Les glucosinolates (GLs) sont des métabolites secondaires très présents dans la famille des Brassicaceae, une famille de végétaux connus dans l’alimentation humaine pour leurs propriétés gustatives et aussi pour leurs effets chimio-préventifs. La myrosinase (E.C. 3.2.1.147) est la seule enzyme capable d’hydrolyser les GLs. Nous nous sommes intéressés aux produits obtenus lors de cette dégradation - les isothiocyanates (ITCs) mais aussi à la fonction thiohydroximate, et plus particulièrement à la synthèse de nitrones thiofonctionnalisées. La synthèse d’analogues d’ITC ainsi que de certains de leurs thio-conjugués a conduit à une large gamme de molécules permettant de cerner les implications chimio-préventives et les potentiels en bio-activité (notamment contre Plasmodium falciparum). Partant d’observations analytiques relatives à certains GLs comme la glucoraphénine, il a été découvert qu’un thiohydroximate pouvait présenter un caractère nucléophile comparable à celui d’une oxime dans un processus cyclisant du type Grigg. Nous avons dès lors cherché à développer de nouvelles voies d’accès à un système fonctionnel très peu décrit dans la littérature : les N-oxydes de thioimidate (OTI). Une étude de réactivité d’un OTI-modèle a révélé des comportements originaux, en particulier dans des réactions de couplage pallado-catalysées qui ouvrent un accès à des nitrones cycliques originales et peu aisées à préparer par des voies plus classiques. / Glucosinolates (GLs) are widespread secondary metabolites in Brassicaceae, a family of vegetables known in human feeding for their tastes and also for their chemo-prevention effects. Myrosinase (E.C. 3.2.1.147) is the only enzyme able to hydrolyze GLs. We have been interested in products acquired during this degradation - isothiocyanates (ITCs) but also in the thiohydroximate function, and more particularly in the synthesis of thiofunctionalized nitrones. The synthesis of ITC analogues as well as some of their thio-conjugates has led to a broad range of molecules allowing to surround chemo-preventive involvements and potentials in bio-activity (namely against Plasmodium falciparum). Starting from analytical observations related to some GLs such as glucoraphenin, it has been disclosed that a thiohydroximate could display a nucleophilic character comparable to that of an oxime in a Grigg-type cyclic process. We have developed new pathways to a scarcely described functional system : thioimidate N-oxides (OTI). Reactivity studies on a model-OTI have revealed an atypical behaviour, notably in palladium-catalyzed coupling reactions : those open the way to original cyclic nitrones which are not easily available via standard methods.

Ces travaux centrés essentiellement sur la fonction thiohydroximate s’articulent selon trois thèmes : (1) les acides thiohydroxamiques, (2) la synthèse et (3) l’étude de réactivité des N-oxydes de thioimidate (TINOs), avec pour objectif l’étude d’une enzyme : la S-glucosyltransférase (S-UGT). La S-UGT est une enzyme clé dans la formation des glucosinolates. Les travaux menés ont tout d’abord été axés sur la synthèse d’acides thiohydroxamiques, substrats de la S-UGT, pour l’étude cinétique de l’enzyme. Dans ce but, nous avons envisagé différentes méthodes et avons pu obtenir les molécules ciblées, permettant ainsi d’obtenir des résultats préliminaires quant à la cinétique de l’enzyme. D’autre part, la synthèse d’iminosucres, inhibiteurs potentiels de l’enzyme, a été envisagée par une double fonctionnalisation d’un N-oxyde de thioimidate (TINO) sur charpente pyranose. Nous avons donc développé une méthode robuste d’accès aux TINOs à partir de différents pyranoses. Le couplage de Liebeskind-Srogl a permis d’accéder aux nitrones. Des additions-éliminations sur les TINOs nous ont donné accès à des N-oxydes d’amidine et à de nouveaux TINOs. Les réactions de réduction et de débenzylation des TINOs ont également été explorées. Des tentatives de cycloaddition 1,3-dipolaire sur une nitrone ont été effectués en vue d’obtenir à des isoxazolidines, précurseurs d’iminosucres Alpha, Alpha-disubstitués. Les résultats concernant la synthèse d’inhibiteurs de la S-UGT restent préliminaires. Toutefois, après déprotection, les différentes molécules synthétisées durant cette thèse pourront être envisagées comme inhibiteurs de glycosidases. / This thesis is articulating into three topics: (1) synthesis of thiohydroxamic acids, (2) the synthesis and (3) the study of the reactivity of thioimidate N-oxides (TINOs). The purpose of these works is the study of the enzyme: S-glycosyltransferase (S-UGT). The enzyme S-UGT is an essential enzyme for the glucosinolate formation.The starting point of these works was focused on the synthesis of thiohydroxamic acids. These products are the substrates of the enzyme S-UGT and could be used for the study of the enzyme kinetic. The second point is to use TINOs anchored on pyranose backbones as precursors of iminosugars which are potential inhibitors of the enzyme S-UGT. An efficient methodology was developed to access a large panel of TINOs from different pyranoses. Liebeskind Srogl coupling allowed us to obtain nitrones. Addition-elimination on TINOs gave us amidine N-oxides and new TINOs. Reduction and debenzylation reactions on TINOs were also explored. 1,3-Dipolar cycloaddition attempts on a nitrone were performed to obtain isoxazolidines which are precursors Alpha, Alpha-disubstituted iminosugars. The results recording the synthesis of S-UGT inhibitors remain preliminary. However, after deprotection, the different molecules, synthesised during this thesis, will be tested as glycosidase inhibitors.

