA, the objective of this chapter was to obtain compound 2 in enantioenriched form, via an enantioselective [2+2]-photocycloaddition of N-allyl-N-(4-methoxyphenyl)acrylamide 1 in the chiral environment of the β-CD cavity.

We first adapted a known synthetic procedure to obtain the substrate 1, then determined the stoichiometry of its complex with β-CD to be 1:1 β-CD/1. NMR spectroscopic studies suggested the formation of a deep-penetration inclusion complex, but with a relatively weak binding constant, estimated to have a value of Kb =14.7 M -1 .

However, when irradiated in water or ether, compound 1 showed no photoreactivity.

Furthermore, in acetone, a photochemical 6π-electrocyclization reaction took place, leading to the formation of compound 5; no photochemical [2+2]-cycloaddition reactivity was in evidence.

At this point, it appeared that appropriate conditions for the proposed enantioselective synthesis were unlikely to be secured. The cumulated difficulties of modest host-guest binding, solvent incompatibility and competitive photochemical reactivity appeared to be insurmountable with our chosen substrate 1.

It seems likely that a different substrate should be envisaged. The use of a carbamate or benzyl group instead of an aryl group on the nitrogen atom might eliminate the unwanted electrocyclization reactivity and allow the [2+2]-cycloaddition reactivity to prevail, while at the same time bestowing reasonable binding affinity of the substrate in the β-CD cavity (see Figure 21). Never the less, we halted our endeavor to access cis-2,3 CB-GABA for the time being, and turned our attention to study the photochemical reactivity of 1,3-dihydro-2Hazepin-2-one a pressure of cis-3,4 CB-GABA

Introduction

We saw in Section 1.3.3 that the synthesis of cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA starting from azepinone 8

begins with the photochemical 4π-electrocyclization of the starting compound, during which two stereogenic centers are created (see Scheme 33).

Scheme 33: The photocyclization reaction of azepinone 8 leading to the formation of the racemic photoadduct 9

Azepinone 8 has 4 electrons in its π system, which by Woodward-Hoffmann rules would undergo a disrotatory photochemical ring closure. Since the diene system of azepinone 8 is non symmetrical, and because the rotation of the orbitals can go both clockwise and anticlockwise with an equal probability; the photoadduct 9 is formed as a racemic mixture of the two enantiomers (See Scheme 34).

We predict that the presence of β-CD during this 4π electrocyclization could provide a chiral environment that would favor one topology of the ring closure over the other, thus allowing preferential access to one of the enantiomers (see Scheme 34). In this chapter we first planned to synthesize azepinone 8, then to a study its host-guest interaction with β-CD, followed by a search for the most productive manner of irradiating the complex in terms of chemical yield and ee of the bicyclic adduct 9. Therefrom, following the previously described procedure for the racemate, enantioenriched cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA could be prepared.

Results and discussion

Synthesis of azepinone 8

Azepinone 8 was prepared following the previously reported method, 102 starting from the commercially available caprolactam 6. (see Scheme 35). α-Dibromination of caprolactam 6

was carried out in the presence of phosphorous pentachloride, zinc chloride and bromine.

The product 7 was isolated in 73% yield and was pure enough to be used without purification. The second step consists of a double elimination of HBr to yield azepinone 8.

This reaction was carried out in the presence of dry lithium chloride in anhydrous DMF at reflux. Azepinone 8 was obtained with 70% yield after chromatography on silica gel.

Scheme 35: Synthesis of azapinone 8 from caprolactam 6.

Formation of the unconjugated compound 8 instead of compound 8' might be attributed to an excessive ring strain in the latter, due to the sequence of five sp 2 hybridized carbons (C2 → C6) with the sp 2 hybridized nitrogen (See Scheme 36).

Scheme 36: Formation of the more stable azepinone 8.

Photoelectrocyclization of azepinone 8 in the absence of β-CD

The photoelectrocyclization of azepinone 8 in the absence of β-CD was studied both in solution as well as in solid state. that the molecule has to be in a quartz reactor if we want it to absorb directly. ( 102)

Scheme 37: The photochemical cyclization of azepinone 8 to give racemic photoadduct 9.

2-Azabicyclo[3.2.0]hept-6-en-3-one 9 was obtained in 97% yield as a brown solid with a very low solubility in ether. As it formed, it stuck on the inner side of the reactor, from which it was collected by scraping it out. The very low solubility of the product precluded further purification, but the 1 H MNR spectrum and HPLC analysis of the crude product showed a nearly pure compound.

Photoelectrocyclization of azepinone 8 in water

Since β-CD is not soluble in ether, we tested the photoelectrocyclization of azepinone 8 in water. The reaction time was the same as in ether (2 h) when the same concentration (7.33 mM) was used. With a higher concentration (91 mM) the reaction time was increased to 15 h.

The irradiated reaction mixture was then lyophilized and the crude cyclobutene product (±)-9 was isolated in a 71% crude yield. Analysis of the crude product by NMR and HPLC showed that it was identical to the sample prepared in diethyl ether. The lower yield in case of water as a solvent is attributed to the loss of product (±)-9 during the lyophilization process. (see Index, section B )

Photoelectrocyclization of azepinone 8 in solid state

In the literature, when CDs were used as a chiral hosts, better enatioselectivity was achieved when photochemical transformations were performed in the solid state rather than in solution. 42 For this reason we also tested the photochemical electrocyclization reaction of azepinone 8 in the solid state.

To irradiate azepinone 8 in the solid state, it was first ground into a fine powder then introduced in a quartz tube. The tube was closed and set to rotate in a Rayonet (R-200)

reactor, in which it was irradiated until no starting material could be observed by 1 H NMR.

The 1 H NMR spectrum of the photoadduct (±)-9 resulting from the irradiation of azepinone 8 in solid state was identical to that obtained in solution.

The reaction time in the solid state, which could range between 6 and 60 h, depended greatly on the amount and the homogeneity of the solid being irradiated. The more evenly the solid was exposed to the irradiation, the less time it needed for total conversion.

Analysis of the enantiomeric excess in photoadduct 9

Before introducing the β-CD to the reaction mixture, a suitable method has to be established to determine the ee of the photoadduct (±)-9. We studied the separation of the enantiomers of (±)-9 by chiral HPLC.

The photoadduct (±)-9 was passed through a normal phase chiral Lux Cellulose-1 column using a 90/10 hexane/isopropanol eluting mixture at 20 o C and a flow rate of 1.5 ml/min.

Under these conditions, the two enantiomers of photoadduct (±)-9 could be separated with 43 sec of difference in retention time. Figure 22 shows an HPLC chromatogram of photoadduct (±)-9 prepared by photoelectrocyclization in the absence of β-CD.

3.2.4

The β-CD/azepinone 8 complex

Formation of the β-CD/azepinone 8 complex:

Both β-CD and azepinone 8 are soluble in water. The β-CD/azepinone 8 complex was prepared by mixing an aqueous solution of the latter with an aqueous solution of β-CD.

Upon stirring at room temperature, a white precipitate started to form gradually, and within few minutes, the clear solution turned into a thick "milky suspension" (See Figure 23). It is important to point out that a minimum concentration of about 6 mM, with respect to β-CD, was needed in order for the precipitate to form; and the lower the concentration of the mixture is, the slower the precipitation process became. For this reason, the volume of the final solution was always chosen to have a concentration just less than 15 mM, which is the maximum solubility of β-CD in water at 25 o C.

Evidence of complexation by powder X-Ray diffraction

To further validate the existence of complexation between the β-CD and azepinone 8, powder X-ray diffraction data was obtained for samples of β-CD alone, azepinone 8 alone, and for the white precipitate "complex" obtained from mixing both components in solution (See Figure 24).

The powder X-ray diffraction data obtained for the β-CD/azepinone 8 complex were clearly different from the data obtained for each component alone. This provides further support for the contention that a genuine complex is implicated, rather than a physical mixture of the two discrete substances.

Figure 24: The powder XRD spectra of azepinone 8 vs β-CD vs β-CD /8 complex.

Determination of the complex stoichiometry

The azepinone 8 to β-CD ratio in the precipitate

In order to determine the stoichiometry of the formed β-CD/azepinone 8 complex, the "milky suspension" (explained in section 3.2.4.1) was filtered out and the ratio of the azepinone 8 to β-CD in the precipitate was found to be around 1:1, even if more than one equivalent of β-CD was used in the above mentioned complexation procedure. This ratio was calculated by comparing the peak areas in an 1 H NMR spectrum of the precipitate with a reference equimolar sample of β-CD/azepinone 8

To further determine the amount of cyclodextrin and lactam in the precipitating solid, the "milky suspension" was filtered and the "uncomplexed" azepinone 8 was extracted out from the aqueous filtrate using ethyl acetate. The organic layer was then concentrated and the aqueous phase was lyophilized to get the uncomplexed β-CD. The masses, summarized in In order to see the effect of adding an excess of β-CD on the complexation equilibrium, the starting "milky suspension" described above was charged with two more equivalents of β-CD. The extra amounts of β-CD added were expected to drive the complexation forward, towards the associated β-CD/azepinone 8 form (See Scheme 38).

Scheme 38: The proposed equilibrium between the free and the associated form of 1,3dihydro-2H-azepin-2-one 8 and β-CD in solution.

The charged suspension was filtered and the "uncomplexed" azepinone 8 was extracted out from the aqueous filtrate using ethyl acetate. The organic layer was then concentrated and the aqueous phase was lyophilized. So the equilibrium has certainly been shifted toward the formation of the β-CD/ 8 complex. Job's method of continuous variation was used to further investigate the stoichiometry of the formed β-CD/azepinone 8 complex. For this, two separate 100 mL (4.10 -2 mmol) mother aqueous solutions were prepared, one containing β-CD and the other azepinone 8. Volumes from each solution were then taken and mixed at different proportions to prepare 11 samples (2 mL) covering the rage of mole ratios

0 <  = [azepinone 𝟖] [azepinone 𝟖] + [𝛽 -𝐶𝐷] < 1
The UV spectra for each of the 11 samples were recorded at λ = 254 nm, and the difference in absorbance, (ΔA), between azepinone 8 alone and in the presence of β-CD multiplied by the molar fraction, (), was plotted against molar fraction () (see Figure 25). The position of the maximum of the curve at  = 0.5 further validates that the stoichiometry of complex formed upon mixing β-CD with azepinone 8 in aqueous solution is of a 1:1 stoichiometry. The rounded shape of the curvature also provides an indication that the equilibrium constant of the complexation reaction is expected to be of a low magnitude. Protons H3 and H5 of the β-CD cavity, showed a gradual up field shift with each addition of the lactam (See Figure 26). At a 1:1 ratio of azepinone 8 to β-CD, the change in the chemical shift reached 0.03 ppm for H3 and 0.07 ppm for H5. On the contrary H1, H2, H4 and H6 of β-CD that are pointing outside the cavity did not show any significant shift throughout the titration.

The protons of the azepinone 8 also showed a gradual up field shift throughout the incremental additions. But unlike the inside protons of the CD cavity, the induced shift in the azepinone 8 protons were of a lower magnitude. The most affected lactam protons are Hb, Hc and Hd (See Figure 27 and table 6). 1 H NMR titration experiments showed that the protons inside the β-CD cavity are the ones interacting with azepinone 8, suggesting that the guest may be bound, totally or partially, inside the hydrophobic cavity. Nevertheless, the absence of an inflection point in the titration curve with a maximum of variation in the chemical shifts, suggest a fast equilibrium between the associated and the free form of the two molecules.

Determination of the binding constant Kb

Since protons H3 and H5 of the β-CD cavity show a chemical shift difference between the free and bound states; one of these protons, H3 whose NMR signal does not overlap with other protons, could be monitored throughout a titration experiment in which the concentration of the guest azepinone 8 was varied incrementally. The data from such a titration experiment allowed to establish a linear plot of 1/ΔδH3 vs. 1/[azepinone 8] (see figure 28). The straight line obtained, has a slope of 1/(Kb × Δδsat) and an intercept at 1/Δδsat, from which Kb was estimated to be 35.4. The relatively low value of Kb for the β-CD/azepinone 8 complex is in accordance with the shape of the Job's plot curve, which shows a gentle rather than a sharp angular curvature (see section 3.2.4.3.2).

2D ROESY NMR studies of the complex

Complexation of molecules to CDs usually occurs through non-covalent interactions between the guest molecule and the CD cavity. This is a dynamic process in which the guest molecule continuously associates and dissociates from the host CD. Although in schematic representations a host/guest complex is usually shown as a single entity, the complex is actually composed of a set of species with the depicted complex representing an average structure. 114 In fact, CD complexes have high conformational flexibility, which can accommodate several modes of complexation. 64 This phenomenon is termed multimodal complexation. [START_REF] Dodziuk | Cyclodextrins and Their Complexes[END_REF] In order to investigate the geometry of the formed complex and propose a mode of complexation between the β-CD cavity and azepinone 8, 2D ROESY NMR experiments were conducted.

A ROESY NMR experiment conducted on an equimolar 1:1 β-CD:azepinone 8 mixture in solution at 45 °C (15 mM, D2O) showed intermolecular cross peaks only with the inner CD protons (H3, H5, H6), (see Figure 29, and 30), which established inclusion of the guest inside the cavity without any evidence for outside interactions Azepinone 8 can possibly approach the β-CD from the wide secondary face; this mode of binding is supported by the correlation of all of its protons with H3 that line up the inner cavity of the CD near the secondary face. The penetration from this face inserts the amide part of the azepinone 8 deep enough inside the cavity, such that all the azepinone 8 protons, except Hd, are in close proximity to correlate with H5 far on the other side of the cavity (see Figure 31). Hd, treated as a "probe hydrogen" never sees H5, but still can clearly see H6

which line up the primary face of the β-CD, suggesting the presence of another mode of complexation in which the penetration of the azepinone 8 occurs from the narrow primary face; but this time the inclusion is not deep enough to put Hd close contact with H5, but only to keep it close to H6. Several selective ROESY experiments are still needed to establish the exact geometry of the azepinone 8 inside the β-CD cavity. Nevertheless, the ROE colorations between the azapinone 8 and the inside protons of the β-CD are clear enough to argue that the azapinone 8 is put in a chiral environment ready for being irradiated.

Photoelectrocyclization of azepinone 8 in the presence of β-CD

Irradiation of azepinone 8 in the presence of β-CD was either done in fluid or in solid state.

Except for one experiment in which the β-CD was manually ground with the lactam without any solvent, all other experiments started by adding a clear solution of azepinone 8 (30-50 mg in 1 ml of water) to a clear aqueous solution containing the desired equivalents of β-CD.

The volume of the final solution was chosen to be at 15 mM with respect to the β-CD. Upon stirring, a white precipitate started to form almost immediately. The formed "milky" suspension was left to stir 2 h at room temperature, and then was treated differently according the desired condition of irradiation (in solution, in suspension, in solid state...).

Our objective was to isolate the resulting bicyclic photoadduct 9 formed by classical extraction methods. But it transpired that this was a major problem.

Recovery of the photoadduct 9

We first attempted to isolate photoadduct 9 from crude irradiated solid by solid-liquid extraction (Soxhlet) using organic solvents (chloroform, acetone…). Due to the low solubility of 9 in such solvents and probably its interaction with the β-CD molecules, it took several days (up to 30 days) to extract the entire amount of 9 out of the irradiated β-CD/azepinone 8 mixture. The speed of extraction was also dependent on the solvent used.

Even though the photoadduct 9 was more soluble in chloroform than in acetone, the latter was better in the continuous extraction process especially if wet acetone was used due to the fact that 9 had a good solubility in water. Due to the prolonged Soxhlet extraction at around 60 o C, the isolated crude 9 was usually a dark viscous oil rather than a light brown solid as it should be. Nevertheless, the 1 H NMR spectrum and the chiral HPLC chromatogram were identical to the photoadduct 9 prepared by irradiation in ether.

Since it was difficult to isolate all of the photoadduct 9 from the irradiated complex, chiral HPLC analysis was used to determine the ee of samples which were partially extracted. The results could not be considered reliable since CDs are known to preferentially hold one enantiomer in their cavity, making the extraction of the other easier. 115 Thus it is possible that the ee observed in a partially extracted sample was due to chiral resolution and was not induced during the photochemical reaction. The difficulty in recovering the photoadduct 9

prompted us to seek a more suitable method to access to the bicyclic cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA precursor.

Reduction of photoadduct 9

In previous work, 102 it was observed that the reduction of the photoadduct 9 gave a product which had a better solubility in organic solvents (See Scheme 39).

Scheme 39: Reduction of photoadduct 9 to 2-azabicyclo [3.2.0]heptan-3-one 10.

Therefore, we reasoned that if we could reduce the photoadduct 9 in the mixture with β-CD, it would be easier to extract and isolate the reduced derivative 10.

The reduction was first attempted on a suspension of photoadduct 9 and β-cyclodextrin in ethyl acetate with 10% w/w Pd/C-10% under a dihydrogen atmosphere. The presence of β-CD slowed down the reaction, and traces of starting photoadduct 9 were still present even after 2 weeks of stirring at room temperature with a large excess of the catalyst (See Scheme 40). Since the irradiated the β-CD/photoadduct 9 complex was soluble in water, we supposed that when it went into solution the complex would dissociate giving more free photoadduct 9. This free form of 9 would be more amenable to reduction.

So the reduction of photoadduct 9 without β-CD was tested in water on an analytical scale.

The reaction worked well under a dihydrogen atmosphere in the presence of 10% w/w Pd/C-10%, and only 2-azabicyclo[3.2.0]heptan-3-one 10 could be observed after stirring overnight at room temperature (See Scheme 41).

Scheme 42: Reduction, in water, of photoadduct 9 in the presence of β-CD to give 2azabicyclo [3.2.0]heptan-3-one,10.

