This study involved the isolation, characterization, quantification and evaluation of the enantiomeric purity of linalool in the essential oil of aromatic species Lippia alba (Mill.) NE Brown (chemotype linalol-1,8-cineole), Ocimum basilicum L., Pelargonium graveolens L'Herit, Cinnamomum camphora Nees and Eberm var. linaloolifera Fujita, Aniba rosaeodora Ducke and Coriandrum sativum L., and carvone and limonene in the essential of Lippia alba (chemotype carvone-limonene). The quantification of monoterpenes was performed by GC-FID method for the internal standard. The analytical curves for quantification were linear in the range 1.0 to 10.0 mg mL-1 and showed appropriate values of correlation coefficients (0.996 to 0.999). The linalool was found with 91.38% (w/w) in essential oil of A. rosaeodora, 84.00% in C. camphora, 79.25% in L. alba, 79.00% in C. sativum, 73.33% in O. basilicum and 13.60% in P. graveolens. The carvone showed 58.13% and limonene showed 31.71% (w/w) of essential oil of L. alba. The enantiomeric purity analysis performed by GC-FID, using a column with enantioselective phase (β-cyclodextrin) and co-injection with commercial patterns, revealed the presence of the enantiomer (S)-(+)-linalool in L. alba and (R)-(-)-linalool in O. basilicum and C. camphora. The two enantiomeric forms of linalool were found in the P. graveolens, A. rosaeodora and C. sativum species. In the L. alba species (chemotype carvone-limonene) was observed a single enantiomeric form (R)-(-)-carvone and limonene showed the enantiomeric forms (S)-(-)-limonene and (R)-(+)-limonene, with predominance of the latter. The linalool was isolated from the essential oil of L. alba by TLC and subjected to analysis of GC-MS, GC-FID, 1H and 13C NMR and [α]D, which elucidated its structure and stereochemistry. The (S)-(+)-linalool isolated from L. alba (> 99%) was used to obtain the analytical curve. This isolation was necessary due to unavailability of acquire the (S)-(+)-linalool commercially pure. / Este estudo envolveu isolamento, caracterização, quantificação e avaliação da pureza enantiomérica do linalol nos óleos essenciais das espécies aromáticas Lippia alba (Mill.) N. E. Brown (quimiotipo linalol-1,8-cineol), Ocimum basilicum L, Pelargonium graveolens L Herit, Cinnamomum camphora Nees e Eberm var. linaloolifera Fujita, Aniba rosaeodora Ducke e Coriandrum sativum L. e da carvona e do limoneno no óleo essencial de Lippia alba (Mill.) N. E. Brown (quimiotipo carvona-limoneno). A quantificação dos monoterpenos foi realizada por CG-DIC pelo método do padrão interno. As curvas analíticas para a quantificação foram lineares no intervalo de 1,0 a 10,0 mg mL-1 e apresentaram valores adequados de coeficientes de correlação (0,996 a 0,999). O linalol foi encontrado com 91,38% (m/m) no óleo essencial de A. rosaeodora, 84,00% em C. camphora, 79,25% em L. alba, 79,00% em C. sativum, 73,33% em O. basilicum e 13,60% em P. graveolens. A carvona apresentou 58,13% e o limoneno 31,71% (m/m) no óleo essencial de L. alba (quimiotipo carvona-limoneno). As análises de purezas enantioméricas realizadas por CG-DIC, utilizando uma coluna com fase enantiosseletiva (β-ciclodextrina) e a co-injeção com padrões comerciais, revelaram a presença do enantiômero (S)-(+)-linalol em L. alba e do (R)-(-)-linalol em O. basilicum e em C. camphora. As duas formas enantioméricas do linalol foram encontradas nas espécies P. graveolens, A. rosaeodora e C. sativum. Na espécie L. alba (quimiotipo carvona-limoneno) foi observada uma única forma enantiomérica (R)-(-)-carvona e o limoneno apresentou as formas enantioméricas (S)-(-)-limoneno e (R)-(+)-limoneno com predominância desta última. O linalol foi isolado do óleo essencial de L. alba por CCD e submetido à análise por CG-EM, CG-DIC, RMN de 1H e 13C e [α]D, as quais elucidaram sua estrutura e estereoquímica. O (S)-(+)-linalol isolado de L. alba (> 99%) foi utilizado na obtenção da curva analítica. Esse isolamento foi necessário devido à indisponibilidade em adquirir o (S)-(+)-linalol comercialmente puro.