This thesis entitled “Design and Access to Disallowed and Unusual Conformers of Peptides in Crystals and in Solution: Structural Consequences of the Imidate and Thioimidate Isosteres for the Peptide bond” is divided into eight chapters. Imidate Modification The range of disallowed dihedral angles for residues in peptides is governed by their local steric and electrostatic clashes. Rare tolerances of violations in these angles are attributed to distortions in both local and global bond characteristics of the peptides. Discerning the origins of such disallowed angles and the consequent distortions in body of the peptides is essential, for a complete understanding of the protein fold, to improve the crystal database for validation of rare but acceptable residue conformations and for validation and improvement of theoretical models that evaluate the interactions that define the Ramachandran space. Unlike for the ordered secondary structures such as β-sheets α-helices and β-turns currently there are no models for residues constrained in disallowed folds. We reasoned that residues may be stabilized in disallowed folds in peptides if a neighbouring group and The range of disallowed dihedral angles ( , ψ) for residues in peptides is governed by their hence its local unfavorable clashes can be selectively modified to a motif that favors such space Steric clashes of the type H•••Xi±n involving the backbone amide hydrogen (H) contribute to ~60% of disallowed ,ψspace. Conversion of an amide to an imidate (A→I) will remove the corresponding H and hence the steric clashes related to it in peptides. Importantly, this will introduce an H-bond acceptor N (of imidate) in place of an H-bond donor NH (of amide), which will allow formation of unusual H-bonding interactions between the imidate N and the neighbouring Hs and hence constrain residues in otherwise inaccessible dihedral angles. The conversion of A→I is challenging owing to difficulties in selective synthesis, stability and purification of the imidate motif. We address all these concerns by the selective conversion of a backbone amide in peptides to the relatively stable cyclic 5,6-dihydro-4H-1,3-oxazine imidate isostere, by an intra¬molecular nucleophilic cyclo-O-alkylation reaction. Chapter 1:SectionB: Autocyclo-O-Alkylation of N-(3-Bromopropyl)amides into 2-Alkyl-5,6-Dihydro-4H-1,3-Oxazinehydrobromides We are describing the reactivity of N-(3-bromopropyl)amides that are precursors for 2-peptide-5,6-dihydro-4H-1,3-oxazine. The starting materials, 3-bromopropylamides, were synthesized in good yields by coupling the corresponding carboxylic acids and anhydrides with 3-bromopropylaminehydrobromide using standard mixed anhydride peptide coupling protocol. N-(3-bromopropyl)-acylamides are unstable during the isolation. Time-dependent 1H NMR of all the acetamides revealed that they underwent clean auto-cyclization to form the corresponding 2-alkyl-5,6-dihydro-4H-1,3-oxazine hydrobromides following first order rate. The salts were easily isolated in high purity by trituration of the mixtures with ether. The t1/2 of autocycliation of decreased upon increase in electron density on the R-carbon. Notably, the tert-butyl substituent cyclized significantly faster than acetamide which have enolizable hydrogens at the R-carbon. Thus, the cyclization rate is affected predominantly by the inductive effect of the R-carbon substituents. The formamide remained stable and unchanged due to the poor electron-donating ability of hydrogen. Chapter 1: Section C: Intramolecular Hydrogen Bond Assistance Improves Autocyclization in N-(3-Bromopropyl)amides The autocyclisation do not go to 100% completion. This is because the released hydrobromic acid quenches the nucleophilicity of amide carbonyl oxygen. In order to scavenge hydro bromic acid, we used 1 equivalent of DIEA base is acting only acid scavenger which conformed by unaffecting the reaction rate upon increasing equivalents of DIEA. We found that autocyclisation of N-(3-bromopropyl)amides rates in peptides involved in intramolecular backbone H-bonding interactions improve the autocyclization rates significantly than unstructured (random coil) peptides. Even with in the ordered structures the rate depends on the proximity of H-bonding distances as well as the H-bond acceptor strength. Half-life of autocyclisation in various peptide secondary structures are determined from time variant 1H NMR studies performed at 60 mM peptide concentration in CDCl3 at 32 oC. Chapter 2: Section A: Synthesis and Isolation of 5,6 Dihydro-4H-1,3-Oxazine Containing Peptidomimetics