Photoelectrocyclization/Reduction of azepinone 8 in presence of β-CD

All the following reactions were carried out according to the one-pot method:

photoelectrocyclization/reduction

Irradiation of β-CD/azepinone 8 complex in fluid state

The "milky suspension" obtained by mixing azepinone 8 and β-CD was treated according to the following methods:

3.2.6.1.1 Experiment A: "1:1 Hot solution"

The "milky suspension" described above, containing one equivalent of lactam to β-CD, was heated up to 45-50 o C; at this temperature it turns into a clear solution. It is worth to note that if the solution was cooled back to room temperature the precipitate was observed again.

The suspension was transferred into a quartz tube fitted with a "heating/cooling finger "which is connected to a temperature control system (see Figure 33). The temperature of the mixture was maintained at 45-50 o C. The clear hot solution was then irradiated in a Rayonet reactor until complete conversion of azepinone 8 (2 h). The resulting yellow solution was cooled down to room temperature, and no precipitation was observed. The 2-azabicyclo[3.2.0]heptan-3-one 10 from experiment A was obtained in 40% crude yield, along with a water soluble solid side product whose 1 H NMR spectrum was not clear enough to identify it. Analysis of the crude 10 by chiral HPLC showed a pure product but no enantiomeric excess (See Figure 34).

Irradiation of β-CD/azepinone 8 complex in solid state

As mentioned in literature, the rigidity of the medium is an important parameter for chiral induction during a photochemical reaction; 41 and good enatioselectivity was achieved when β-CD/pyridone complexes were irradiated in the solid state. 42 3.2.6.2.1 Irradiation of β-CD/azepinone 8 complex in solid state powder form

Experiment D: Mechanically ground mixture of β-CD and azepinone 8

One equivalent of azepinone 8 was mechanically ground with β-CD in a mortar. The powder mixture was then transferred to a quartz tube which was then closed and set to slowly rotate while being irradiate inside a Rayonet reactor (See Figure 47). Four experiments were done in these conditions and the crude chemical yield was around 70%, although the ee was not constant, varying from 0 to 40%. The variable enantioselectivity suggested that mechanical grinding of azepinone 8 with β-CD does not give a sufficiently homogeneous mixture and thus can not be considered as a reproducible way to prepare the β-CD/azepinone 8 complex.

Experiment E: "β-CD/8 complex, filtered then irradiated in powder form"

The β-CD/azepinone 8 milky suspension was filtered out and the solid residue was left to dry in open air, then finely crushed in a mortar.

The fine solid "complex" was introduced into a rotating quartz tube as illustrated in experiment D (see Figure 37 section 3.2.6.2.1.1). The rotation of the tube was intended to serve to stir the complex as it was being irradiated, but when performing the experiment, the finely crushed solid always aggregated into small balls that need to be crushed regularly to achieve complete conversion (See Figure 38). To avoid the aggregation, the fine powder was spread on a glass plate and irradiated inside the Rayonet reactor. Even though the exposed area was larger on the plate, the solid was standing still and should be homogenized by agitating it regularly using a spatula, in order to achieve complete conversion. The need for manual agitation rendered the experimental set-up unpractical, especially on larger scale.

The time needed for complete conversion of the starting material during the powder solid state experiments varied largely, from 6 to 60 h depending on the amount of the irradiated solid, its homogeneity and the surface area exposed to the irradiation.

When no starting materials were observed in the 1 H NMR spectrum, the irradiated solid was dissolved in water to obtain a clear solution and the photoadduct 9 was then reduced with dihydrogen in the presence of 10% w/w Pd/C-10%. The 2-azabicyclo[3.2.0]heptan-3-one 10 was extracted from the aqueous phase with ethyl acetate. The organic phase was dried over Na2SO4 then concentrated under vacuum to give 10 as a yellow oil.

Crude 10 from experiment E was obtained in 71% yield (for two steps) with respect to the amount of azepinone in the irradiated precipitate; the crude product was analyzed by chiral HPLC and showed an ee of 36%. The peak at 9.71 min corresponded to caprolactam 6

formed by the reduction of a small amount of the β-CD/azapinone 8 complex which had not been transformed by the electrocyclisation reaction (See Figure 39). No remaining drawbacks to performing the irradiation in powder form. In order to perform the irradiation in a more homogenous solid state and thus decrease the time needed for complete conversion, the starting milky suspension described above was spread on a glass plate and left to dry in open air to give a consistent white film. The film was irradiated directly in the Rayonet reactor (See Figure 40). The reaction course was followed by 1 H NMR until complete conversion, which was achieved within 5 h. The solid was then dissolved in water to obtain a clear solution and the photoadduct 9 was reduced with dihydrogern in the presence of 10% w/w Pd/C-10%. The resulting 2azabicyclo[3.2.0]heptan-3-one 10 was then extracted from the aqueous phase with ethyl acetate. The organic phase was dried over Na2SO4 then concentrated under vacuum to give 10 with a crude yield of 77% over two steps.

The β-CD/8 complex film texture

Although the ee values in all three experiments on film type complexes was similar, a change in the texture and consistency of the film was observed when the concentration of β-CD was changed. A film made from a 1:1 mixture of β-CD and azepinone 8 at 15 mM (experiment F) was smooth and could detach itself from the glass plate, whereas at 5 mM (experiment H) the film always stuck to the glass and could only be scraped off (see Figure 44 and45). Moreover, the presence of two equivalents of β-CD at 15 mM (experiment G) gave a ragged, crispier, film with clear crystals all over it. Single crystal X-ray diffraction of the crystals showed only β-CD with water inside and outside the cavity (see Figure 49).

Scanning electron microscopy (SEM) of β-CD/azepinone 8 complex films

The solid films of all experiments along with one film made from azepinone 8 alone and another one from β-CD alone, were subjected to analysis by scanning electron microscopy.

The pictures showed a well-organized fibrillar morphology in all films, in contrast with azepinone 8 alone and β-CD alone, which each showed only amorphous block structures (See Figure 47).

The absolute configuration of compound 10

In all the above-described experiments, the same major enantiomer of 10 was in evidence.

To determine the configuration of this predominant enantiomer, a sample of 10 (from experiment G, 42% ee) was transformed into the N-Boc derivative of cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA 12

following the known procedure. 102 The sample of 12 thus obtained had an optical rotation value of + 33 (c 0.5, CHCl3, at 25 o C). The literature value for a sample of (R,R)-12 with

Chiral resolution of ()-cis-3,4 CB-GABA by HPLC

Our efforts to obtain enantiomerically pure cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA through the enantioselective photoelectrocyclization of azepinone 8 in the presence of β-CD as a chiral host, followed by reduction of the photoadduct, provided samples of 2-azabicyclo[3.2.0]heptan-3-one 10 with 45% ee at best.

In order to have a more suitable method to access the target amino acid cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA in enantiomerically pure form, we decided to develop a chiral resolution strategy for its N-Boc derivative using semi-preparative HPLC. Such a procedure would have several advantages when compared to the currently employed resolution methodology, which relies on chiral derivatization using an oxazolidinone, as we mentioned before. 102

Preparation of the Boc protected amino acid ()-12

The multi-step synthesis of the N-Boc derivative of racemic cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA, previously described by our collaborated groups, as we mentioned before. 102 We decided to reproduce this synthesis to obtain ()-12 on gram scale.

Photoelectrocyclization of azepinone 8 in ether

A solution of azepinone 8 was irradiated for 3 h with a 400 W mercury lamp under argon in a reactor fitted with a quartz filter and cooled by an external water/ice bath. The crude product was obtained after evaporation of the solvent, with a yield of 97%, as a brown solid (See Scheme 44). Since the photoadduct (±)-9 is extremely insoluble in organic solvents, no purification was carried out. The 1 H NMR spectrum in CDCl3 of the obtained compound (±)-9

is consistent with the literature. 102 These conditions were then applied on a semi-preparative scale, a saturated solution of (±)-12

in acetonitrile was introduced at a rate of 50 μl each 15 min (50 μl/injection). The eluted products were collected over three fractions; one that contained the first enantiomer, one that contained the second enantiomer, and a small middle fraction containing both (See Figure 50).

Switching to the N-Boc benzylic ester (±)-13

In order to circumvent these problems, we considered HPLC chiral resolution of the benzyl ester of (±)-12. The ester (±)-13, (see Scheme 48) was expected to be more soluble in organic solvents, which should improve the rate of the separation. Furthermore, it was proposed that ester (±)-13 could be resolved using a normal phase column, thus avoiding the use of water as an eluant.

Preparation of N-Boc benzyl ester (±)-13

To obtain ester (±)-13, compound (±)-12 was reacted with one equivalent of benzyl alcohol in the presence of DMAP and DCC in dichloromethane, overnight. The reaction mixture was then filtered and the filtrate was concentrated under vacuum. The crude product was purified over silica to obtain (±)-13 as a white solid in 90% yield (see Scheme 50).

Scheme 48: Synthesis of benzyl ester (±)-13 from (±)-12.

Chiral HPLC resolution of N-Boc benzylic ester (±)-13

The N-Boc protected benzyl ester (±)-13 was passed through a normal phase chiral Lux Cellulose-2 column using a 97/3 hexane/isopropanol eluting mixture, at 30 o C and a flow rate of 1 ml/min. Under these conditions, the two enantiomers of 13 were separated with a retention time difference of 2.4 min. Figure 51 shows a chromatogram of racemic 13 eluted on an analytical HPLC. These conditions were then applied on semi-preparative scale. A 166 mg/ml solution of (±)-13

in isopropanol was introduced at a rate of 120 μl each 17 min (120 μl/ injection). The eluted products were collected over three fractions: one that contained the first enantiomer, one that contained the second enantiomer and a small middle fraction containing the second enantiomer along with traces of the first (See Figure 52). The absolute configuration of the first enantiomer was determined by x-ray analysis and was found to have a (3R,4R) configuration. The second enantiomer was thus deduced to be the (3S,4S) enantiomer (see Figure 54).

Conclusion

The 2+2 photoelectrocyclization of 1,3-dihydro-2H-azepin-2-one (8) was studied in the presence of β-CD as a chiral host. The formation of a precipitate upon mixing 8 with β-CD in aqueous solution suggested an interaction between the two components. 1 H NMR titration experiments were conducted to explore this interaction and showed that the protons inside the β-CD cavity interacted with azepinone 8, suggesting that the guest was bound, totally or partially, inside the hydrophobic cavity. Nevertheless, the absence of an inflection point in the titration curve with a maximum of variation in the chemical shifts, and a low Kb value of 35 suggested a fast equilibrium between the associated and the free form of the two molecules.

The photoelectrocyclization reaction of the formed β-CD/azepinone 8 precipitate was performed at different ratios of both components, in hot solution, in cold suspension, in solid powder form, as well as in a solid thin film form. The isolation of the photoadduct 9 from β-CD after the irradiation was not practical so it was reduced into 2-azabicyclo[3.2.0]heptan-3-one 10 in water in the presence of β-CD then extracted in an average chemical yield of 75% over two steps. While no enantiomeric excess was observed in hot solution, up to 45% enantiomeric excess of the (R,R) enantiomer could be reached in cold (5-8 o C) suspension and up to 42% in solid form.

The presence of β-CD provided a chiral environment for the photoelectrocyclization of azepinone 8 into photoadduct 9, and its reaction into 2-azabicyclo[3.2.0]heptan-3-one 10 could be performed on a preparative scale, with a reasonable yield of 75% over two steps, but at best a 45% enantiomerically enriched mixture was obtained.

We established a more suitable method to access both enantiomers of cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA via a semi-preparative HPLC fitted with a chiral column. The N-Boc benzyl ester derivative (±)-13

was most amenable to this procedure. The use of semi-preparative HPLC circumvented the long procedure of chiral resolution using an oxazolidinone and provided both enantiomers of cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA in a conveniently diprotected form.

Chapter 4: Synthesis and Folding Pattern of γ/α-Hybrid Peptides

Introduction

The diversity of protein structures depends on the amino acid polypeptide backbone which can be induced to adopt a very large range of compact conformation. Privileged conformations are often stabilized by short-or long-range non-covalent interactions, leading to well-defined molecular architectures. Substantial progress has been made in establishing peptidomimic architectures, called foldamers, displaying helices, sheets and ribbon shapes.

The folding pattern of homogeneous oligomers of α, β, or γ amino acids have been more

extensively studies than folding pattern of mixed hybrid α/β, α/γ and β/γ-oligomers of amino acids. The most common regularly-folded architectures are helical: they include the 14-helix and the 9-helix for γ-peptides, the 13-helix and the 11/13-helix for β,γ-peptides, the 12-helix and the 12/10-helix for α,γ-peptides.

Many of these secondary structures along with other helical manifolds have been anticipated

and simulated by theoretical calculations of systematic conformational analysis. 116,117 Mixed α/γ-peptides manifolds merit further experimental investigations, to explore if indeed their folded architecture are as suggested by the Hoffman's group theoretically studies.

The Hoffman group 117 suggested through theoretical studied that in a hybrid α/γ peptide a θ dihedral angle around 33 o , for the γ-amino acid component, is ideal for the formation a 12/10 helix. The X ray crystal structure of the cis 3,4 CB-GABA, which was synthesized in chapter three, showed that this molecule has an angle θ around 27 o which makes it a suitable building block for a hybrid α/γ-peptide that could fold into a 12/10 helical secondary structure.

In this chapter we aim to synthesize, characterize, and study the three dimensional folding behavior of mixed α/γ-peptides composed of alternating, enantiomerically pure cis 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA as the constrained γ-amino acid component and D-Alanine (D-Ala) as the α-amino acid component. Two series of mixed α/γ-peptides were studied one that has the (R,R)-cis 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA enantiomer as the γ-amino acid component and one that has the (S,S)-cis 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA enantiomer as the γ-amino acid component.

The Three dimensional conformation of polypeptides

In order to understand the three dimensional structure of a given polymer, two essential types of information should be known. The first one is the configuration of the monomer unit itself, and the second, is the relative orientation of the units at each linkage.

The conformation of the peptide link

In the case of polypeptides, the dimensions of the peptide unit itself has been determined by Pauling and Corey,119,120 although slight variations are observed in certain structures, it is established that the peptide link or amid unit form a considerably rigid plane. The origin of this planer organization of the amid unit, is the resonance of the double bond between the carbonoxygen and carbon-nitrogen positions which leads to a partial double bond character between the O-C-N atoms (see Figure 55). 119 As a consequence, the set of atoms Cαn-C'O-NH-Cαn+1, starting from one α-carbon and going to the next α-carbon atom, forms a planer group.

The conformation around the peptide bond

When two peptide groups are linked together, it is possible to have a rotation of the planer amid units around the bonds N-Cαn and CαC', and thus the relative configuration of the two groups about the Cα atom can be indicated by two parameters φ and ψ. 118 To properly visualize the two rotations around Cα, the two planer amid units are first kept in the same plane, fully stretched with the carbonyls pointing roughly in opposite directions (see This procedure could be further applied for describing the conformation of a complete polypeptide chain containing "n" amino acid residues by first keeping them all stretched in the same plane, and then applying, the series of rotations (φ1, ψ1), (φ2, ψ2). . . up to (φn, ψn) at each successive α-carbon. The obtained set of n pairs of dihedral angles contains the most important information needed for describing the conformation of the backbone for the entire polypeptide chain. 121 The φ and, ψ parameters would be enough to fully describe the conformation of a polypeptide chain only when each peptide unit is considered as an ideal plane with the same dimensions at every α-carbon. In reality this is never the case, and the dimensions at each peptide unit can vary. In order to compensate for these variations, it is necessary to have further parameters to completely describe the conformation of a polypeptide chain.

To describe the deviation of a peptide unit from being an ideal plane, we need to define the dihedral angle ω. It represents a rotation around the peptide bond C'-N1 and may be defined as the angle between the planes Cα-C'-N1 and C'-N1-Cα (See Figure 59). Another parameter that affects the overall conformation of a poly peptide chain is denoted by χ, which describes the conformation of the atoms in the side chain, and is denoted by a series of dihedral angles χ1, χ2, etc., that describe the rotations about the bonds (Cα-Cβ), (Cβ-Cγ), etc.

Nevertheless, it is important to realize that, although these φ, ψ, ω, and χ parameters are all needed to describe the exact conformation of a polypeptide chain. The most important parameters are the φ and ψ angles.A good knowledge about the conformation of the polypeptide chain may be obtained from a set of these two dihedral angles.

The folding pattern in natural peptides

Conformation of natural peptides

Proteins can have four different structural levels. The most basic one is the linear arrangement of amino acid residues in a polypeptide, which defines its primary structure. Due to stabilizing hydrogen bonds between certain residues, parts of this linear amino acid sequence can assume local structural conformations. These different spatial arrangements define the protein's secondary structure. Peptides secondary structure includes: helices, strands and loops. The folding of a polypeptide into its secondary structure puts some specific amino acids in a close proximity. This can lead to a further folding of the protein into its tertiary structure, which is defined as the overall three-dimensional shape of the entire protein molecule. Some proteins can show an additional level of organization, when they interact with other proteins and arrange themselves to form an aggregate protein complex called the protein quaternary structure. The final structure of the protein is stabilized by different interaction between its constituents (see Figure 58). 122 Helices are the most abundant secondary structures found in globular proteins. 123 They are named p(n)-helix according to the number of atoms (n), and the number of residues (p) for each turn. For example, a 3(10)-helix has 10 atoms and 3 residues per turn.

As mentioned in section 4.1.1.2 above, the secondary structure i.e. the three dimensional folding pattern of a polypeptide is governed by the repeated display of a particular set of backbone dihedral angles φ and ψ. The three types of helix whose φ and ψ are presented Table 7, are populated differently in natural proteins. 123,124 The 3.6( 13)-helix also known as the right handed α-helix was discovered by L. Pauling and is the most abundant helix in natural proteins. 119,125 About 32-38% of residues are involved in α-helices. The 3.0(10)-helix also known as the 310-helix is a rare secondary structure of natural proteins. It accounts for only 3.4% of all residues. The 310helices are typically very short structures, found at the beginning or the end of some α-helices.