This thesis describes the synthesis of novel ligands that include enantiomerically pure acetal and oxazoline moieties. These ligands are utilised in a number of metalmediated asymmetric syntheses. All asymmetric acetals and pyridine based acetals are synthesised in good yield in a single step from their corresponding enantiomerically pure diols. C2 symmetric bisacetals are investigated as ligands in the organolithium and Grignard additions to benzaldehyde with promising results. C2 symmetric bisacetals and pyridine based acetals are tested for their ability to induce asymmetry in copper(l) catalysed cyclopropanation of styrene using ethyl diazoacetate and the lanthanide(lII) catalysed Diels-Alder cycloaddition involving Danishefsky's diene with little success. Enantiomerically pure phosphinooxazoline ligands are available in good yield in two steps from their corresponding enantiomerically pure aminoalcohols. Enantiomerically pure acetal substituted pyridines and phosphinooxazoline ligands are considered in the rhodium (I) catalysed hydrosilylation of ketones. Reaction conditions for the more successful phosphinooxazoline ligands are optimised. Using these ligands a range of enantiomerically enriched alcohols is presented in good yield and enantiomeric excess. Novel phosphinooxazoline ligands are applied to the palladium(O) catalysed allylic substitution reaction with excellent enantioselectivities of the substitution product.

No description available.

Understanding the relation between amino acid sequences and protein structures is one of the most important problems in modern molecular biology. However, due to the complexities in the protein structure, this task is really daunting. Hence, understanding the structural features of proteins and the rules of folding is central to the design of novel and more effective biomaterials. With the inception of the de novo design of synthetic mimetics for protein structural elements, the study of designed peptides is a subject of intense current research. The de novo design of polypeptide structures provides insights into the factors that govern the folding of peptides and proteins. The rational design of synthetic peptide models for secondary structural motifs in proteins depends on the ability to control the polypeptide chain stereochemistry. An approach, which seems to be useful, is the introduction of constrained genetically coded amino acids like Proline or the introduction of non-protein constrained amino acids like Aib which are capable of restricting the range of available backbone conformations of the polypeptide chain. The use of such residues would then permit the design of well defined and intended structural motifs like the β-turns which serve as chain reversal areas of the polypeptide chain. Templates incorporating multiple repeats of such conformationally constrained residues would in turn further enhance the choice of conformational parameters for the polypeptide chain towards folding. Crystal structure determination of the oligopeptides by X-ray diffraction gives insight into the specific conformational states, modes of aggregation, hydrogen bond interactions and solvation of peptides. Precise structural analysis and good characterization of geometrical parameters and stereochemical details of these molecules provide valuable inputs for peptide design and are indispensable for exploring strategies to design peptide sequences which serve as synthetic mimics for folding motifs in proteins. Many of the above points have been investigated in this thesis which incorporates study of designed peptides containing heterochiral and homochiral diproline segments followed by protein database analysis. This thesis reports results of x-ray crystallographic studies of twenty two (22) oligopeptides containing heterochiral or homochiral diproline segments. Apart from the crystal data, protein database analysis has also been carried out to investigate what actually is found in nature. Given in brackets are the compound names used in the thesis for the peptides solved. 