We have introduced 5,6-Dihydro-4H-1,3-oxazine as the imidate isostere at C-terminus of a number of peptides through NaH (base) mediated intramolecular cyclo-O-alkylation of N-(3-bromopropyl)amides. The amide to imidate (A→I) modification reaction is faster (1-5.5 h), Exhibiting no electronic and structural effects under these conditions. The side product NaBr can be easily separated by filtration through celite. No side products were observed and there is no need of further purification to get pure 1,3-oxazines in quantitative yields in all the peptidomimetics. Using this synthetic protocol we have synthesised a variety of 1,3-oxazine containing peptide analogues including aliphatic, branched aliphatic, polar side chains and larger peptides. We show that the retention of configuration at Cαof peptides during the base mediated cyclo-O-alkylation reaction. that the C5i.structures are more populated at Aib due to operation of The Thorpe-Ingold effect. The strength of hydrogen bonding interaction in C5i structure is similar to those of the highly buried backbone hydrogen bonding interaction found in the middle of a model 310-helical peptide as indicated by DMSO titration experiments. Chapter 3: Section A: Consequences of "Disallowed" Conformations on Constrained β-Turn Peptides Here we are describe the consequence of disallowed conformations the on a C-terminus of a type-II β-turn. We choose stereochemically constrained Type-II β-turn Pro-Aib dipeptide analogue which is the ideal model to mirror the structural effects of introducing the A→I modification at the C-terminus. The imidate containing peptidomimetic crystallised in dichloromethane and hexane mixture. Analysis of crystal structure revealed that Aib NH is involved in 3-centered H-bonding interactions with the N of oxazine and N of proline. This constrains Aib in a conformation that is natively disallowed to it. The (, ) angles of Aib residue fall in the (180,0) region which is strictly disallowed for natural peptides due to steric clashes involving the back bone amide N-H. More importantly there are two C•••O interactions which are accomidated in the crystal structures. Both oxygen‟s were place in staggered orientation of the Pro oxygen (OPro) between the two β-CH3 groups of Aib, which is again strictly disallowed in natural peptides due to strong C•••Oi-1 hard sphere clashes. However no vdW space violations are observed between these atoms. Chapter 3: Section B: Conformational Effects of “Disallowed Aib on a 310-Helical peptide In order to investigate the origins and consequences of “disallowed” conformations on a folded helical peptide body, the conformationally stable peptide sequence Boc-Leu1-Aib2-Ala3-Leu4-Aib5-Ala6-Phe7-Aib8-OMe (310-helix-OMe)was chosen which is known to adopt 310-helix in crystal structure. Analyses of the ROESY spectra, DMSO titration experiments, and CD spectra of 310-helix-OMe and its Oxa analogue reveal that their solution conformations are identical to those of the crystal structure of 310-helix-OMeSix sequential i+3→i intramolecular backbone H-bonds stabilize the 310-helical peptide fold in both peptides in solvents of varying polarity. The N-terminal and central segments of the helical molecules are quite structurally rigid and are not deformed. The presence of the disallowed Aib*8 residue in Oxa analogue has a clear conformational effect mainly on the residue Phe7. It looks like the Phe7 amide H is involved in shielding, the Aib*8 amide H through a bifurcated hydrogen bonding interactions with the nitrogen of oxazine and carbonyl oxygen of Ala6 residue. Maximum structural distortion effect on the registers closest to the putative imidate bond. Our results show that “disallowed folds need not denature order in the peptide fold”. Chapter 4: Synthetic Methods for Introducing the A → I Modification anywhere along the Peptide Chain Here we describe the incorporation of imidate isostere in the middle of any peptide sequence. In Oxa selectivity is towards 5-exo-cyclo-O-alkylation in 1 : 4. In Thi it is towards 6-exo-cyclo-S-alkylation in 3 : 1 ratio. This is because of better nucleophile of sulphur (S). We saw that Thi is stable to peptide coupling, N-and C-terminus protection, deprotection conditions and can be easily incorporated in middle of peptide. Chapter 5: Section A: Cis-trans Isomerism in the X-Pro Peptide Bond In tertiary amides like X-Pro peptides having high propensity to access cis conformations due to similar environment in both cis and trans around the Cof X. X-Pro peptide bonds, constrained in s-cis conformations are