The π-helix, or 4.4( 16)-helix, has been recently discovered and is an extremely rare protein secondary structure that is found at the end of certain α-helix. 126 The left-handed 3.6( 13)-helix and the 2.2(7), 4.3(14) or 4.4( 16)-helices, have been predicted but never yet found in natural proteins. 127

The folding pattern in unnatural peptides

During the past few decades, chemists have been inspired by the ability of natural peptides to fold into a well-defined helical secondary structure. These self-organized systems have gained an increasing attention, and have found various applications, like molecular recognition, peptidomimetics, and therapeutics. 128,129 (128) (129) The term "foldamers" was first coined by

Gellman 130 and defines "oligomers with a strong tendency to adopt a specific conformationally ordered structure in solution, stabilized by a set of noncovalent interactions between nonadjacent monomer units". 131 These noncovalent interactions include steric repulsions, hydrogen-bonding, electrostatic and π-π interactions, coordination to metal ions, as well as solvophobic effects. Out of these interactions, hydrogen-bonding are the most versatile in creating intra-strand connections that could stabilize a well-defined three dimensional structure adopted by an oligomeric peptide chain. 129 The early studies of Gellman 130, 132 and Seebach 133,134 and the later contributions of Hofmann 116,117,135 have certainly initiated the rapidly evolving field of foldamers. In this context, several homo and hybrid peptides of α, β, and γ-amino acids have been studied for their preferential three dimensional folding pattern. inserted between the carboxylic acid and the amino group gives to β-peptides an additional dihedral angle θ, and thus a higher conformational flexibility than α-peptides (see Figure 59).

This additional torsional freedom does not prevent the formation of ordered secondary structures in -peptides. 132,136 Figure 59: The dihedral angels in a β-peptide.

The Hofmann group 135,136 predicted via ab initio calculations the different types of helices that could be adopted by β-peptide. So far, seven different helices have been experimentally observed (see Figure 60, solid arrows); the 8-, 137 10-, 138

Example and application of the β-Peptides

The 12-and 14-helical structures adopted by β-peptides are the most important ones. The helicity in these two structures is comparable to the one of a native α-helix (13-helix). This has led researchers to study β-peptides as scaffolds to mimic certain native α-helices in terms of structure and biological activity. 129 One example is the 17-residue β-peptide prepared by the Gellman group. 143 Their peptide was composed of (R,R)-trans-2-aminocyclopentanecarboxylic acid (trans-ACPC) and trans-4aminopyrrolidine-caboxilic acid (trans-ACP) (see Figure 61). The trans-ACP served as a cationic residue giving an amphiphilic property to the formed 12-helix, which was essential to mimic the biological activity of amphiphilic cationic antimicrobial peptides found in nature. which makes γ-peptides more flexible than their α-and β-counterparts (see Figure 62). The Hofmann group predicted via ab initio calculations that a hexamer γ-peptide has the ability to fold into eight different helical secondary structures. 144 So far only the 7-, 145 9-, 146 and 14helices 147 have been experimentally observed (see Figure 63, solid arrows). One should note that the folding pattern with a 7-membered pseudocycle arising from intraresidual H-bond formation is viewed as a 7-ribbon rather than a helix due to its small pitch and flattened structure.

Figure 63: The different types of helices that could be adopted by a γ-peptide, as predicted by the Hofmann group. Solid arows indicate the so far experimentally observed ones.

Examples and application of the γ-peptides

Unlike β-peptides, γ-peptides did not receive any attention in the field of biological applications. The prepared γ-peptides were only a subject of structural and conformational behavior studies. Hanessian 147 and Seebach 148,149, 150 groups independently studied one example of substituted γ-peptides that fold into a stable 14-helix (see Figure 64). The research groups also pointed out the effect of having a substituent at the C-4 position in promoting the formation of a helical structure in γ-peptides. For instance, γ 4-, γ 2,4-, γ 2,3,4-peptides all adopted a 14-helical structure, whereas γ 2-, γ 3-peptides folded into a flexible, hard to determine structure.

Folding pattern in hybrid peptides

Hybrid peptides include α/β, α/γ, and β/γ-peptides. They have drawn much attention due to their ability to further expand the scope of foldamers in terms of variations in the backbone pattern compared to their homo-peptides counterparts. Furthermore, incorporating unnatural amino acids such as β or γ-amino acids into an α-peptide sequence with a proteinogenic side chain needed for biological activity, could enhance its in vivo proteolytic stability, due to the absence of suitable enzymes. This heterogeneous structure could so allow the peptide to keep its biological function without being easily hydrolyzed by the protease enzymes. 151,152 In hybrid peptides, the formed hydrogen-bonding interactions between the non-neighboring amino acids that define their helical structures, could be categorized into three types: hydrogen bonds formed in a backward direction along the sequence, hydrogen bonds formed in a forward direction of the sequence, and hydrogen bonds formed alternately in backward and forward direction (see Figure 65). 116

Mixed α/β-peptides

The Hofmann group studied the possible helical secondary structures that could be adopted by a, free of strain, α/β-octapeptide composed of alternating glycine and β-alanine. 116 The authors found via ab initio calculations that such a peptide could fold into seven distinct stable helices which are : 9/11-, 11/9-, 11-, 12/13-, 14/15, 16/18-and 18/16-helices. So far, only the 11/9-, 153 11-, 154 12/13-, 155 and 15/14-helices 156 have been experimentally observed.

Examples of α/β-peptides and applications

One example of α/β-hybrid peptide, was studied by the Gellman group. 157 The authors were able to design a chimeric peptide composed of an N-terminal α/β-fragment and a C-terminal αfragment. The α/β-fragment which was made of trans-ACP and trans-ACPC folded into a 15/14-helical structure whereas the α-fragment composed of proteinogenic amino acids adopted a native α-helix structure (see Figure 66). This α/β+α-peptide had the ability to bind, with an inhibitory effect, to its target transmembrane protein (Bcl-xL). This demonstrates the potential use of specially designed peptide foldamers to inhibit certain protein-protein interactions.

Examples and applications

Out of the eleven stable helical structures predicted by the Hofmann group, the 12-, the 12/10and most recently the 9-and the 9/12-helix have been experimentally observed in solution and in solid state as illustrated below.

The Balaram group studied α/γ-hybrid peptides composed of alternating α-aminoisobutyric acid (Aib) and gabapentin (Gpn). 160 The conformational restrictions, imposed by the constrained Aib and Gpn residues on the dihedral angles, allowed the α/γ-nonapeptide to adopte a 12helical structure in solution but, more interestingly the authors were able to characterize C9, C7, C12, and C17 hydrogen-bonded turns in the crystalline form, in the shorter tetrapeptide;

which emphasizes the fragility of structuration in short oligomers compared to longer ones (See Figure 70). A 12-helix structuration in solution, as well as in crystalline form, was also observed by the Gopi group. 161 An α/γ-peptides composed of alternating α-aminoisobutyric acid (Aib) and γ 4phenyl alanine showed a 12-helical structure which was favored by gauche conformation adopted by the dihedral angels θ, and ζ of the γ 4 -phenyl alanine component. Moreover, the obtained 12-helical structure presented a good correlation between its backbone, with its side chain projections, and a native α-helix (RMSD = 0.8 Å); signifying the possibility of employing specially designed α/γ-peptides for natural peptides mimicry (see Figure 71). On the other hand the 12/10 secondary helical structure in solution was first reported by the Kunwar's group, 162 the authors prepared a α/γ-hybrid tetrapeptide ,composed of alternating Lalanine and C-linked carbo-γ-amino acid of D-mannose, whose tetra and hexamers folded into a 12/10-helix in solution (see Figure 72). Similarly, the Gellman's group studied the conformational behavior of a α/γ-hybrid hexapeptide composed of alternating L-alanine and (1R,2R,3S)-2-(1-aminopropyl)cyclohexanecarboxylic acid (APCH). 163 The conformationally restricted backbone of the (APCH) residue, strongly enforced the formation of a 12/10-helical structure with an alternating direction of the hydrogen bond pattern in solution as well as in crystalline state (see Figure 73).

(ATCs) as the restricted γ-amino acid component. The authors found that ATCs induced a highly stable 9 pseudocycle (see Figure 75).

Stereochemistry effect on the peptide secondary structure

The stereochemistry of each building block of a peptide sequence has an important effect on the nature and the conformation of the formed secondary structure. 166 For instance, an oligomer composed of alternating homochiral cis-1R,2S-aminocyclopentanecarboxylic acid (cis-ACPC) is found to adopt a strand secondary conformation, 167 whereas an alternation of heterochiral 1R,2S-and 1S,2R-cis-ACPC oligomer folds into a stable 10/12-helix. 139 The change in the relative stereochemistry of amino acid residues changed the secondary structure from a strand to a helix, revealing a profound correlation between the stereochemistry of the amino acid residues and the conformation of the adopted secondary structure (See Figure 76). Aside from the more popular 12-and the 12/10-helical folding, the Maillard's group 165,168 prepared an α/γ-hybrid peptide, containing enantiomeric pure 4-amino(methyl)-1,3-thiazole-5carboxylic acids (ATCs) as the restricted γ-amino acid component, (see Figure 77

Objectives of this work

Studies on α/β-, β/γ-, and α/γ-hybrid peptides provided a rich pool of foldamers having helical secondary structures. These foldamers have attractive features, which give them applications in various fields of molecular recognition, peptidomimetics, and therapeutics. 128,129 Over the last few years, the Aitken research team has become specialized in the synthesis of unnatural amino acids, especially those incorporating small constrained rings and the development of oligomers based on these building blocks. Oligomers of these constrained amino acids present specific dihedral angles that confer to them the ability to promote stable three dimensional secondary conformations. Even though, there is a lot of theoretical knowledge that correlates between structure of amino acids and peptides folding pattern, a lot remains to be explored experimentally before one can intentionally manipulate the design of a peptide sequence to promote a specific desirable folding pattern in a bottom-up approach. 92,158 The ab initio theoretical studies done by the Hoffman group suggested that in an α/γ-hybrid peptide a θ dihedral angle around 33 o , for the γ-amino acid component, is ideal for the formation a 12/10-helix. 117 The crystal structure of the cis 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] cyclobutane -aminobutyric acid (cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA), which was synthesized in chapter three, showed that this molecule has an θ angle around 27 o (see Figure 78) which makes it a suitable building block for a α/γ-hybrid peptide that could fold into a 12/10-helical secondary structure. In this chapter, we aimed to validate the cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA as a γ-amino acid component capability to induce a 12/10-helical folding pattern in mixed γ/α-peptides. We also studied the effect of reversing the stereochemistry of the cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA, on the three dimensional folding pattern of these γ/α-peptides. Thus we synthesized and characterized, γ/α-hybrid peptides composed of alternating, enantiomerically pure either (S,S)-or (R,R)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA as the constrained γ-amino acid component and D-Alanine (D-Ala) as the α-amino acid component. The three dimensional folding behavior of these peptides was subsequently studied (see Figure 79).

Results and discussion

The starting amino acids: Enantiomerically pure protected γ-and αamino acids

The synthesis of mixed γ/α-peptides requires access to enantiomerically pure γ-and αamino acid monomers.

The enantiomerically pure Boc-cis-3,4 CB-GABA-OBn

The diprotected Boc-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA-OBn was prepared in its racemic form following by the semi-preparative Chiral-HPLC separation method as described in chapter three, to obtain both enantiomers in pure form (see Scheme 49).

Scheme 49: The synthesis of Boc-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA-OBn in its racemic form (top); separation of the two enantiomers via the semi-preparative Chiral-HPLC resolution method (bottom).

The Enatiomerically pure D-Alanine

The 12/10 helical structure of a α/γ-peptides, generated by the theoretical calculations of the Hofmann group, 117 suggested that, if we used the (S,S)-cis 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA as the γ-amino acid component and a R configuration around the Cα of the α-amino acid component, the α-amino acid side chain and the cyclobutane ring would be in a plane perpendicular to the helix axis and would not disturb the helical structure formation. On these bases D-Alanine was chosen to be the α-amino acid component in our studied α/γ-peptides.

The

The general Synthetic method 4.2.2.1 The activation/ coupling strategy

Peptide synthesis via amino acids coupling involves two steps (see Scheme 51). The first step is the activation of the carboxylic acid group of one amino acid residue using a coupling agent.

The second step involves the nucleophilic addition of the amino group of the other amino acid derivative at the activated carboxylic acid.

Scheme 51: The steps of peptide synthesis via amino acid coupling.

A suitable coupling method has to form peptide bonds in high yields and to maintain the configurational integrity of the carboxylic component. 169,170 Racemization at the α-carbon usually occurs during the activation of the carboxylic acid function if the amine part is bearing an electron withdrawing group (see Scheme 52).

Scheme 52: The two possible explanations for the racemization at the α-carbon during peptide coupling.

It is important to note that even though our convergent strategy in building up the mixed γ/αpeptides involved an activation of a carboxylic acid group and an amine protected with an electron withdrawing group, no racemization of the D-alanine was observed in any of the synthesized peptides.

Choice of the coupling agent

The carboxylic acid activation, first step in peptide coupling, is crucial and one should experiment in order to find the best coupling agent and reaction conditions for a given peptide synthesis. Several coupling reagents are available. For instance, carbodiimide reagents, like DCC, have been used as peptide coupling agents for a long time.

In the recent years, two classes of coupling reagents became popular, the phosphonium type reagents such as BOP, and PyBOP, and the aminium-(uronium) type reagents such as TBTU, HBTU, and HATU (see Figure 80). These compounds achieve high coupling rates accompanied by few undesired side reactions. In contrast with carbodiimide based reagents, activation by these coupling agents require the presence of a base like diisopropylethylamine (DIPEA) for the activation reaction to proceed (see Scheme 53). 170

Scheme 53: The general mechanism of peptide coupling using HATU.

In our coupling reactions we followed the reaction conditions established previously in our group for β/γ-hybrid peptides preparation. 158 Thus we used HATU, as the coupling agent, which was easy to handle and provided high coupling yields, without any unwanted racemization.

The dipeptide coupling reactions were performed by first cleaving the Boc protecting group of the amine partner using TFA in DCM. Then the activation of the carboxylic acid residue of the acid partner, using HATU in the presence of DIPEA, generates the activated carboxylate in a mixture of DCM and DMF. (Scheme 5 below) The DCM/DMF mixture allowed a better dissolution of the HATU, as well as higher order peptides, which have low solubility. The activation of the carboxylic acid residue could be confirmed by the darkening of the reaction mixture (from light brown to dark brown) due to the 1-hydroxy-azaenzine-triazole anion (HOAt) released by HATU (see Scheme 55). Once the activation was complete, within 10 min, a solution of the amine partner, in DCM, with a sufficient amount of DIPEA to neutralize the excess of TFA, was added to the HATU activated reaction mixture, the resulting reaction mixture was then left to stir at room temperature, under argon, for 24 hours, after which it was purified to give the desired dipeptide.

Convergent peptide synthesis

The general synthetic strategy we followed in building up the mixed γ/α-peptides was a convergent one. This strategy seems to us more adequate than the linear synthesis of peptides, where the monomers are successively coupled one after the other until the target molecule is reached, with the yield dropping quickly as the number of steps increases. A convergent synthesis is a strategic approach in which fragments of the peptide are first prepared and then combined together to complete the target molecule (see Figure 81). In a convergent strategy, fewer steps than in the linear one are needed to reach the same molecule, thus enhancing the yield and saving time.

Synthesis of the Boc-[(S,S)-cis-3,4 CB-GABA/(R)-Ala]-OBn peptide p1

The C-terminal benzyl protecting group, of Boc-(S,S)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA-OBn was first removed in a quantitative yield by catalytic hydrogenolysis under dihydrogen atmosphere in the presence of 10% Pd-C (10% w/w) for 4 hours to obtain the free acid intermediate (see Scheme 54). The N-terminal Boc protecting group of Boc-D-Ala-OBn was cleaved by treatment with a large excess of TFA for 1 hour to give the corresponding amine partner as a TFA salt.

The obtained free acid was then activated with HATU and reacted with the amine partner in the presence of DIPEA for 24 hours at room temperature, under argon. to afford dipeptide p1 in an 85% yield after purification.

Synthesis of the Boc-[(S,S)-cis-3,4 CB-GABA/(R)-Ala]2-OBn peptide p2

One equivalent of dipeptide p1 was selectively deprotected at the N-terminal with large excess of TFA for 1 hour. Another equivalent of dipeptide p1 was selectively deprotected at the Cterminal by catalytic hydrogenolysis under dihydrogen atmosphere in the presence of 10% Pd-C (10% w/w) for 4 hours. The two monoprotected dipeptides were then mixed together in the presence of excess of DIPEA before HATU was added. The coupling reaction was left to stir for 24 hours, at room temperature, under argon to afford tetrapeptide p2 in a 66% yield.

In is coupling reaction, the free acid and the free amine partners were mixed together along with enough DIPEA to make the reaction mixture basic before adding the HATU to avoid the risk of racemization of the activated dipeptide (see section 4.2.2.1), by minimizing the time of it being activated without the presence of the nucleophilic amine partner.

Synthesis of the Boc-(R,R)-cis-3,4 CB-GABA-(R)-Ala-OBn peptide p3

The C-terminal benzyl protecting group, of Boc-(R,R)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA-OBn was first removed in a quantitative yield by catalytic hydrogenolysis under dihydrogen atmosphere in the presence of 10% Pd-C (10% w/w) for 4 hours to obtain the free acid intermediate (see Scheme 55). The N-terminal Boc protecting group of Boc-D-Ala-OBn was cleaved by reaction with a large excess of TFA for 1 hour to give the corresponding amine partner as a TFA salt.