1) Piv-DPro-LPro-NHMe ( DPPN ) [C16H27N3O3 ] 2) Piv-DPro-LPro-LVal-OMe ( DPPV ) [C21H35N3O5 . 0.09 H2O] 3) Piv-DPro-LPro-LPhe-OMe ( DPPF ) [C25H35N3O5 . H2O] 4) Piv-DPro-LPro-DAla-OMe ( DPPDA ) [C19H31N3O5] 5) Piv-LPro-DPro-LAla-OMe ( PDPA ) [C19H31N3O5] 6) Piv-DPro-LPro-LVal-NHMe ( DPPVN ) [C21H36N4O4 . H2O] 7) Piv-DPro-LPro-LLeu-NHMe ( DPPLN ) [C22H38N4O4 . 0.34H2O] 8) Piv-DPro-LPro-LPhe-NHMe ( DPPFN ) [C25H36N4O4 . H2O] 9) Piv-DPro-LPro-Aib-NHMe ( DPPUN ) [C20H34N4O4] 10) Piv-DPro-LPro-DAla-NHMe ( DPPDAN ) [C19H32N4O4] 11) Piv-DPro-LPro-DVal-NHMe ( DPPDVN ) [C21H36N4O4 .1.43 H2O] 12) Piv-DPro-LPro-DLeu-NHMe ( DPPDLN ) [C22H38N4O4 . H2O] 13) Piv-LPro-DPro-LAla-NHMe ( PDPAN ) [C19H32N4O4] 14) Piv-LPro-DPro-LVal-NHMe ( PDPVN ) [C21H36N4O4] 15) Piv-LPro-DPro-LLeu-NHMe ( PDPLN ) [C22H38N4O4 . H2O] 16) Piv-LPro-DPro-LVal-OMe ( PDPVO ) [C21H35N3O5 . H2O] 17) Racemic mixture of Piv-DPro-LPro-DVal-NHMe + Piv-LPro-DPro-LVal-NHMe ( PPVVN ) [C21H36N4O4 . 0.74H2O] 18) Racemic mixture of Piv-DPro-LPro-DLeu-NHMe + Piv-LPro-DPro-LLeu-NHMe ( PPLLN ) [C22H38N4O4 . H2O] 19) Racemic mixture of Piv-DPro-LPro-DPhe-NHMe + Piv-LPro-DPro-LPhe-NHMe ( PPFFN ) [C25H36N4O4 . 2 H2O] 20) Piv-LPro-LPro-LPhe-OMe ( PPFO ) [C25H35N3O5 . 0.5 H2O] 21) Piv-LPro-LPro-LVal-NHMe ( PPVN ) [C21H36N4O4 . H2O] 22) Piv-LPro-LPro-Aib-NHMe ( PPUN ) [C20H34N4O4. H2O] Results from the X-ray crystallographic analysis of the above peptides provided substantial information regarding role of diproline templates on the folding of the polypeptide chain. The thesis is divided into the following eight chapters : Chapter 1 gives a general introduction to the stereochemistry of polypeptide chains and the secondary structure classification: helices, β-sheets and β-turns. This section also provides a brief overview of the use of non standard and D-amino acids into peptide design. Discussions on DProline, puckering states of the Proline ring, diproline segments and racemic mixtures of peptides are also presented. A brief discussion on X-ray diffraction and solution to the phase problem is also given. Chapter 2 describes the structural characterization in crystals of the five following designed peptides: Piv-DPro-LPro-NHMe (DPPN), Piv-DPro-LPro-Xxx-OMe [Xxx = LVal (DPPV); LPhe (DPPF); DAla (DPPDA)] and Piv-LPro-DPro-LAla-OMe (PDPA) containing the heterochiral diproline segment with an aim towards understanding the directive influence of short range interaction on polypeptide folding. Except PDPA, in all the structures, a type II’ β-turn was observed at the DPro-LPro segment with the formation of a 4→1 intramolecular hydrogen bond between the atoms of the polypeptide backbone. In PDPA, the expected type II β-turn occurred at the LPro-DPro segment. Thus, the DPro-LPro segment preferably adopts a type II’ β-turn conformation when present at the C-terminus which is mimicked by the methyl ester group. The use of pivalyol group at the N-terminus is to ensure the trans geometry of the peptide bond between pivalyol and the first Proline. Crystal parameters DPPN: C16H27N3O3; P21; a = 10.785(1) Å, b = 15.037(1) Å, c = 11.335(1) Å; β = 109.96(1)°; Z = 4; R = 0.0388, wR2 = 0.1047. DPPV: C21H35N3O5 . 