prevalently found in the turn regions of peptides with the residue „X‟ in the i+1position and Pro at the i+2position of the β¬turn. These types of turns are termed as the type VI β-turns. For better understanding of the molecular recognition at specific cis X-Pro peptide bonds in biological events, the structure and dynamics of various constrained cis X-Pro peptide bond analogues with varying steric and electronic perturbations have been studied. Many models have been developed for stabilizing cis conformer by perturbation of molecular recognition surface of proline by employing steric and electronic interaction. In biological functions proline molecular recognition surface and cis X-Pro peptide bond more important. There is need of novel method for stabilizing X-Pro peptide bond in cis conformer without modifying the pyrolidine ring in proline. Chapter 5: Section B: Biasing the cis/trans Equilibrium in X Pro Peptides using Reverse ni → ni-1 * Interactions Here we present our findings that peptidomimetics containing the 5,6-dihydro-4H-1,3¬oxazine (Oxa) and 5,6-dihydro-4H-1,3-thiazine (Thi) functional groups at the C-terminus of Pro selectively and remotely stabilize the s-cis rotamers of the preceding pyrrolyl (Xaa-Pro) 3° amide bonds, while conserving these recognition elements. The cis/trans equilibrium of Xaa-Pro peptide bonds is shifted significantly in favor of the satirically disfavored cis isomers in these peptidomimetics (upto ~90%). We also provide evidence for the influence of an unusual n→ πi-1 * interaction in the cis, and the n)(n repulsion in the trans, conformers of these molecules to beat the origin of such the origin of such cis stabilization. Chapter 6: Steric Interactions in the cis Piv-Pro Peptide Bond The inaccessibility of cis Piv-Pro rotamer in any peptide is believed to be because the steric clashes between substituents on CX and CPro are unavoidable in this conformer. Here we access the cisPiv-Pro conformer in crystal structure of Piv-Pro-Aib-OMe and that it is sufficiently flexible to undergo bond distortions and avoid all steric clashes between substituents on CPiv and CPro . It is however the unavoidable distortions in the dihedral angle of the Prothe cisPiv-Proconformer. The cisPiv -Pro conformer is indeed accessible, if such distortions are accommodated in the peptide. Chapter 7: Steric and Electronic Interactions in the cis Isomer of Piv-Pro Peptide Bond in Solution We have studied the electronic and steric interactions and the conformational equilibrium in two sets of homologous peptides, X-Pro-Aib-OMe (which contain Aib) and X-Pro-NH-Me, where X is acetyl, propionyl, isobutyryl and pivaloyl, in solvents of varying polarities consisting of carbontetrachloride, chloroform or dimethylsulfoxide, by means of their 1H and 13C-NMR, and FT-IR spectra. Formation of n * interactions between the carbonyls that flank the Aib residue, influences the alleviation of steric interactions that are believed to preclude access to the cis conformer of the Piv-Pro peptide bond. The cis Piv-Pro conformer is observable in the Aib containing peptides, at ambient conditions by FT-IR and at temperatures as low as 273 K by NMR. We estimate that the steric interactions contribute < 0.5 kcal/mol to the conformational free energy of X-Pro peptide bond isomerism, irrespective of the steric bulk on the acyl (X) group. The relative strengths of intramolecular hydrogen bonding interactions involving the X-Pro peptide motif in different conformers of these peptides influence their relative conformational stabilities. Chapter 8: Remote Effect of Oxa and Thi Functional Groups on cis-trans Isomerism at X-Pro Peptide Bonds The C5a interaction at Pro residue occurs in the transition states for the intramolecular acid catalysis of cis → trans isomerization in peptidyl prolyl isomerases (PPIs) and enables the decrease in transition energy barrier for the isomerization process. We show that the NPro….HAib interactions in C5a structures can be remotely effected in order to control in equilibrium constant values of the cis/trans isomerism (Kc/t) in X-Pro¬Aib-Oxa and Thi containing peptides. By this method we observed improvement in Kc/t values from 0.18 in esters to 0.56 in Thi and 0.66 in Oxa containing peptides. Analyses of the ROESY spectra, DMSO titration experiments, variable temperature experiments and FT-IR spectra of R-CO-Pro-Aib-Oxa (R = Me, Et, iPr) and its Thi analogues reveals that both interactions (C5a and C5i) are persistent in cis and trans conformers of this peptidomimetics. (for structural formula pl. see the abstract.)