The obtained free acid was activated with HATU and reacted with the amine partner in the presence of DIPEA for 24 hours at room temperature, under argon to afford dipeptide p3 in a 92% yield. The dipeptide p3 was selectively deprotected at the C-terminal by catalytic hydrogenolysis under dihydrogen atmosphere in the presence of 10% Pd-C10% (w/w) for 4 hours to give its free acid from, p3-OH. The analysis of a single crystal of dipeptide p3-OH by X-ray diffraction shows no intra residual hydrogen bonding in solid state, but rather an infinite array of intermolecular hydrogen bond interactions implicating three hydrogen bond donors and two acceptors that stack the dipeptide p3-OH molecules in an anti-parallel fashion (see Figure 82).

Synthesis of the Boc-[(R,R)-cis-3,4 CB-GABA-(R)-Ala]2-OBn peptide p4

One equivalent of dipeptide p3 was selectively deprotected at the N-terminal with large excess of TFA for 1 hour. Another equivalent of dipeptide p3 was selectively deprotected at the Cterminal by catalytic hydrogenolysis under dihydrogen atmosphere in the presence of 10% Pd-C (10% w/w) for 4 hours. The two monoprotected dipeptides were then mixed together in the presence of excess of DIPEA before HATU was added. The coupling reaction was left to stir for 24 hours, at room temperature, under argon to afford tetrapeptide p4 in a 40% yield

The tetrapeptide p4 exhibited low solubility in organic solvents such as chloroform, and displayed a tendency to form a gel, which made its purification problematic. The preparation of the higher order hexapeptide was subsequently inaccessible. Thus the conformational analysis was performed on the tetrapeptide.

Structural and Conformational analysis in solution 4.2.5.1 General methods for structural and conformational analysis

The linear structure and the three dimensional conformation of the synthesized peptides were studied, in solution, using different experimental and theoretical techniques.

Structural analysis

The linear structure of the synthesized di-and tetrapeptides was confirmed by a combination of a series of 1D and 2D NMR experiments ( 1 H, 13 C, TOXY, COSEY, HSQC, and HMBC).

These spectroscopic techniques allowed the unambiguous assignment of all the protons and carbons of the studied peptides, except for some ones, which appeared overlapped with each other. Here again ROESY was chosen over NOESY due to its better sensitivity in detecting NOE colorations in molecules of molecular masses above 1 KDa (see Section 1.2.3).

Molecular modeling

Molecular modeling studies for both tetrapeptides (p2 and p4) were performed using a hybrid Monte Carlo Multiple Minima (MCMM). The three dimensional conformational examination was carried out in a chloroform medium using Macromodel 04 from Schrödinger software and the Merck Molecular Force Field (MMFF). Out of the 10 000 conformers that were generated by MCMM, the 100 lowest energy conformers (E=10KJ/mol) were retained and classified into different conformational families.

The performed molecular modeling was done without any constraints from the experimental data. Thus, the modeling data could be considered as a theoretical tool that is completely independent from experimental one.

Determining the linear structure of the peptides p2 and p4

The linear structure of the tetrapeptides p2 and p4 was confirmed by a combination of a series of 1D and 2D NMR experiments. The chemical shift of the carbamate N-H always relatively more shielded than the other N-Hs was easily attributed. An analysis combination of COSY, HSQC and HMBC experiments allowed the unambiguous assignment of all the protons and carbons of the studied peptides. A TOXY experiment allowed us to distinguish between the γand αamino acid protons especially when they appeared to overlap with each other.

DMSO-d6 titration experiment

The DMSO-d6 NMR titration experiment was performed on CDCl3 solutions of the tetrapeptides p2 (1.3 mg/ 0.3 ml, 7.5 mM) and p4 (1.5 mg/ 0.4 ml, 6.5 mM) at 300 K. The difference in chemical shifts Δδ of the N-Hs between 0% and 50 or 10% of added DMSO-d6

were determined (see The tetrapeptide p2 showed two N-H (4 and 11) with relatively high DMSO-d6 titration coefficient (red color) and two N-H with relatively low coefficients (15 and 22) (blue color).

The low mobility of N-H 15 and 22 is most likely an indication of their involvement in intramolecular hydrogen bonding.

On the other hand, tetrapeptide p4 showed three N-H (4, 11, and 22) with relatively high DMSO-d6 titration coefficient (red color) and only one N-H with relatively low coefficients (N-H 15) (blue color). This preliminary observation hints a difference in the preferential folding pattern of the two diastereomeric tetrapeptides in solution, which will be further investigated via two dimensional NMR experiments and molecular modeling.

Conformational analysis of the peptides p2 and p4

4.2.5.3.1 2D ROESY NMR experiment 2D ROESY NMR experiments were done on a CDCl3 solution (5 mg/ 0.5 ml) for each of the tetrapeptides p2 and p4. Best ROE colorations were detected when the spinlock parameter during the experiment was set to 400 ms. The most significant ROE correlations and the deduced hydrogen bonding patterns are presented in Figure 83 for tetrapeptide p2 and in Figure 84 for tetrapeptide p4.

The tetrapeptide p2 generated ROE correlations, which suggest the presence of an alternating 12 and 10-membered pseudocyles, indicating the tendency of this peptide to have a 12/10 helical secondary structure (see Figure 84), which is in accordance with the Hofmann group predictions. 117 Unlike the tetrapeptide p2, ROEs correlations observed for the tetrapeptide p4 do not suggest the presence of a 12/10 folding pattern. They rather indicate the occurrence of an alternating 7and 9-membered pseudocycles (see Figure 84). This kind of experimentally uncharted, secondary structure results from interactions between adjacent peptide bonds rather than non-nearest neighbour ones, which is a competitive possibility as suggested by the Hofmann group. 117

Molecular modeling

An MCMM (Monte Carlo Multiple Minima) calculation was performed on the tetrapeptides p2 and p4 to investigate their conformational landscape in chloroform. The lowest energy conformations, were arranged according to the hydrogen-bonded ring systems they form.

Table 9 shows the list of predicted conformations by the MCMM calculations for tetrapeptide p2 along with their percentage abundance. The discreet, successive hydrogen bonded rings formed between a carbonyl oxygen and an amide hydrogen are separated by a hyphen '-', while the ones implicating a carbonyl oxygen that is bifurcated between two amide hydrogens are separated by a coma ','.

β/γ-peptide composed of (1R,2R)-2-aminocyclobutane carboxylic acid (trans-ACBC) and GABA, that fold with an alternating 9-, 8-hydrogen bonding pseudocycles. 158 Table 10 shows the list of predicted conformations by the MCMM calculations of tetrapeptide p4 along with their percentage abundance.

Conformations Relative abundance of each conformation [9,12-9] 7% [9-7,12] 27% [15-13-7] a 9% [9-7-9] 15% [9,12-12] 20% [7-13-7] 2% [7,12-10] 3% [9-12-10] 9% [10-12-10] 5% [7-10,7] 2%

Table 10: The list of conformations for tetrapeptide p4 as predicted by the MCMM calculations. a 15 and 7 rings are from the same carbonyl

The experimentally found [9-7-9] conformer is the third most abundant (15%) and has the third lowest energy. Figure 89 shows the hydrogen bonding pattern in the most abundant [9-7,12] and the lowest energy [9,12-9] conformers as calculated by MCMM, and the experimentally found [9-7-9] conformer.

Boc-(S,S)-cis-3,4 CB-GABA/(R)-Ala-NHBn peptides

Having a terminal amide group in a peptide should provide an extra hydrogen bond acceptor, thus increasing the chance of a relatively short tetrapeptide to adopt a well-defied secondary structure. For this, we opted to prepare an analog of the tetrapeptide p4 with a benzyl amide terminal rather than a benzyl ester.

The synthesis of Boc-[(S,S)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA-(R)-Ala]n-NHBn peptides p5 and p6 was carried out following a convergent strategy, starting from the Boc-(S,S)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA-OBn enantiomer (-)-13 as the γ-amino acid partner, and the Boc-D-Ala-NHBn (+)-16 as the α-amino acid partner (see Scheme 58).

Scheme 56: The convergent synthesis of Boc-[(S,S)- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA-(R)-Ala]n-NHBn peptides p5 and p6. The obtained free acid was then activated with HATU and reacted with the amine partner in the presence of DIPEA for 24 hours, at room temperature, under argon, to afford dipeptide p5 in 85% yield. The analysis of a single crystal of dipeptide p5 by X-ray diffraction shows no intra residual hydrogen bonding in solid state, but rather an infinite array of intermolecular hydrogen bond interactions implicating three hydrogen bond donors and three acceptors that stack the dipeptide p5 molecules in a parallel fashion (see Figure 87).

Conclusion

In conclusion two diastereoisomeric γ/α-hybrid tetrapeptides composed of alternating cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA and D-Alanine were synthesized and fully characterized and their three dimensional folding pattern in solution was investigated. Even though, more conformational analysis including infrared spectroscopy and DFT calculations are still needed to further validate the folding behavior of the two diastereoisomeric γ/α-hybrid tetrapeptides, 2D ROESY NMR experiments and molecular modeling data revealed the tendency of these peptides to fold into a 12/10 helix in solution when the (S,S)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA was used as the γ-amino acid component. These experimental results are in correlation with the Hofmann group theoretical calculations. 117 On the other hand when the chirality of the γ-amino acid component was switched to (R,R)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA, the peptide showed a tendency to adopt an unprecedented 9/7 folding pattern interpreted by observing the intra residual ROEs colorations displayed by tetrapeptide p4.

Despite the fact that the 9/7 folding pattern was only the third abundant one as calculated by the preliminary molecular modeling of p4 in CDCl3, the switching from a 12/10 structuration observed for its diasteriomeric analog p2 emphasizes the importance of the side chain configuration as a tool to design peptides in a deliberate secondary structure.

Perspective and Overall Conclusion

Our goal in this work was to prepare two cyclobutane-constained -amino acids, cis-2,3 CB-GABA and cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA, in an enantioselective manner, and use these building blocks for the synthesis of -hybrid peptides that are likely to adopt helical structuration. We focused our interest on a supramolecular photochirogenesis method, adopting -CD as the chiral guest, since it appeared easily adaptable to our proposed photochemical reactions.

We synthesized N-allyl-N-(4-methoxyphenyl)acrylamide 1 and assumed its photocycloaddition reaction would lead to the cycloadduct 2, a precursor of cis-2,3 CB-GABA. The complexation of compound 1 with -CD was studied by 1D and 2D 1 H NMR experiments which showed diagnostic interactions between protons of both host and guest. The complex was identified as having a 1:1 (-CD/1) ratio and had a low binding constant (Kb =14.7).

A photocyclization reaction test performed with compound 1 (without -CD) did not lead to the expected photochemical [2+2]-cycloaddition product but gave instead 1-allyl-6-methoxy-3,4dihydriquinolin-2(1H)-one 5, resulting from a photochemical 6-electrocyclization followed by a 1,5-sigmatropic shift.

Then we turned our attention to the photoelectrocyclization of 1,3-dihydro-2H-azepin-2-one 8

in the presence of β-CD which was expected lead to an enantiomerically enriched photoadduct 9, a precursor of cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA. The complexation of compound 8 with -CD was studied by 1D and 2D 1 H NMR experiments which showed diagnostic interactions between protons of both host and guest. The complex was identified as having a 1:1 (-CD/8) ratio and had a moderate binding constant (Kb = 35.4).

Irradiation of the -CD/8 complex in aqueous solutions, suspensions, or in the solid state, resulted in the electrocyclization of azepinone 8 to give the photoadduct 9. This later compound had a very low solubility in organic solvent and was immediately reduced in a one-pot reaction into the 2-azabicyclo[3.2.0]heptan-3-one 10 with an yield up to 79% from azepinone 8. The highest ee of 45% was obtained when the irradiation of the -CD/8 complex was performed in suspension form.

Continuing our efforts to have access to (R,R) and (S,S)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA, we then established a resolution method for the N-Boc benzyl ester derivative on a semi-preparative HPLC instrument fitted with a chiral column, which furnished both enantiomers in pure form and on a gram scale.

Enantiomerically pure (-) and (+)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA derivatives were used thereafter to synthesize two diastereoisomeric -peptides. These hybrid peptides were composed of alternating either (-)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA or (+)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA with D-Alanine. Two dipeptides, Boc-S,S-GABA-R-AlaOBn and Boc-R,R-GABA-R-AlaOBn, as well as two tetrapeptides, Boc-(S,S-GABA-R-Ala)2OBn and Boc-(R,R-GABA-R-Ala)2OBn, were prepared by a convergent synthesis and fully characterized.

The conformational analysis of both diastereoisomeric dipeptides by single crystal X-ray diffraction showed no intramolecular interactions but a network of intermolecular hydrogen bonding. 1D and 2D NMR experiments showed that the tetrapeptide, Boc-S,S-GABA-R-Ala)2OBn, adopted a 12/10 helical conformation, whereas its diastereomeric analog, Boc-(R,R-GABA-R-Ala)2OBn, displayed evidence of an unprecedented 7/9 folding pattern in solution.

As a perspective of this work, the [2+2] photocycloaddition of N-allyl acrylamide could be favored by the use of a benzyl group on the nitrogen atom in place of an aryl group, thereby suppressing the conjugated 6π system responsible of the 6π-electrocyclization.

Photochirogenesis using β-CD in the electrocyclization of azepinone 8 could be improved by use of a benzyl or tert-butyloxycarbonyl group attached on the nitrogen, to strengthen the -CD/azepinone complexation.

-tetrapeptides, Boc-(S,S-GABA-R-Ala)nNHBn, could be prepared and their conformational preferences studied, since the amide on the C-terminal is able to provide additional H-bond interactions to enhance folding propensity.

Experimental Part 3-(phenylsulfonyl)propanoic acid [2]

To a stirring solution of sodium benzenesulfinate (4.00 g, 24.39 mmol) in 25 ml distilled water, acrylic acid (1.67 ml, 1 eq.) was added, and the reaction mixture was left to stir overnight. The aqueous solution was then acidified with 2 M HCl and the desired product was extracted with diethyl ether (4 x 25 ml). The organic layer was concentrated under vacuum to obtain the desired product as white solid (3.90 g, 18.29 mmol). The crude product was pure enough to be used in the next step after drying under high vacuum.

Yield: 75%

TLC Rf (EtOAc): 0.6

1 H NMR (300 MHz, CDCl3): δ = 2.80 (t, 2H, H-2), 3.42 (t, 2H, H-1), 3.76 (s, 3H, H-13), 7.56-7.62 (m, 2H, H-8), 7.66-7.71 (m, 1H, H-9), 7.90-7.94 (m, 2H, H-7) Hz, J = 5.9 Hz, J = 2H, H-3), 3.78 (s, 3H, H-13), 4.63-4.41 (m, 2H, H-10), 5.37-5.02 (m, 2H, H12), 5.88 (ddt, J = 17.2 Hz, J = 10.0 Hz, 4.7 Hz, 1H, H-11), 6.79-6.67 (m, 1H, H-9, 1H, H-7), 7.12-6.84 (m, 1H, H-6).

3,3-Dibromoazepan-2-one [7]

A solution of caprolactam 6 (5.00 g, 44.25 mmol) in chloroform (125 mL) was cooled to 4 °C in an ice bath. Phosphorus pentachloride (18.39 g, 89.01 mmol, 2 eq.) was then added followed by dry zinc iodide (0.56 g, 1.75 mmol). Bromine (4.5 mL, 88.5 mmol) was then added slowly over 15 min. The reaction mixture was allowed to warm to room temperature and left to stir overnight. The reaction was quenched with an ice-water mixture (200 mL). The organic layer was separated and washed with water (3 × 100 mL) and with a 0.5 M solution of sodium bisulfite (3 × 100 mL), dried over sodium sulfate, and concentrated under reduced pressure.

The crude yellow solid was washed with water to obtain compound 7 as white solid (8.30 g, 30.86 mmol).

Yield: 70%.

TLC Rf (PE/EtOAc 50/50): 0.60.

1 H NMR (400 MHz, CDCl3): δ = 1. 67-1.75 (m, 2H, H-6), 1.97 (ddd, J = 8.4, 7.2, 4.7 Hz , 2H, H-5), 2.72-2.75 (m, 2H, H-4), 3.35-3.44 (m, 2H, H-7), 6.59 (bs, 1H, H-3).

2,3-Dihydro-1H-azepin-2-one [8]

To a solution of compound 7 (5.80 g, 21.56 mmol) in dry DMF (130 mL), dried anhydrous LiCl (2.71 g, 63.93 mmol) was added. The reaction mixture was heated to reflux under argon atmosphere for 5 h, then left to stir at room temperature overnight. The solvent was then distilled out under reduced pressure. Water 50 ml was added, and the aqueous layer was extracted with CH2Cl2 (5 X 50ml). The organic layer was dried over NaS2O4, filtered and concentrated to obtain crude 8 as dark brown oil that was purified by flash chromatography on silica (gradient eluent from 80/20 to 50/50 PE/EtOAc). Compound 8 was obtained as yellow solid which was recrystallized from pentane to give white crystals (1.63 g, 14.65 mmol).

Yield: 69%.

TLC Rf (PE/EtOAc 50/50): 0.53. Hz, 1H, H-6), 5.84 (dd, J = 5.1 Hz, J = 8.9 Hz, 1H, H-4), 6.17-6.23 (m, 1H, H-5, 1H. H-3), 8.17 1 H NMR (300 MHz, D2O): δ =1. 75-1.85 (m, 1H, H-3, 1H, H-4), 2.2 (d, J = 17.7 Hz, 1H, H-6), 2.18-2.35 (m, 1H, H-3, 1H, H-4), 2.51 (dd, J = 8.7 Hz, J = 17.7 Hz, 1H, H-6), 3.03-3.12 (m, 1H, H-1), 4.03-4.12 (m, 1H, H-2).

179.3 (C-7). LiOH (4.08g, 9.71 mmol, 10 eq.) was added to a solution of the compound (±)-11 (2.052 g, 9.72 mmol), in a 1/1 THF / H2O mixture (30 mL). The reaction mixture was left to stir overnight at room temperature. The THF was evaporated under reduced pressure. The residual aqueous phase was acidified with a solution of HCl (0.5 M) to pH = 1 and then saturated with solid NaCl. It was then extracted with AcOEt (5 x 30 mL). The combined organic phases were dried over MgSO4, filtered and concentrated under reduced pressure. The crude product was then purified by flash chromatography on silica (gradient from 50/50 to 0/100 PE/EtOAc). The N-Boc protected amino acid (±)-12 was obtained in the form of a white solid (1.58 g, 6.89 mmol).

IR

Yield: 71%.

TLC Rf (EP/EtOAc 1/1): 0.28

(R)-2-((tert-butoxycarbonyl)amino)propanoic acid [(+)-14)]

To a solution of D-alanine (1.00 g, 11.2 mmol, 1 eq.) in ultrapure water (25 ml), at 0 o C, were added dropwise NaOH 1 M (22.5 ml) (24.5 mmol, 2 eq.) and a solution of Boc2O (3.60 g, 16.5 mmol, 1.5 eq.) in dioxane (25 ml). After the addition, the reaction mixture was left to stir overnight at room temperature. The dioxane was then evaporated under vacuum and the To a solution of (+)-14 (600 mg, 3.17 mmol, 1eq.), benzyl alcohol (0.33 ml, 1.02 eq.)

and DMAP (0.038 mg, 0.1 eq.) in DCM (15 ml) at 0 o C, was added DCC (0.719 g, 1.1eq.). The reaction mixture was warmed to room temperature and left to stir overnight. The solvent was then evaporated to dryness and the crude product was then purified by flash chromatography on silica (8/2 PE/EtOAc) to obtain Boc-(R)-Ala-OBn (+)-15 (663 mg, 2.37 mmol) as a colorless solid.

Crude yield: 75%

TLC Rf (PE/EtOAc 8/2): 0.44 [𝜶] 𝑫 𝟐𝟎 = +13 (c. 0.5 in CHCl3)

(R)-tert-butyl (1-(benzylamino)-1-oxopropan-2-yl)carbamate [(+)-16]

To a solution of (+)-14 (100 mg, 0.52 mmol, 1eq.), benzyl amine (14 µl, 1.02 eq.) and DIPEA (275 µl, 1.58 mmol, 3 eq.) in DCM (15 ml) was added HATU (0.72 g, mmol 1.05 eq.). The reaction mixture was stirred for 48 h and the solvent was then evaporated to dryness. Ethyl acetate was added to solubilized the solid products, and the organic layer was then successively washed with a saturated solution of bicarbonate (10 ml), brine, HCl 1M (10 ml), and then brine (10 ml). The organic layer was dried over MgSO4 then concentrated under vacuum. The crude product was purified by chromatography on flash silica (PE/EtOAc 8/2) to obtain Boc-(R)-Ala-NHBn (+)-16 (117 mg, 0.42 mmol) as a white solid.

Yield: 80%

TLC Rf (PE/EtOAc 1/3): 0.75 [𝜶] 𝑫 𝟏𝟖 = + 24 (c. 0.5 in CHCl3)

Boc-(S,S)-GABA-(R)-Ala-OBn [p1]

Following the general procedure "III" dipeptide p1 was prepared from Boc-(S,S)-cis- 1, 128.4, 128.6, (C-17, C-18, C-19), 135.2 (C-16), 155.4 (C-3), 171.9 (C-10), 172.9 (C-14).

Boc-[(S,S)-GABA-(R)-Ala]2-OBn [p2]

Following the general procedure "IV" the benzyl ester of dipeptide p1 (86 mg, 0.

Yield: 40%

TLC Rf (DCM/MeOH 100/4): 0.7

Boc-(S,S)-GABA-(R)-Ala-NHBn [p5]

Following the general procedure "III" dipeptide p5 was prepared from Boc-(S,S)-cis-

L'objectif de ce travail

Dans ce travail, nous avons cherché à explorer la possibilité d'utiliser la photochimie supramoléculaire asymétrique comme stratégie de synthèse alternative pouvant fournir un accès plus rapide et / ou plus efficace au cis-2,3 CB-GABA et au cis- Nous avons envisagé la synthèse énantiosélective des dérivés du cis-2,3 CB-GABA et de cis-

La réactivité photochimique de la 1,3-dihydro-2H-azépine-2-one 8

Dans cette partie du projet de recherche, nous avons cherché à établir un accès synthétique aux dérivés du cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA, sur la base de l'électrocyclisation photochimique intramoléculaire énantiosélective du composé 8 en présence de β-CD.

Nous supposions que la présence de β-CD au cours de cette électrocyclisation 4π pourrait fournir un environnement chiral qui favoriserait une topologie de fermeture du cycle par rapport à l'autre, permettant ainsi un accès préférentiel à l'un des énantiomères (schéma e).

Dans ce chapitre, nous avons d'abord planifié la synthèse de l'azépinone 8, l'étude de l'interaction hôte-invité 8/β-CD, les meilleurs conditions d'irradiation du complexe en termes de rendement chimique et d'excès énantiomérique en produit 9. Par la suite, le cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA énantioériquement enrichi pourraient être préparés en suivant le mode opératoire décrit pour la forme racémique.

La synthèse de l'azépinone 8

L'azépinone 8 a été préparée selon un procédé précédemment décrit. Elle est ainsi obtenue par la dibromation du caprolactame 6 commercial conduisant au composé 7 avec un rendement de 73%, qui par double élimination de HBr produit l'azépinone 8 avec 70% de rendement (schéma f). Afin de contourner ce problème, nous avons effectué le dédoublement de l'ester benzylique (±)-13, qui présentait une plus grande solubilité dans les solvants organiques (Schéma j).

Schéma j: Synthèse de l'ester benzylique (±)-13 à partir de (±)-12

normale. La figure j montre le chromatogramme en HPLC analytique de (±)-13.

Conclusion et Perspective

Le but de ce travail était de préparer les deux -amino acides cyclobutaniques contraints, cis-

Scheme 12 :

 12 Scheme 12: Asymmetric Norrish Type II / Yang cyclization reaction of chiral N,Ndiisopropyl-2-oxo-2-arylacetamide derivatives in the presence of β-CD and chirally modified β-CD.

Figure 2 :

 2 Figure2: The most common binding stoichiometries between CDs and guest molecules.[START_REF] Linde | Changes to Taste Perception in the Food Industry[END_REF]

Figure 5 :

 5 Figure 5: A plot of 1/Δδ vs 1/[G], based on the Benesi-Hildebrand equation.

Figure 6 :

 6 Figure 6: The variation of the 1 H/ 1 H nuclear Overhauser effect (NOE) and rotating-frame Overhauser effect (ROE) with respect to correlation time τc.

  H5' of p-nitrophenol shows a ROE correlation with H3 and H5 of the β-CD indicating that former set of protons are in close proximity to both H3 and H5, placing this part of the pnitrophenol molecule deep inside the β-CD cavity (see Figure 7 spectrum B). On the other hand, selective excitation of H2' and H6' of the p-nitrophenol shows a correlation only with H3 indicating that this part of the p-nitrophenol molecule is located in a more shallow manner, towards the secondary face of the β-CD. (see Figure 8 spectrum C).

Figure 7 :

 7 Figure 7: The proposed β-CD/p-nitrophenol complex (left), 1D selective ROE spectra (right).

Figure 8 :

 8 Figure 8: The proposed inclusion complexes formed between β-CD and vanillylmandelic acid VMA (left), and the 2D ROESY spectrum leading to this proposal (right)

Figure 9 :

 9 Figure 9: The α-, β-and γ-amino acid backbones.

Figure 10 :

 10 Figure 10: Three different structural sub-classes of γ-amino acids.

80

 80

1. 3 . 1 . 1

 311 Constrained -amino acids -Amino acids can be considered as doubly-homologated α-amino acids, and as such the two extra carbons between the amine and carboxyclic acid functions provide the backbone with more flexibility when compared to α-and β-amino acids. Consequently, GABA can adopt many low-energy conformations, each one having its own specific biological relevance.

Figure 12 :

 12 Figure 12: Example of different sized cyclic-constrained -amino acids used as building blocks for peptide foldamers.

Scheme 26 :

 26 Scheme 26: Synthesis of 3-(phenylsulfonyl)propanoic acid, 3, from sodium benzenesulfinate and acrylic acid.

Scheme 28 :

 28 Scheme 28: The synthesis of N-allyl-N-(4-methoxyphenyl)acrylamide, 1, from N-(4methoxyphenyl)-3-(phenylsulfonyl)propanamide, 4.

  Unlike common examples of β-CD complex formation, 109 mixing N-allyl-N-(4methoxyphenyl)acrylamide 1 with β-CD in water at a 15 mM concentration, did not result in the formation of a precipitate. In order to prepare a β-CD/1 complex, we therefore added one equivalent of solid compound 1 to a clear (5 ml, 15 mM, 1 eq) aqueous solution of β-CD. The mixture was heated to 35-40 o C and left to stir for 5 min, during which time 1 dissolved completely. The resulting clear solution was stirred at room temperature for 2

Figure 14 :

 14 Figure 14: The white film (complex) of N-allyl-N-(4-methoxyphenyl)acrylamide with β-CD.

Figure 15 :

 15 Figure 15: Powder X-ray diffraction spectra of β-CD, compound 1, and β-CD/1 complex

Figure 16 :

 16 Figure 16: Job's plot of UV absorbance (λ =228 nm) at different β-CD:1 molar ratios.

Figure 17

 17 Figure 17: 1 H NMR (in D2O solution) of β-CD showing the shift of H3 and H5 with each increase in the percentage of compound 1 to β-CD.

Figure 18 : 1 H

 181 Figure 18: 1 H NMR (in D2O solution) of N-allyl-N-(4-methoxyphenyl)acrylamide (1) (bottom), and at 100% of 1 relative to β-CD (top).

Figure 19 :

 19 Figure 19: The linear plot of 1/ΔδH3 vs. 1/ [N-allyl-N-(4-methoxyphenyl)acrylamide]

  Scheme 29.

Figure 20 :

 20 Figure 20: The X ray crystal structure of compound 5.

Figure 21 :

 21 Figure 21: The benzyl (left), and carbamate (right) analogues of compound 1.

Scheme 34 :

 34 Scheme 34: The proposed enantioselective photoelectrocyclization of azepinone 8 induced by β-CD

Figure 22 :

 22 Figure 22: HPLC chromatogram of racemic (±)-9 on normal phase chiral Lux Cellulose-1.

Figure 23 :

 23 Figure 23: (a) A clear aqueous solution of azepinone 8 at 0.45 M, (b) a clear aqueous solution of β-CD at around 15 mM, (c) the "milky suspension" formed after stirring at room temperature for 30 sec, (d) 1.5 min, (e) 5 min.

Figure 25 :

 25 Figure 25: Job's plot of UV absorption (λ =254 nm) at different β-CD:azepinone 8 molar ratios.

58

 58

3. 2 . 4 . 4 1D

 244 NMR studies of the β-CD/azepinone 8 complexTrying to understand the nature of the interactions between azepinone 8 and β-CD, an NMR titration experiment was done. A 15 mM solution of β-CD in D2O was titrated by adding an incremental amount of a D2O solution of azepinone 8 (10 µl at 73 mM / addition), up to 7 equivalents. The temperature of the experiment was maintained at 40-45 o C to have a clear solution throughout the experiment.

Figure 27 :

 27 Figure 27: 1H NMR of azepinone 8 with its incremental additions to a solution of β-CD.

Figure 28 :

 28 Figure 28: The linear plot of 1/ΔδH3 vs. 1/[azepinone 8].

Figure 31 :

 31 Figure 31: The proposed mode of complexation between β-CD and azepinone 8. Deep penetration of the unsaturated part of azapinone 8 via the two β-CD faces (top left, top right) or a shallow penetration of the amid part of the azapinone from the primary face of β-CD (bottom left) or a deep penetration from the secondary one (bottom right).

Scheme 40 :

 40 Scheme 40: Reduction of photoadduct 9 in EtOAc in the presence of β-CD to give 2azabicyclo[3.2.0]heptan-3-one 10.

Figure 33 :

 33 Figure 33: A schematic draw of the Rayonet reactor (left) showing the reaction tube fitted with a "heating/cooling" finger connected to the temperature control system (right)

Figure 34 :

 34 Figure 34: Chromatogram of 10 obtained from Experiment A

Figure 35 :

 35 Figure 35: HPLC chromatogram of enantiomerically enriched 2-azabicyclo[3.2.0]heptan-3-one 10, obtained from experiment B.

Figure 36 :

 36 Figure 36: Chromatogram of enantiomerically enriched 2-azabicyclo[3.2.0]heptan-3-one 10, obtained from experiment C.

Figure 37 :

 37 Figure 37: A photo of the quartz tube containing the solid reactants inside a Rayonet reactor.

Figure 38 :

 38 Figure 38: Small balls of β-CD/azepinone 8 formed when the complex was irradiated in powder form in a rotating quartz tube.

Figure 39 :

 39 Figure 39: Chromatogram of enantiomerically enriched 2-azabicyclo[3.2.0]heptan-3-one 10, obtained from experiment E.

Figure 40 :

 40 Figure 40: Schematic draw of β-CD/azepinone 8 complex being irradiated as a solid film form inside the Rayonet reactor.

Figure 41 :

 41 Figure 41: Chromatogram of crude enantiomerically enriched 2-azabicyclo[3.2.0]heptan-3one 10, obtained from experiment F.

Figure 42 :Figure 43 :

 4243 Figure 42: Chromatogram of crude enantiomerically enriched 2-azabicyclo[3.2.0]heptan-3-one 10, obtained from experiment G

Figure 44 :

 44 Figure 44: Photo showing films of; (a) experiment F, (b) azepinone 8 alone , (c) β-CD alone, (d) experiment H.

Figure 45 :

 45 Figure 45: Photo of 1/1 β-CD/azepinone 8 complex film (experiment F) as it detaches itself from the glass plate after 2 days (left), and after 4 days (right).

Figure 46 :

 46 Figure 46: Photo showing film of experiment G

Figure 47 :

 47 Figure 47: SEM images of β-CD alone (top left), azepinone 8 alone (top right), film of experiment G: before irradiation (bottom left), and after irradiation (bottom right)

Figure 49 :

 49 Figure 49: HPLC chromatogram of racemic 12 showing separation of the two enantiomers on an analytical column.

Figure 50 :

 50 Figure 50:HPLC chromatogram of racemic 12, separation of the two enantiomers on an a semi-preparative column.

Figure 51 :

 51 Figure 51: HPLC Chromatogram of racemic 13, separation of the two enantiomers on an analytical HPLC.

Figure 52 :

 52 Figure 52: HPLC chromatogram of racemic amino acid (±)-13, separation of the two enantiomers on a semi-preparative column.

Figure 53 :

 53 Figure 53: Chromatogram of the pure enantiomers (+)-13 (top) and (-)-13 (bottom)

Figure 54 :

 54 Figure 54: X-ray diffraction structure of the (3R,4R) enantiomer of the protected (+)-cis-3,4 CB-GABA.

Figure 55 :

 55 Figure 55: The resonant structures of the peptide unit.

Figure 56 )

 56 Figure 56). Now, the O-C''-N0 residue (blue) is held in plane, and the other unit (red) as a whole is given a rotation of an angle φ about the N0-Cα bond, in an anticlockwise sense looking from N0 towards Cα. This bond, N0-Cα, is then tightened and the O-C''-N0 residue (blue) is then rotated through the angle ψ about the Cα-C' bond, in a clockwise sense, looking from C' towards Cα. The resulting dipeptide has now a conformation which can be described by the dihedral angles (φ, ψ) .

Figure 56 :

 56 Figure 56:The dihedral angles (φ, ψ, ω, and χ) that describe the conformation of two peptide units.

4. 1 . 2 . 1 57 )Figure 57 :

 1215757 Figure 57: The Ramachandran plot for all residues, except glycine, with the permitted combinations of torsional angles shown as colored regions.

Figure 58 :

 58 Figure 58: The Primary, secondary, tertiary, and quaternary structures of protein 122

  acids are homologated analogous of α-amino acids. The additional backbone carbon

Figure 60 :

 60 Figure 60: The different types of helices that could be adopted by a β-peptide, as predicted by the Hofmann group. Solid arows indicate the so far experimentally observed ones.

Figure 61 :

 61 Figure 61: The Gellman's group 12-helix antimicrobial β-peptide.

Figure 62 :

 62 Figure 62: The dihedral angels in a γ-peptide.

Figure 64 :

 64 Figure 64: One example of the γ 4 -peptides that fold into a stable 14-helix, from Seebach's group.

Figure 65 :

 65 Figure 65: The three possible directions of hydrogen bonding in an α/β-hybrid peptide.

Figure 66 :

 66 Figure 66: The Gellman's group chimeric α/β+α-peptide.

Figure 69 :

 69 Figure 69: The resemblance between a theoretically predicted 12/10-helix for a α/γ-hybrid octapeptide (bottom), and that of the experimentally found for a beta peptide (top), with a three dimensional superimposition of both helical structures (right).

Figure 70 :

 70 Figure 70: The folding pattern in the Balaram's group α/γ-hybrid nonapeptide (top), and tetrapeptides (bottom)

Figure 71 :

 71 Figure 71 : The Gopi's group α/γ-hybrid peptide that folds into a 12-helix (left), and a three dimensional superimposition of it with a native α-helix (right).

Figure 72 :

 72 Figure 72: The Kunwar's group α/γ-hybrid tetrapeptide that folds into a 12/10-helix.

Figure 75 :

 75 Figure 75: The Maillard's group peptide that show a 9-membered hydrogen bonding pseudocycle.

Figure 76 :

 76 Figure 76: The effect of changing the chirality of an amino acid componant on the over all structuration in an ACPC ologomer.

  ), which showed unique conformational properties resulting from its aromatic ring. These ATC γ-amino acids, having a ζ torsion angle locked around 0 o , are structurally similar to Z-vinylogous γamino acids. The ATC acted as a strong turn inducer for folding. The folding pattern depended as well of the absolute configuration of the γ-amino acid. The alternating homochiral (S)-α/(S)-ATC dipeptide exhibited a C-9 foldwhile the heterochiral (S)-α/(R)-ATC dipeptide displayed ribbon structure stabilized by unusual C-9/12 bifurcated hydrogen bonds

Figure 77 :

 77 Figure 77: The 9,12-bifurcated hydrogen bonding pattern recently found by the Maillard's group in γ/α-hybrid peptides.

Figure 78 :

 78 Figure 78: The crystal structure of the cis-3,4 cyclobutane -aminobutyric acid (cis-3,4 CB-GABA) (left), the torsional angel needed to obtain a 12/10 helical folding pattern in mixed α/γpeptides, as predicted by the Hoffman group (right)

Figure 79 :

 79 Figure 79: The target γ/α-hybrid peptide, the (S,S/R)-series (left), and the (R,R/R)-series right.

  Scheme 50. The synthesis of compounds 15 and 16 from commercial D-Alanine.

Figure 80 :

 80 Figure 80: Examples of the phosphonium (top), and aminium-uronium (bottom) type coupling reagents.

Figure 81 :

 81 Figure 81: The linear (top) v.s. the convergent (bottom) startegie for γ/α-hybrid peptide synthesis.

Figure 82 :

 82 Figure 82: The X ray crystal structure of peptide p3-OH (top), and its intermolecular hydrogen bonding interactions viewed from the side (left), front (middle), and top (right).

4. 2

 2 .5.1.2 DMSO-d6 NMR titration experiments DMSO-d6 NMR titration experiments were done to assign the free and bonded N-H in peptides.Incremental amounts of DMSO-d6 were successively added to an NMR tube containing a peptide in CDCl3 (6-7 mM), and the 1 H spectra were recorded after each addition. Addition of the DMSO-d6 into the tube increases the polarity of the environment around the peptide. This change in polarity induced a significant change in the chemical shift on the free N-H while it had less effect on the bonded ones. 4.2.5.1.3 Conformational analysis 2D ROESY NMR experiments were used to understand the three dimensional molecular conformation of the synthesized peptides, in solution, by detecting through-space dipolar magnetic interactions known as the Nuclear Overhauser Effect (NOE). By looking at the intramolecular NOE correlations within a peptide molecule, one could gain insight into the overall folding pattern of this peptide.

Figure 83 :

 83 Figure 83: Tetrapeptide p2 showing ROE correlations characteristic of a 10-membered (blue) and 12-membered (green) hydrogen-bond rings (above) and the overall deduced hydrogen bond pattern (below). The fade colored arrow corresponds to a weak ROE correlation.

Figure 84 :

 84 Figure 84: Tetrapeptide p4 showing ROE correlations characteristic of a 9-membered (orange) and 7-membered (pink) hydrogen-bond rings (above) and the deduced overall hydrogen bond pattern (below). The fade colored arrow corresponds to a weak ROE correlation.

Figure 86 :

 86 Figure 86: The hydrogen bonding pattern in the most abundant (left), the lowest energy (middle), and the experimentally found (right), conformers of peptide p4, as predicted by the MCMM calculations.

  terminal benzyl protecting group of Boc-(S,S)-cis-3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA-OBn was first removed in a quantitative yield by catalytic hydrogenolysis under dihydrogen atmosphere in the presence of 10% Pd-C (10% w/w) for 4 hours to obtain the free acid intermediate. The N-terminal Boc protecting group of Boc-D-Ala-NHBn, was cleaved by treating it with a large excess of TFA for 1 hour to give the corresponding amine partner as a TFA salt.

Figure 87 :

 87 Figure 87:The X ray crystal structure of peptide p5 (top), and its intermolecular hydrogen bonding interactions viewed from the side (left), front (middle), and top (right).

  1(44 mg, 0.20 mmol) in 10 ml degassed acetone in a Pyrex tube was irradiated for 9 h inside a Luzchem (LCZ-4V) reactor fitted with 14 UV-B lamps. The solvent was then evaporated under reduced pressure and the crude was purified by chromatography on silica (gradient from 90/10 to 70/30 hexane/EtOAc) to obtain compound 5 as a white solid (22 mg, δ = 2.67 (dd, J = 8.7 Hz, J = 5.8 Hz, 2H, H-2), 2.89 (dd, J = 8.8

  : m/z calcd for [C13H15NO2+Na] + 240.0995 ; found 240.0994.

  IR (solid): (C-Br) 769, (C-C) 1326, (C=O) 1665, (C-H) 2940, (NH) 3090, (NH) 3207 cm -1 HR-MS: m/z calcd for [C6H9Br2NO+Na] + 291.8943; found 293.8918 (2.5 ppm).

1 H

 1 NMR (360 MHz, CDCl3): δ= 2.91 (d, J = 6.8 Hz, 2H, H-7), 5.61 (dt, J = 9.3 Hz, J = 6.8

  photoelectrocyclisation/reduction.

  (solid): (C-C) 1287, (C-N) 1310, (C=O) 1677, (C-H), 2939, (NH) 3237 cm -1 . HR-MS: m/z Calcd for [C6H9NO+H] + 112.0757; found 112.0761 (-3.5ppm). General procedure "II" for photoelectrocyclisation in solid state / Reduction: All of the next experiments in solid state started by adding a clear aqueous solution of azepinone 8 to a clear aqueous solution containing the desired equivalents dried β-CD. The volume of the final solution was always chosen to be just below 15 mM with respect to the hostβ-CD. Upon stirring, a white precipitate starts to form almost immediately. The suspension was left to stir for 2 hours at room temperature, and then treated accordingly to obtain a white solid (powder or film). The resulting solid was irradiated in Rayonet RPR-200 equipped with 16 lamps [λ = 250 nm (RPR-3000 Å, Rayonet)]. The course of the reaction was followed by 1 HNMR. When no more azepinone 8 starting material could be detected, the reaction was considered complete and the irradiated solid was transferred into a round bottom flask, dissolved in ultra-pure water then reduced overnight, at room temp, under dihydrogen atmosphere with 10% w/w of Pd/C-10%. The catalyst was then filtered out and the crude 10 was extracted with ethyl acetate until no more of it could be detected in the aqueous phase by1

  C-2),79.4 (C-7), 155.2(C-6), 177.7(C-10). solution of amino acid (±)-7(400 mg, 17.46 mmol), and benzyl alcohol (0.185 ml, 1.02 eq.) in dichloromethane (26ml) was added DMAP (21.2 mg, 0.1 eq.). The reaction mixture was then cooled to 0 o C and DCC (400 mg, 1.1 eq.) was added. The reaction mixture was stirred for 10 min at 0 o C overnight at room temperature. The formed precipitate side product was removed by filtration, and the filtrate was concentrated under vacuum. The crude product was then purified by flash chromatography on silica (gradient from 90/10 to70/30 PE/EtOAc) to obtain (±)-8 (490 mg, 1.53 mmol) as a white solid.Yield: 90%.TLC Rf (PE/EtOAc 70/30): 0.85.

  aqueous solution was cooled to 0 o C, and acidified with 1 M HCl to pH = 1. The aqueous layer was then extracted with ethyl acetate (4 x 50 ml) and the combined organic layers were washed with brine, dried over MgSO4 and concentrated under vacuum to afford crude Boc-(R)-Ala-OH (+)-14 (1.90 g, 10.1 mmol) as white solid. The crude product was pure enough to be used in the next step without any purification.Crude yield: 90%TLC Rf (EtOAc): 0.44 1 H NMR (360 MHz, DMSO): δ = 1.21 (d, J = 7.3 Hz, 3H, H-6), 1.37 (s, 9H, H-1),3.91 (p, J = 7.4 Hz, 1H, H-5), 7.10 (d, J = 7.6 Hz, 1H, H-4).

  22 mmol) was hydrolysex in 4 h to afford Boc-(S,S)-GABA-(R)-Ala-OH (65 mg, 0.21 mmol) (crude yield 95%)Following the general procedure "V" peptide p2 was prepared from Boc-(S,S)-GABA-(R)-Ala-OH(44.60 mg, 0.15 mmol, 1eq.), DIPEA (50 µl, 2 eq.), HATU (60 mg, 1.05 eq.) in a mixture of DCM/DMF (3/1) (2 ml) and the corresponding TFA salt of Boc-(S,S)-GABA-(R)-Ala-OBn (58.0 mg, 0.15 mmol), DIPEA (0.155 ml, 6 eq.) in dry DCM (2 ml), over 24 h. The resulting yellowish crude solid dissolved in DCM (25 ml) and successively washed with a saturated bicarbonate solution (10 ml), brine (10 ml), a 1 M HCl solution (10 ml), then brine (10 ml). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting crude was purified by flash chromatography over silica (gradient eluent of 100/1 to 100/4 DCM/MeOH) to afford Boc-[(S,S)-GABA-(R)-Ala]2-OBn p2 as white solid (57.(400 MHz, CDCl3): δ= 1.37 (d, J = 7.1 Hz, 3H, H-13), 1.43 (s, 9H, H-1), 1.45 (d, J = 7.5 Hz, 1H, H-24), 1.68 -1.57 (m, 1H, H-7), 1.91 -1.80 (m, 1H, H-6), 2.07 -1.92 (m, 3H, 1H, Following the general procedure "IV" the benzyl ester of dipeptide p3 (80 mg, 0.20 mmol) was hydrolysex in 4 h to afford Boc-(R,R)-GABA-(R)-Ala-OH (60 mg, 0.20 mmol) (crude yield 100%)Following the general procedure "V" peptide p4 was prepared from Boc-(R,R)-GABA-(R)-Ala-OH (34.0 mg, 0.11 mmol, 1eq.), DIPEA (40 µl, 2 eq.), HATU (45 mg, 1.05 eq.) in a mixture of DCM/DMF (3/1) (2 ml) and the corresponding TFA salt of Boc-(R,R)-GABA-(R)-Ala-OBn(44.1 mg, 0.11 mmol), DIPEA (0.118 ml, 6 eq.) in dry DCM (2 ml), over 24 h. The resulting yellowish crude solid was washed with water and filtered on a sintered glass funnel (size 4), the residue was washed with ether, dried under vacuum then purified by flash chromatography over silica (gradient eluent of 100/1 to 100/4 DCM/MeOH) to afford Boc-[(R,R)-GABA-(R)-Ala]2-OBn as white solid (25.2 mg, 0.044 mmol).

3, 4

 4 Scheme a: Réactions photochirogéniques proposées en présence de β-CD, pouvant conduire au cis-[START_REF] Avalos | [END_REF]3 CB-GABA (en haut) et au cis-3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA (en bas) énantioenrichis

Figure a :

 a Figure a: Structure par diffraction aux rayons X du composé 5.

Figure b :

 b Figure b: Analogues benzyl (left), and carbamate (right) analogues of compound 1.

8

 8 Schema f: Synthèse de l'azapinone 8 à artir du caprolactame 6 Préparation et caractérisation du complexe β-CD / La β-CD et l'azépinone 8 sont solubles dans l'eau. Le complexe β-CD / azépinone 8 a été préparé en mélangeant une solution aqueuse de cette dernière avec une solution aqueuse de β-CD. Sous agitation à la température ambiante, un précipité blanc se forme pour conduire à une «suspension laiteuse» épaisse. Il est important de souligner qu'une concentration minimale d'environ 6 mM par rapport à la concentration de β-CD, est nécessaire pour que le précipité se forme; et plus la concentration du mélange est faible, plus le processus de précipitation est lent. Pour cette raison, le volume de la solution finale a toujours été choisi pour avoir une concentration juste inférieure à 15 mM, ce qui correspond à la solubilité maximale de la β-CD dans l'eau à 25 °C. L'apparition d'un précipité lors du mélange du lactame 8 avec la β-CD suggère la présence d'une interaction entre ces deux composés. Cela a été validé par l'analyse du précipité par diffraction aux rayons X sur poudre (PXRD). Les données obtenues pour le complexe β-CD / 8 étaient effectivement différentes de celles du lactame 8 et de la β-CD seules. Nous avons ainsi eu une indication supplémentaire de l'implication d'un véritable complexe, plutôt que d'un mélange physique de deux substances distinctes (Figure c).

Figure c :

 c Figure c: Spectre PXRD de l'azépinone 8 vs β-CD vs complexe

Figure d :

 d Figure d: Images MEB de la β-CD seule (en haut à gauche), de l'azepinone 8 seule (en haut à droite), du complexe β-CD/8 avant irradiation (en bas à gauche), et après irradiation (en bas à doite).

Figure

  Figure e: Job's plot de l'absorption UV (λ =254 nm) d'une solution β-CD:azepinone 8 à différents rapports molaires.

Figure f :

 f Figure f: Spectres RMN 1 H de la β-CD montrant la variation de déplacement chimique de H3 et H5 avec chaque addition d'azépinone 8

Figure

  Figure g: variation de 1/ΔδH3 en function de 1/[azepinone 8].

Figure h : 4 Entrée

 h4 Figure h: Agrandissement du spectre RMN ROESY à 600 MHz d'une solution 1:1 de β-CD/8 (15 mM in D2O, 40 °C) (en haut). β-CD (en bas à gauche)et lactame 8 (en bas à droite).

Figure i :

 i Figure i: Dédoublement par HPLC semi-préparative du N-Boc-cis-3,4 CB-GABA 12 racémique.

Figure j :

 j Figure j: Chromatogramme en HPLC analytique de (±)-13

Figure k :

 k Figure k: Chromatogramme des énantiomères (+)-13 (en haut) and (-)-13 (en bas) purs.

Figure l :

 l Figure l: Structure par diffraction aux rayons X du (+)-(3R,4R)-13.

Figure m :

 m Figure m: Structure cristalline du (+)-(3R,4R)-13 with the θ value.

Figure o :

 o Figure o: Les quatre peptides hybrides γ/α préparés, en série (S,S/R) (à gauche), en série (R,R/R) (à droite).

Figure p :

 p Figure p: Structure par diffraction aux rayons X du peptide p3 (enhaut), réseau de liaisons hydrogène intermoléculaires, vue de côté (à gauche), de face (au milieu), de dessus (à droite).

Figure q :

 q Figure q: Structure par diffraction aux rayons X du peptide p1 (enhaut), réseau de liaisons hydrogène intermoléculaires, vue de côté (à gauche), de face (au milieu), de dessus (à droite).

Figure r :

 r Figure r: Corrélations ROE caractéristiques sur le tetrapeptide p2 (en haut) montrant la présence de pseudocycles (en bas) à 10 atomes (bleu) et à 12 atomes (vert). la flèche bleu claire correspond à une corrélation ROE faible.

Figure s :

 s Figure s: Corrélations ROE caractéristiques sur le tetrapeptide p4 (en haut) montrant la présence de pseudocycles (en bas) à 9 atomes (orange) et à 7 atomes (rose). La flèche orange claire correspond à une corrélation ROE faible.\

2, 3

 3 photochimique.

  1:2, where two guest molecules are (totally or partially) included inside the same CD cavity;52 2:1, where two CD molecules encapsulate a single guest; 53 or a 2:2 complex made of 2 CD molecules and 2 Guest molecules.54

1.3.3 Literature synthesis of cis-and trans-3,4 CB-GABA

	2,3 CB-GABA. Conditions:
	a), hν, acetone, 9 h; b) (i) Na, NH3 (l), t-BuOH; (ii) Boc2O, DMAP, CH3CN; c) (i) LiOH,
	H2O, THF; (ii) H3O + ; d) 6 M NaOH, MeOH, Δ, 16 h e) (i) TFA, CH2Cl2; ii) Dowex-H +

Chapter 2: The Photochemical Reactivity of N- Allyl-N-(4-methoxyphenyl)acrylamide 2.1 Introduction

	Organic chemists are constantly on the lookout for new methodologies for the synthesis of
	compounds with biological significance. N-allylacrylamide derivatives have been studied
	for the construction of heterocyclic compounds with multiple stereocenters, of interest as
	drugs or bioactive reagents, using regioselective ring-closing metathesis reactions
	(RCM) 104 or palladium-catalyzed tandem cyclization reactions. 105 Enantioselective
	photochemical cycloaddition reactions of N-allylacrylamide derivatives have not been
	previously studied, but might constitute a "green" synthetic methodology for preparing
	heterocyclic compounds. Bicyclic lactams, such as the particular intermediate we target
	below, are of considerable synthetic interest in this respect.

3.2.2.1 Photoelectrocyclization of azepinone 8 in solution 3

  .2.2.1.1 Photoelectrocyclization of azepinone 8 in etherThe photochemical electrocyclization reaction of 8 was first performed in ether, as previously reported in the literature (See Scheme 37). 102 A solution of azepinone 8 in anhydrous diethyl ether was irradiated for 2-3 h with a 400 W mercury vapor lamp, in a

reactor fitted with a quartz filter and cooled by an external water bath. The choice of a quartz filter was made on the basis of a previous analysis of the UV absorbance spectrum of 8 in acetonitrile (0.11 mM), which showed a maximum absorbance at 269 nm, indicating

table 3 ,

 3

	Mass of dry	Mass of dry	Mass of dry	Mass of remaining	Mass of remaining
	β-CD used	azepinone 8	precipitating	azepinone 8 extracted	β-CD extracted
		used	solid complex	from the filtrate	from the filtrate
	1.04 g	100 mg	790 mg	30 mg	321 mg

show that about 70% by mass of β-CD and azepinone 8 are precipitating out in a 1:1 ratio.

Table 3 :

 3

Distributiuon of azepinone 8 and β-CD between the precipitate and the filtrate after complexation.

  The masses, summarized in table4, show that about 90% by mass of the starting azepinone 8 is precipitating with β-CD.

	Mass of dry	Mass of dry	Mass of dry	Mass of remaining	Mass of remaining
	β-CD used	azepinone 8	residue	azepinone 8 extracted	β-CD extracted
		used		from the filtrate	from the filtrate
	3.12 g	100 mg	2.16 g	10 mg	960 mg

Table 4 :

 4

Distribution of azepinone 8 and β-CD between the precipitate and the filtrate after complexation. 3.2.4.3.2 The Job's method of continuous variation:

Table 6

 6

	: The change in chemical shift (Δδ) of the azepinone 8 protons at 100%, 200%,
	700% of it relative to β-CD.

Table 7

 7

	shows the (φ, ψ) values that define the three types of

Table 7 :

 7 The (φ, ψ) values that define the three types of natural protein helices.

  10/12-, 139 12-, 140 12/10-, 141 14-, 133 and 18/20-helices. 142

Table 8

 8

			)
		50%	10%	Tetrapeptide p2
		DMSO-d6	DMSO-d6
	Δδ(NH4)	1.25	0.73
	Δδ(NH11)	1.25	0.73
	Δδ(NH15)	0.008	-0.02
	Δδ(NH22)	0.75	0.17
				Tetrapeptide p4
	Δδ(NH4)	1.42	0.81
	Δδ(NH11)	1.40	0.84
	Δδ(NH15)	0.39	0.27
	Δδ(NH22)	1.38	0.82

Table 8 :

 8 The results of the DMSO-d6 NMR titration experiment for tetrapeptides

p2, and p4.

1 H NMR (360 MHz, CDCl3) δ=

	1.39 (d, J = 7.1 Hz, 3H, H-6), 1.41 (s, 9H, H-1), 4.16-4.32 (m,
	1H, H-5), 4.44 (s, 2H, H-9), 5.16 (bs, 1H, H-4), 6.76 (bs, 1H, H-8), 7.42 -7.19 (m, 5H, H-Ar).
	13 C NMR (90 MHz, CDCl3): δ= 18.371 (C-6), 28.27 (C-1), 43.35 (C-9), 50.13 (C-5), 80.13
	(C-2), 127.43, 127.56, 128.66, (C-11, C-12, C-13), 138.10 (C-10), 155.59 (C-3), 172.63 (C-7).
	MP: 104-106 ºC.
	HR-MS: m/z calcd for [C15H22N2O3+Na] + 301.1523 ; found 301.1520.

  Avec une telle approche, la chiralité peut être induite par l'interaction intramoléculaire entre le centre chiral, agissant comme un «auxiliaire chiral», et le fragment réactif. La deuxième catégorie, la photochirogenèse supramoléculaire, peut être décrite comme une interaction hôte-invité entre le réactif, en tant qu'invité, et une molécule hôte qui fournit un environnement chiral pour que la réaction photochimique se déroule de manière asymétrique.

		une importance industrielle, en raison du besoin de molécules stéréochimiquement pures dans
		le domaine de la médecine et de la science des matériaux.
	NNT : 2018SACLS186	Au cours des dernières décennies, la synthèse asymétrique de composés organiques a été traditionnellement réalisée par des transformations catalytiques ou enzymatiques, et un large éventail de méthodes a été développé pour obtenir un contrôle stéréochimique sur diverses transformations chimiques. La photochimie s'est révélée être un outil polyvalent en synthèse organique et fait l'objet d'une attention encore plus grande aujourd'hui, car elle est considérée comme une méthode de préparation de nouveaux matériaux dans un contexte «respectueux de
		l'environnement», appelé chimie verte. Néanmoins, les transformations photochimiques
		asymétriques ne sont pas très largement étudiées. Le terme «photochirogenèse» est assez
		récent. Elle a été inventée en 1996 et est conceptuellement synonyme de «photochimie
		asymétrique» ou «photochimie chirale», l'accent étant mis sur la création d'une chiralité
		moléculaire via l'utilisation d'un photon.
		Yield: 82%
		TLC Rf (PE/EtOAc 1/3): 0.53
		1 H NMR (400 MHz, CDCl3): δ= 1.40 (d, J = 7.0 Hz, 1H, H-13), 1.43 (s, 2H, H-1), 1.51-1.62 (m, 1H, H-7) 1.78-1.90 (m, 1H, H-6), 1.90-2.03 (m, 1H, H-7), 2.24 (dd, J = 7.7, 14.5Hz, 1H, H-Introduction
		9), 2.28 -2.39 (m, 1H, H-6, overlapped with H-9), 2.45 (dd, J = 7.1, 14.5Hz, 1H, H-9), 2.76-La majorité des molécules naturelles et biologiquement actives ont une configuration bien
		2.87 (m, 1H, H-8), 4.18-4.30 (m, 1H, H-5), 4.41 (d, J = 6.8 Hz, 1H, H-16), 4.44 (d, J = 7.0 Hz, définie en lien avec leur fonction moléculaire. Il est étonnant de voir comment les organismes
		1H, H-16), 4.50 (p, J = 7.1 Hz, 1H, H-12), 5.19 (s, 1H, H-4), 6.44 (d, J = 6.4 Hz, 1H, H-11), vivants ont la capacité de choisir ou de biosynthétiser de manière sélective un seul énantiomère
		6.71 (s, 1H, H-15), 7.22-7.35 (m, 1H, H-Ar). d'une molécule donnée. Les chimistes cherchent depuis toujours à imiter les systèmes Ici, la chiralité est transférée d'une molécule à une autre par le biais d'interactions
		biologiques; ainsi, la synthèse asymétrique de composés organiques est devenue un terrain de intermoléculaires entre le substrat et une molécule voisine possédant certaines propriétés
		prédilection pour la recherche. La préparation énantiosélective de molécules revêt également chirales.

3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF]

GABA-OH (50.0 mg, 0.22 mmol), DIPEA (75 µl, 2 eq.), HATU (87 mg, 1.05 eq.) in a mixture of DCM/DMF (4/1) (2 ml), and the corresponding TFA salt of Boc-(R)-Ala-NHBn

(60.7 mg, 0.22 mmol)

, DIPEA (0.23 ml, 6 eq.) in dry DCM (24 ml), overnight. After workup the resulting crude was purified by flash chromatography over silica (1/3 PE/EtOAc) to obtain Boc-(S,S)-GABA-(R)-Ala-NHBn p5 as white solid (72.2 mg, 0.18 mmol).

La recherche en matière de photochirogénèse a fait de grands progrès quantitatifs et qualitatifs grâce aux nouvelles méthodologies développées pour contrôler les processus impliqués. Il est essentiel pour obtenir un contrôle stéréochimique d'une réaction photochimique, de s'assurer que la molécule « substrat » réside déjà dans un environnement chiral pendant l'étape d'excitation. À cet égard, la photochirogenèse peut être facilement divisée en deux catégories principales. Le premier comprend les réactions dans lesquelles la chiralité est transférée à une molécule réactive par la présence d'un substituant chiral, lié de manière covalente ou ionique au réactif.

C NMR (75 MHz, CDCl3): δ = 37.2 (C-6), 114.2 (C-3), 121.1 (C-1), 125.4 (C-2), 127.6 (C-

C NMR (75 MHz, CDCl3): δ=25.2 (C-4), 26.3 (C-3), 33.1 (C-1), 37.1 (C-6), 54.2 (C-2),

C NMR (90 MHz, CDCl3): δ = 21.2 (C-4), 27.5(C-3), 28.4 (C-8), 34.5 (C-9), 37(C-1), 46.8

Acknowledgments

First, I would like to express my gratitude to Pr. Mahmoud Faraj, the person who triggered my interest in organic chemistry, and Pr. Olivier Piva, for being reporters for my PhD

The asymmetric photocyclization of 4-(but-3-enyloxy)quinolone.

The same research group also utilized thiourea based molecules as templates to host, by the same logic, another enantioselective intramolecular [2+2] photocycloaddition reaction with ee up to 91% (see Scheme 9). 22 Scheme 9: The asymmetric photocycloaddition of a 2,3-dihydropyridone-5-carboxylate derivative. Scheme 32:The intramolecular [2+2]-cycloaddition N-allyl-N-benzylcinnamamide.

As far as we are aware, the molecular structure of 1 is the first to provide a formal test case for the competition between the two different photochemical reactivity types,

[2+2]-cycloaddition vs. 6π-electrocyclization. In the present case, the latter reaction mode prevails. Further addition of azepinone 8 up to 7 equivalents induces a further increase in the Δδ values up to 0.064 and 0.012 for H 3 and H5 respectively (see table 5). This increase is almost linear and no significant inflection point could be detected indicating that the complexation of azepinone 8 and β-CD is a dynamic process of fast exchange between the complexed and the free form of the two molecules relative to the NMR time scale, and the observed chemical shifts at a certain moment are the weighted average of the chemical shifts for the free and complexed form coexisting at that moment. 69 (69) Correia 2002. β-CD proton 1 2 3 4 5 6

Δδ (ppm) 0-100% 0.000 0.000 0.030 0.000 0.068 0.000 Δδ (ppm) 0-200% 0.000 0.000 0.043 0.000 0.087 0.000 Δδ (ppm) 0-700% 0.000 0.000 0.064 0.000 0.120 0.000 Scheme 41: Reduction of photoadduct 9 in water.

Analysis of the enantiomeric excess compound (±)-10

To determine the optimal separation conditions of the two enantiomers of (±)-10 on an analytical HPLC system, several tests were done and the most satisfying result was achieved with a normal phase chiral Lux Cellulose-2 column, using a 90/10 hexane/ethanol eluting mixture at 20 o C and a flow rate of 2 ml/min. Under these conditions, the two enantiomers of (±)-10 were separated with 42 sec of difference in retention time. Figure 32 shows a HPLC chromatogram of (±)-10. Scheme 46: Protection of 2-azabicyclo [3.2.0]heptan-3-one (±)-10 with a carbamate group.

Basic hydrolysis of (±)-11

A solution of (±)-11 in a 50/50 mixture of THF-H2O was stirred in the presence of an excess of LiOH at room temperature overnight (See Scheme 47). THF was then evaporated and the remaining aqueous solution was acidified with 0.5 M HCl. The crude product (±)-12 was extracted with ethyl acetate and purified by flash chromatography on silica. The N-Boc protected amino acid (±)-12 was obtained as a white solid with a yield of 71%.

Scheme 47: Basic hydrolysis of (±)-11.

Chiral HPLC resolution of N-Boc amino acid (±)-12

We first tried to resolve the N-Boc amino acid (±)-12 on an analytic chiral HPLC column. A solution of (±)-12 in acetonitrile was passed through a reversed phase chiral Lux Cellulose-3 column with a 15/85 acetonitrile/water (0.1% TFA) eluting mixture, at 30 o C and a flow rate of 1 ml/min. Under these conditions, the two enantiomers of (±)-12 were separated with a retention time difference of 1.5 min. Figure 49 shows a chromatogram of racemic (±)-12 eluted on an analytical HPLC.

Mixed β/γ-peptides

The β/γ-peptides form another class of hybrid peptides, which consist of alternating β-and γamino acids. β/γ-peptides are interesting due to their ability to adopt helical secondary structures that mimic those of the native α-peptides, such as the 13-or α-helix.

The Hofmann group studied via ab initio calculations the different possible helical structures which could be adopted by a β/γ-octapeptide composed of alternating β-Glycine and GABA. 117

The authors found ten distinct stable helical forms: 11-, 11/13-, 13/11-, 13-,15/16-, 18/17-, 20-, 20/22-, 22/20-and 22-helices. Out of the ten stable helical structures predicted by the Hofmann group, the 11/13-helix has been experimentally observed in solution and the 13-helix has been observed both in solution and in solid state.

Recently the Aitken group were the first to established the ability of β/γ-peptide composed of (1R,2R)-2-aminocyclobutane carboxylic acid (trans-ACBC) and GABA, to fold into a 9/8ribbon, with alternating 9-, 8-hydrogen bonding pseudocycles. 158

Examples of β/γ and applications

As mentioned above the β/γ-hybrid peptides are important because they are able to fold into a 13-helix similar to the natural α-helix. The Gellman group, 96 based on the Hoffman group calculations, 117 have designed a β/γ-hybrid peptide composed of (R,R)-trans-ACPC and ethylcis-2(aminocyclohexane)acetic acid ((R,R,R)-EtACHA). The hybrid hexapeptide adopted a regular 13-helix in solution as well as in solid state (see Figure 67). More recently the Aitken group designed an α/β/γ-hybrid peptide using the trans-ACBC as the key β-amino acid component and γ [START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] -amino acids as the γ-amino acid components. 159 The α/β/γhybrid peptide folded into a 12,13-helix in solution, (see Figure 68) which resembles the native α-helix, and was able to act as functional and selective α-helix-mimetic inhibitor of the p53/hDM2 interaction, validating the importance of designed peptide foldamers for applications in the field of protein-protein interactions.

Figure 68: The Aitken's group α/β/γ-hybrid peptide that folds into a 13-helix.

Mixed α/γ-peptides

Another class of hybrid peptides, α/γ-peptides, are composed of alternating α-and γ-amino acids. α/γ-peptides dimer units, with their proteinogenic α-amino acid component, may properly replace a dipeptide unit in the nonproteinogenic β-peptides. 117 This could open the door to a pool of foldamers having a secondary structure that resembles the folding pattern of β-peptides but with proteinogenic component needed for biological activity.

The Hofmann group studied via ab initio calculations the different possible helical structures which could be adopted by an α/γ-octapeptide composed of alternating glycine and GABA. 117

The authors found eleven distinct stable helical forms: 10-, 10/12-, 12/10-, 12-, 15/17-, 18-, 18/20-, 20/18-, 20-, 21/23-and 26-helices. They also suggested that α/γ-hybrid peptides have Most recently the Gopi's group reported a 12/10 folding pattern in α/γ-hybrid peptides composed of alternating alanine (Ala), or leucine (Leu), and 4-aminoisocaproic acid (Aic). 164 The authors were able to flip from a 12/10-into a 12-helical structure when the α-amino acid component was changed into the achiral 1-aminocyclohexane-1-carboxylic acid (Ac6c), thus presenting the influence of small structural variations in the α-amino acid structure on the overall helicity of an α/γ-hybrid peptide. (See Figure 74). Aside from the 12 and the 12/10-helical structuration, The Maillard's group, 165 prepared α/γhybrid peptide containing enantiomeric pure 4-amino(methyl)-1,3-thiazole-5-carboxylic acids

Synthesis of Boc-(S,S)-cis-3,4 CB-GABA/(R)-Ala-OBn peptides

The synthesis of Boc-[(S,S)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA-(R)-Ala]n-OBn peptides p1 and p2 was carried out following a convergent strategy, starting from the Boc-(S,S)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA-OBn enantiomer (-)-13 as the γ-amino acid partner, and the Boc-D-Ala-OBn (+)-15 as the α-amino acid partner (see Scheme 54).

Scheme 54: The convergent synthesis of Boc-[(S,S)-cis-3,4 CB-GABA-(R)-Ala]n-OBn peptides p1

and p2.

Synthesis of Boc-(R,R)-cis-3,4 CB-GABA/(R)-Ala-OBn peptides

In order to investigate the effect of reversing the stereochemistry of the cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA component on the three dimensional folding pattern of our α/γ-hybrid peptide, we prepared a di and a terrapeptide composed of alternating (R,R)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA and D-Alanine.

The synthesis of Boc-[(R,R)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA-(R)-Ala]n-OBn peptides p3 and p4 was carried out following a convergent strategy, starting from the Boc-(R,R)-cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA-OBn enantiomer (+)-13 as the γ-amino acid partner, and the Boc-D-Ala-OBn (+)-15 as the α-amino acid partner (see Scheme 55).

Scheme 55: The convergent synthesis of Boc-[(R,R)- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA-(R)-Ala]n-OBn peptides p3 and p4.

Conformations Relative abundance of each conformation [10-12-10] 9.3% [12-10] 3.5% [7,7-9] 6.4% [7,12-10] 25% [7,7-13-9] 44.4% [7,7-10] 5% [7-10] 5.8% [12-13] 0.6%

Table 9: The list of conformations for tetrapeptide p2 as predicted by the MCMM calculations

Even though the most abundant conformation is found to be the [7,7-13-9] (44.4%), the lowest energy conformer corresponds to the experimentally found [10-12-10]. The Figure 85 below

shows the hydrogen bonding pattern in the most abundant [7,7-13-9] and the lowest energy [10-12-10] conformers as calculated by MCMM. On the other hand, for the tetrapeptide p4 the most abundant conformation is found to be the [9-7,12] (27%), and the lowest energy conformer is the [9,12-9], this conformer is similar to the one recently reported by the Maillard group for α/γ-hybrid peptides containing enantiomeric pure ATCs (See Figure 75, and 77). 165,167 Such short range H-bonds are reminiscent of the Aitken group for homologous γ-peptides of cis-2,3 CB-GABA, which showed short range consecutive intramolecular 7-membered hydrogen bonds topology in dilute solution; 94 and for

N-(4-methoxyphenyl)-3-(phenylsulfonyl)propanamide [4]

To a solution of 3 (4.00 g, 18.69 mmol) in dry dichloromethane 25 ml, thionyl chloride was added (14 ml, 10 eq.). the reaction mixture was left to reflux overnight. The solvent along with excess thionyl chloride were distilled out under vacuum. The solid residue was left to dry under high vacuum then dissolved in 150 ml dry dichloromethane. The solution was cooled to 0 O C and p-anisidine (2.30 g, 1 eq.) was added followed by trimethylamine (2.5 ml 1.2 eq.). The reaction mixture was left to stir for 3 hours at 0 o C then washed with 1 molar HCl (2 x 20 ml), followed by brine (2 x 20 ml). The organic layer was dried over MgSO4 and concentrated to half under reduced pressure. To the resulting solution petroleum ether 300 ml was added to precipitate the desired product 4, which was filtered off over a sintered glass funnel, porosity 3.

The crude product was pure enough to be used in the next step. It could also be recrystallized from dichloromethane/ hexane to get clear crystals (5.06 g, 15.88 mmol).

Yield: 85%

TLC Rf (Hex/EtOAc 2/1): 0.25 -12), 166.9 (C-3).

MP: 118-119 ºC.

HR-MS: m/z calcd for [C16H17NO4S+Na] + 342.0770 ; found 342.0761

N-allyl-N-(4-methoxyphenyl)acrylamide [1]

To a solution of 4 (3.00 g, 9.4 mmol) in 100 ml dry THF, at 0 o C, potassium tertiary butoxide (1.04 g 1 eq.) was added. The reaction mixture was left to stir for 1 hour at 0 o C after which another equivalent of potassium tertiary butoxide was added followed by allyl bromide (0.8 ml, 1eq.) the reaction mixture was left to stir at 0 o C for 3-4 h. The THF was removed under vacuum and ethyl acetate (100 ml) was added. The organic layer was washed with brine (5 x 30 ml). then dried over MgSO4 and reduced under vacuum. The crude was purified by chromatography on silica (80/20 hexane/EtOAc) to obtain compound 1 as a clear oil. Diethyl ether was added to co-evaporate ethyl acetate and obtain a white solid (1.22 g, 5.6 mmol). Note that the product could polymerize if heated and left as an oil under high cavum.

Yield 60%

TLC Rf (PE/EtOAc 7/3): 0.54 A solution of 8 (200 mg 1.834 mmol) in diethyl ether (250 mL) was degassed with an argon stream in a cylindrical water-cooled reactor during 15 min, and then irradiated for 2 h with a 400 W medium-pressure mercury lamp fitted with a Quartz filter, while the reactor was cooled with an external ice bath. The solvent was evaporated under reduced pressure to obtain crude (±)-9 (195 mg 1.788 mmol). The crude product was pure enough and was not further purified Crude yield: 97%.

TLC Rf (Et2O): 0.11.

Photocyclization of azepinone 8 in water without β-CD:

A solution of azepinone 8 (500 mg, 4.58 mmol) in 50 ml of ultra-pure water was introduced in a quartz tube and irradiated inside a Rayonet for 15 h at 10°C. The reaction mixture was then lyophilized to obtain crude (±)-9 (356 mg, 3.26 mmol). The crude product was pure enough and

was not further purified. The spectral data were identical to those previously reported on page 145.

Crude yield: 71%.

Photoelectrocyclization of azepinone 8 in solid state without β-CD:

Azepinone 8 (30.00 mg, 0.275 mmol) was mechanically ground in mortar and introduced in a quartz tube. The tube was closed and set to rotate in a Rayonet in which it was irradiated for 7

h. The crude product was then completely dissolved in ultra-pure water and stirred overnight, without degassing, at room temperature, under dihydrogen atmosphere with 10% w/w Pd/C-10%. The catalyst was then filtered out and the product (±)-10 was extracted with ethyl acetate (12 x 25 ml). The organic layer was dried over Na2SO4 then concentrated under reduced pressure to obtain crude (±)-10 (12.22 mg, 0.110 mmol). The crude product was of enough purity and no further purification was done. Spectral data were identical to those previously reported.

Crude yield over 2 steps: 40%.

Photoelectrocyclisation in the presence

Photoelectrocyclisation of β-CD/azepinone 8 complex in hot water / reduction

Experiment A

According to general procedure "I" azepinone 8 (50.00 mg, 0.458 mmol) was complexed with 1 equivalent of β-CD (0.52 g, 0.458 mmol) in 31 ml of water. The suspension was transferred to a quartz tube, and then heated up to 45 °C using a temperature control system (heating cooling finger). The hot solution was then irradiated in the Rayonet for 2 hours at 45 °C. The resulting yellowish clear solution was transferred to a flask. The cycloadduct 4 was reduced and extracted from the aqueous phase with ethyl acetate (13 x 40ml). The organic phase was dried over Na2SO4 then concentrated under vacuum to obtain crude 10 (12.22 mg, 0.110 mmol). The product was of enough purity and no further purification was done. Spectral data were identical to those previously reported.

Crude yield: 40%. ee 0%.

Experiment B

According to general procedure "I" azepinone 8 (30.00 mg, 0.275 mmol) was complexed with 1 equivalent of β-CD (0.32 g, 0.282 mmol) in 19 ml of water. The suspension was cooled to 5 °C, and the cold suspension was irradiated for 2 h at 5°C. The resulting clear solution was subjected to reduction extracted with ethyl acetate (12 x 25 ml). The organic phase was dried over Na2SO4 then concentrated vacuum to obtain crude 10 (24.01 mg, 0.216 mmol). The crude product was of enough purity and no further purification was done. Spectral data were identical to those previously reported.

Crude yield: 79%. ee 38%.

Experiment C

According to general procedure "I" azepinone 8 (30 mg, 0.275 mmol) was complexed with 1 equivalent of β-CD (0.32 g, 0.282 mmol) in 19 ml of water the suspension was charged with extra 2 equivalents β-CD during the stirring time. The suspension was cooled to 5 °C, and the cold suspension was irradiated for 3.5 hours at 5 °C. The resulting clear aqueous solution was subjected to reduction and extracted with ethyl acetate (12 x 25 ml). The organic phase was dried over Na2SO4 then concentrated under vacuum to obtain crude 10 (23.02 mg, 0.207 mmol). The crude product was of enough purity and no further purification was done. Spectral data were identical to those previously reported.

Crude yield: 75%. ee 45%.

Photoelectrocyclisation of β-CD/azepinone 8 "3/1 complex" in solid state / Reduction

Experiment D

Mechanically ground:

One equivalent of azepinone 8 (30.00 mg, 0.275 mmol) was mechanically ground with β-CD (0.32g, 0.282mmol) in a mortar. The solid was transferred to a quartz tube which was set to slowly rotate while being irradiate inside a Rayonet. When no starting material can be observed by 1 H NMR (7 hours), the irradiated solid was dissolved in ultra-pure water (10 ml) and subjected to reduction. The aqueous phase was extracted with ethyl acetate (12 x 25ml). The organic phase was dried over Na2SO4and concentrated under vacuum to obtain crude 10 (21.30 mg, 0.191 mmol). The crude product was of enough purity and no further purification was done. Spectral data were identical to those previously reported.

Crude yield: 70%. ee 0-40%.

Photoelectrocyclisation of β-CD/azepinone 8 in powder form / Reduction: Experiment E

According to general procedure "II" azepinone 8 (50.00 mg, 0.458 mmol) was complexed with 1 equivalent of β-CD (0.52 g, 0.458 mmol) in 31 ml ultra-pure water. The suspension was then filtered on a sintered glass funnel (porosity 3) and washed with 10 ml of ultra-pure water. The uncomplexed azepinone 8 was extracted with ethyl acetate from the filtrate, and 20 mg of it could be recovered. The white complex was dried in air, finely crushed in a mortar and transferred into a quartz tube. The tube was closed and set to rotate inside a Rayonet in which it was irradiated for 20 hours. The irradiated solid was dissolved in ultra-pure water, reduced, and then extracted with ethyl acetate (13 x 40 ml). The organic phase was dried over Na2SO4 and concentrated under vacuum to obtain crude 10 (21.30 mg, 0.191 mmol). The product was of enough purity and no further purification was done. Spectral data were identical to those previously reported.

Crude yield: 71% (with respect to the remaining 30 mg in the complex). ee 36%.

Potoelectrocyclisation of β-CD/azepinone in a 1:1 film, made from a 15 mM suspension /Reduction

Experiment F

According to general procedure "II" azepinone 8 (50.00 mg, 0.458 mmol) was complexed with 1 equivalent of β-CD (0.52 g, 0.458 mmol) in 31 ml of water the suspension was then spread on a glass sheet and left to dry in open air for 3 days. The film that forms was irradiated in a

Rayonet for 5 hours. The irradiated solid was dissolved in ultra-pure water, reduced, and then extracted with ethyl acetate (13 x 40 ml). The organic phase was dried over Na2SO4 then concentrated under vacuum to obtain crude 10 (39.20 mg, 0.323 mmol). The product was of enough purity and no further purification was done. Spectral data were identical to those previously reported.

Crude yield: 77%. ee 41%.

Reduction Experiment G

According to general procedure "II" azepinone 8 (33.60 mg, 0.308 mmol) was complexed with 2 equivalents of β-CD (0.702 g, 0.619 mmol) in 41 ml of ultra-pure water. The suspension was then spread on a glass sheet and left to evaporate in open air for 3 days. The film that forms was irradiated in a Rayonet for 5 h. The irradiated solid was put in solution, reduced, and then extracted with ethyl acetate (12 x 30 ml). The organic phase was concentrated under vacuum to obtain crude 10 (26.34 mg, 0.237 mmol). The crude product was of enough purity and no further purification was done. Spectral data were identical to those previously reported.

Crude yield: 77%. ee 42%. Crude yield: 78%.

Potoelectrocyclisationof β-CD

e.e 41%.

MP:117 ºC.

HR-MS: m/z calcd for [C11H19NO4+Na]+ 252.1206 ; found 252.1212 (-2.4 ppm).

2-((1R,2R)-2-((tert-butoxycarbonyl)amino)cyclobutyl)acetic acid [(+)-17]

To a solution of Boc-(R,R)-cis- 34 GABA-OBn (+)-13 (154 mg, 0.48 mmol) in 40 ml of 20/5 DCM/ethyl acetate was added 10% Pd-C (10% w/w) and the black suspension was stirred under dihydrogen atmosphere. When no starting material could be detected by TLC the reaction mixture was filtered (through a PVDF 0.45 μm filter) and concentrated under reduced pressure to afford Boc-(R,R)-cic-34 GABA-OH (+)-17 in quantitative yields. The acid (+)-17

was pure enough to be used in the coupling reactions without any purification.

The analysis is identical to that reported in literature 102

2-((1S,2S)-2-((tert-butoxycarbonyl)amino)cyclobutyl)acetic acid [(-)-17]

Same procedure than for compound (+)-17

The analysis is identical to that reported in literature 102

General procedure "III" for the synthesis of the dipeptides (p1, p3, p5)

Step one

In flask A, Boc-(R)-Ala-OBn, or (Boc-(R)-Ala-NHBn for peptide p5) (1eq.), was dissolved in dry DCM (4 ml), TFA (30 eq.) was then added, and the reaction mixture was stirred at room temperature. When no Boc-(R)-Ala-OBn starting material could be detected by TLC, the reaction was considered complete and the DCM was evaporated under reduced pressure. The excess TFA was co-evaporated with DCM several times, and then left under high vacuum for 2 h. the resulting crude TFA salt was then dissolved in dry DCM, and DIPEA (6 eq., sufficient quantity to reach PH 9) was added and the mixture was left to stir at room temperature.

Step two

In flask B, Boc-cis-34 GABA-OH (1eq.) was dissolved in a mixture of DCM/DMF (4/1), DIPEA

(2 eq.) was then added followed by HATU (1.05 eq.) and the resulting mixture was stirred for 10 min at room temperature during which the color changes from light to dark brown.

Step three

The content of flask A was then transferred to flask B and the resulting reaction mixture was stirred at room temperature for 24 h. The reaction mixture was concentrated under reduced pressure and toluene was added to co-evapourate DMF. The crude solid was dissolved in Ethyl acetate, or DCM (for p5), (50 ml) and successively washed by a saturated bicarbonate solution (20 ml), brine (20 ml), a 1 M HCl solution (20 ml), then brine (20 ml). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure, and the resulting crude was purified accordingly.

General procedure "IV" for the hydrogenolysis of the benzyl ester prodecting group

To a solution of Boc-peptide-OBn in 25 ml DCM/Ethyl acetate (20/5), 10% Pd-C (10% w/w) (125 ml/mmol) was added and the black suspension was stirred under dihydrogen atmosphere;

when no Boc-peptide-OBn starting material was detected by TLC the reaction was considered complete. The reaction mixture was filtered through a (PVDF 0.45 μm filter), concentrated under reduced pressure to afford Boc-peptide-OH which was of sufficient purity to be used in the coupling reactions.

General procedure "V" for the synthesis of the tetrapeptides (p2, p4, p6)

Step one

In flask A, Boc-peptide-O(or NH)Bn (1 eq.) was dissolved in dry DCM (2 ml), TFA (30 eq.)

was then added, and the reaction mixture was stirred at room temperature. When no Bocpeptide-O(or NH)Bn starting material could be detected by TLC, the reaction was considered complete and the DCM was evaporated under reduced pressure. The excess TFA was coevaporated with DCM several times, and then left under high vacuum for 2 h. the resulting crude TFA salt was then dissolved in dry DCM, and DIPEA (6 eq., sufficient quantity to reach PH 9) was added and the mixture was left to stir at room temperature.

Step two

In flask B, Boc-peptide-OH (1eq.) and DIPEA (2 eq.) was dissolved in a dry mixture of DCM/DMF (3/1). The content of flask A was then transferred into flask B, then HATU (1.05 eq.) was introduced to the reaction mixture, which was left to stir at room temperature for 24 h during which a precipitate forms. The solvent was removed under reduced pressure and the remaining DMF was co-evaporated with toluene. And the resulting crude was purified accordingly.

MP: 155-157 ºC.

HR-MS

Index

A-Determination of the binding constant Kb for a 1:1 complex

The equilibrium constant, K, for a complex formation between a host (H) and a guest (G) can be formulated from the general equilibrium reaction:

when the stoichiometry of the formed β-cycldextrin/ guest complex is 1:1, the desired equilibrium or binding constant Kb is given by:

Where [G] is the concentration of free guest, [H] is the concentration of free host, i.e. (β-CD),

and [HG] the concentration of the formed host/guest complex.

Because the complexation process is a dynamic equilibrium, the individual concentration of each species is not readily measurable. Instead of measuring separate concentrations, a wellknown approach to determine the molecular binding constant, is to generate the binding curve and then to extract K via a regression analysis. 65 To apply this approach, we need to define the total guest concentration [H]t, which is always equal to the concentration of the host in the free form added to the concentration of the Host in the bound form, equation ( 2):

In addition, we have the expression that indicates the fraction of the host that is bound Xb, which is the concentration of the bound host at a given time divided by the total concentration of the host at that time:

Substituting equations ( 2) and ( 3) in equation ( 1) and rearranging for Xb, we obtain equation ( 4)

which is known as the binding isotherm: 66

In order for equation ( 4) to be useful, Xb needs to be described in terms of a measurable property whose magnitude varies as a function of the amount of Host bound, i.e. complex formation. 65 One of the most common properties whose variation could be studied as a function complex formation is the NMR shift of specific protons located on the host or the guest molecules.

So we first need to relate Xb to the chemical shift variation of a specific proton on the host or the guest. To be able to establish such a relation one should be aware that the rates of complex formation and dissociation are usually faster than the NMR time scale, and the observed signals are actually an average between the chemical shifts in the free and the bound form of the host and the guest molecules. [START_REF] Dodziuk | Cyclodextrins and Their Complexes[END_REF] Thus if δH is the chemical shift of the monitored proton in the free state, and δHG is the chemical shift of the same proton in the bound state, then the observed chemical shift, δobs, is given by: 66

In equation ( 5) Xf and Xb represent the fraction of the host in the free and bound form respectively. Now since Xf + Xb = 1 equation ( 5) could be written as

The expression δobs -δH represents the variation in the chemical shift of the monitored proton Δδ, and the δHG -δH represents the maximum change in chemical shift which is attained at saturation Δδsat. using this notation equation 6' becomes:

Substituting equation 6'' into equation 4 we get:

The reciprocal of equation 7 gives:

A plot of 1/Δδ vs [G] establishes a straight line whose slop is directly related to the value of K (see Figure 88). B-In a subsequent control reaction, we observed that up to half the amount of cyclobutene product (±)-9 was lost when an equimolar solution of this compound and β-CD in water was lyophilized for 3 days.

Summary in French (Résumé français)

Nouvelles Transformations Enantioselective Induites par une Cyclodextrine: Applications pour la Preparation de Blocs de Construction Moleculaire d' Intérêt Biologique

La configuration absolue du composé 10

Toutes les expériences décrites ci-dessus ont conduit au même énantiomère majoritaire de 10.

Pour déterminer la configuration de cet énantiomère majoritaire, un échantillon de 10 (avec ee = 42%) a été transformé en dérivé N-Boc 12 du cis- 3,[START_REF] Gotor | Asymmetric Organic Synthesis with Enzymes[END_REF] CB-GABA, selon une procédure déjà cyclization of N-allyl-N-(4methoxyphenyl)acrylamide, we only obtained a δlactam resulting from a 6π; electrocyclization, whereas the electrocyclization of 1,3-Dihydro-2H-azepin-2-one allowed access to a 45% enantiomerically enriched bicyclic γlactam precursor of (+)-cis-3,4CB-GABA. The second strategy was based on a racemic synthesis of N-Boc-cis-3,4CB-GABA followed by a separation of the two enantiomers using a semi-preparative HPLC fitted with a chiral column. This allowed access to optically pure (-) and (+)-cis-3,4CB-GABA, on a gram scale. Furthermore, the enantiomerically pure (-) and (+)cis-3,4CB-GABA, were used to synthesize, and fully characterize two series [the (S,S/R) and the (R,R/R)] of short diasteriomeric hybrid γ/α-peptides composed of alternating cis-3,4CB-GABA and D-Alanine. Analysis of the conformational behavior of the dipeptides from both series by X-Ray diffraction on a single crystal, showed no intramolecular interactions but rather an array of intermolecular hydrogen bonding between the dipeptide molecules. On the other hand, a series of 1D and 2D NMR experiments showed that the tetrapeptide of the (S,S/R)-series could attain a 12/10 helical structuration, whereas its diasteriomeric analog of the (R,R/R)-series, displayed evidence of an unprecedented 7/9 folding pattern in solution.