0.09 H2O; P212121; a =10.676(1) Å, b = 16.608(1) Å, c = 39.887(1) Å, Z = 12; R = 0.0688, wR2 = 0.1701. DPPF: C25H35N3O5 . H2O; P21; a = 9.538(1) Å, b = 10.367(1) Å, c = 13.102(1) Å; β = 93.04(1) °; Z = 2; R = 0.0504, wR2 = 0.1455. DPPDA: C19H31N3O5; P21; a = 11.269(1) Å, b = 9.945(1) Å, c = 18.550(2) Å; β = 97.46(1)°; Z = 4; R = 0.0563, wR2 = 0.1249. PDPA: C19H31N3O5; P212121; a = 9.043(1) Å, b = 10.183(2) Å, c = 23.371(1) Å; Z = 4; R = 0.0753, wR2 = 0.1603. Chapter 3 describes the crystal structures of the four following designed peptides containing the heterochiral diproline segment followed by a L-residue or an achiral amino acid residue like Aib : Piv-DPro-LPro-Xxx-NHMe [Xxx = LVal (DPPVN); LLeu (DPPLN); LPhe (DPPFN) and Aib (DPPUN)]. In the first three peptides the DPro-LPro segennt adopts a type II’ β-turn conformation with the formation of a type I β-turn at the LPro-Xxx segment. The peptide backbone overall therefore adopts a consecutive β-turn structure. When the L-amino acids at the C-terminus are replaced by the achiral amino acid Aib, the overall folded structure adopted by the peptide backbone still remains unchanged with the formation of a consecutive β-turn. All the structures are stabilized by two intramolecular 4→1 hydrogen bonds between the C=O group and the nitrogen atom of the polypeptide backbone. Crystal parameters DPPVN: C21H36N4O4 . H2O; P21; a = 9.386(1) Å, b = 12.112(1) Å, c = 10.736(1) Å; β = 99.53(1) °; Z = 2; R = 0.0528, wR2 = 0.1337. DPPLN: C22H38N4O4 . 0.34H2O; P21; a =9.231(1) Å, b = 17.558(1) Å, c = 15.563(1) Å; β = 91.94(1) °; Z = 4; R = 0.0555, wR2 = 0.1422. DPPFN: C25H36N4O4 . H2O; P212121; a = 10.473(1) Å, b = 15.980(1) Å, c = 15.994(1) Å; Z = 4; R = 0.0620, wR2 = 0.1826. DPPUN: C20H34N4O4; P212121; a = 10.571(2) Å, b = 11.063(1) Å, c = 18.536(1) Å; Z = 4; R = 0.0578, wR2 = 0.1256. Chapter 4 describes the crystal structures of the seven designed peptides containing heterochiral diproline segment. Three of these contain sequences of the type DPro-LPro-DXxx [DXxx = DAla (DPPDAN); DVal (DPPDVN); DLeu (DPPDLN)] and three contains the enantiomeric peptides of the ones that are mentioned earlier in sequences of the type LPro-DPro-LXxx [LXxx = LAla (PDPAN); LVal (PDPVN); LLeu (PDPLN)]. In order to investigate the effect of the C-terminal protecting group, a final peptide Piv-LPro-DPro-LVal-OMe (PDPVO) was crystallographically characterized. All the peptides containing the DXxx residues adopted different backbone conformations. For DAla, a structure simultaneously having a β-turn and an α-turn was obtained which is the first example in designed peptides of an isolated α-turn. In the case of DVal, an open / extended structure devoid of any intramolecular hydrogen bonding was obtained whereas for DLeu, type II β-turn occurred at the LPro-DLeu segment instead of the expected type II’ turn at the DPro-LPro segment. In the case of enantiomeric peptides, all the three peptides adopted folded structures with exact mirror image conformation being generated for LAla and nearly identical mirror image conformation in the case of LLeu. The enantiomeric peptide of DVal which contained LVal residue following the diproline segment also adopted a folded conformation with the formation of type II β-turn at the LPro-DPro segment as expected. X-ray crystallographic characterization of PDPVO resulted in the peptide adopting an overall extended / open structure. Thus, the chirality of the C-terminal residue seems to have a profound effect on the conformation of the heterochiral diproline segments. The role of the C-terminal protecting group cannot also be undermined. Crystal parameters DPPDAN: C19H32N4O4; P1; a = 5.964(1) Å, b = 9.354(1) Å, c = 9.961(1) Å; α = 75.44(1), β = 78.90(1) °, γ = 77.04(1); Z = 1; R = 0.0728, wR2 = 0.1528. DPPDVN : C21H36N4O4 .1.43 H2O; P212121; a = 8.744(8) Å, b = 11.609(1) Å, c = 23.577(2) Å; Z = 4; R = 0.0625, wR2 = 0.1856. DPPDLN : C22H38N4O4 . H2O; P212121; a = 10.531(3) Å, b = 11.659(3) Å, c = 20.425(6) Å; Z = 4; R = 0.0444, wR2 = 0.1239. PDPAN: C19H32N4O4; P1; a = 5.964(1) Å, b = 9.354(2) Å, c = 9.961(2) Å; α = 75.44(1), β = 78.90(1) °, γ = 77.04(1); Z = 1; R = 0.0745, wR2 = 0.1572. PDPVN : C21H36N4O4; P212121; a = 9.743(1) Å, b = 11.423(1) Å, c = 21.664(3) Å; Z = 4; R = 0.0803, wR2 = 0.1899. PDPLN : C22H38N4O4 . H2O; P212121; a = 10.462(4) Å, b = 11.572(4) Å, c = 20.262(7) Å; Z = 4; R = 0.0968, wR2 = 0.2418. PDPVO : C21H35N3O5 . H2O; P212121; a = 8.784(4) Å, b = 11.587(5) Å, c = 23.328(1) Å; Z = 4; R = 0.0888, wR2 = 0.1465. Chapter 5 describes the crystal structures of the three designed peptides containing racemic mixtures [Racemic mixture of Piv-DPro-LPro-DVal-NHMe + Piv-LPro-DPro-LVal-NHMe (PPVVN); Racemic mixture of Piv-DPro-LPro-DLeu-NHMe + Piv-LPro-DPro-LLeu-NHMe (PPLLN); Racemic mixture of Piv-DPro-LPro-DPhe-NHMe + Piv-LPro-DPro-LPhe-NHMe (PPFFN)] having the heterochiral diproline segment in their sequences and three peptides having a homochiral diproline segment [Piv-LPro-LPro-LPhe-OMe (PPFO); Piv-LPro-LPro-LVal-NHMe (PPVN); Piv-LPro-LPro-Aib-NHMe (PPUN)]. The inability of the pure enantiomers to crystallize in the case of Phe (chapter 4) invoked the use of peptide racemates for obtaining a crystal state conformation for the said compound. In all the cases, the L-enantiomer of Xxx crystallized in the asymmetric unit. A type II β-turn was obtained in the case of PPVVN at the LPro-DPro segment and a type II’ β-turn was obtained for PPLLN at the DPro-LLeu segment. in the case of Phe, an open structure devoid of any intermolecular hydrogen bonding an very similar to DPPDVN (chapter 4) was obtained. In the case of homochiral diproline segment containing peptides, PPFO crystallized with two molecules in the asymmetric unit, both of which adopted a type VIA1 hydrogen bonded β-turn conformation with a cis peptide bond between the diproline segment. In the case of Valine (PPVN) however, a structure devoid of any intramolecular hydrogen bonding was obtained. In the final peptide PPUN, a type II β-turn conformation is observed at the LPro-Aib segment. The analysis revealed that the hydration of the peptide can cause dramatic changes in its backbone conformation. In homochiral LPro-LPro sequences, the tendency to form hydrogen bonded turns competes with the formation of semi-extended polyproline structures. The results also emphasize the subtle role of sequence effects in modulating the conformations of short, constrained peptide segments. The possibility of trapping distinct conformational segments of the diproline segments in crystals by generating racemic centro-symmetric crystals in which packing effects may be appreciably different from those observed in the crystals of individual pure enantiomeric peptides has been clearly exploited in this chapter to obtain a crystal in the case of Phe. These results suggest that the energetic differences between these states is small. Conformational choice can therefore be readily influenced by environmental and sequence effects. Crystal parameters PPVVN: C21H36N4O4 . 0.74H2O; C2/c; a = 36.667(17) Å, b = 10.092(5) Å, c = 13.846(6) Å; β = 107.27(1) °; Z = 8; R = 0.1317, wR2 = 0.3141. PPLLN: C22H38N4O4 . H2O; P21/c; a = 10.555(1) Å, b = 11.687(1) Å, c = 20.108(2) Å; β = 95.47(1) °; Z = 4; R = 0.0761, wR2 = 0.2034. PPFFN: C25H36N4O4 . 2 H2O; P21/c; a = 8.883(5) Å, b = 18.811(10) Å, c = 16.033(9) Å; β = 96.28(1) °; Z = 4; R = 0.1218, wR2 = 0.2848. PPFO : C25H35N3O5 . 0.5 H2O; P212121; a = 10.199(1) Å, b = 20.702(2) Å, c = 23.970(2) Å; Z = 8; R = 0.0716, wR2 = 0.1901. PPVN : C21H36N4O4 . H2O; P212121; a = 9.454(1) Å, b = 11.119(1) Å, c = 23.021(2) Å; Z = 4; R = 0.0551, wR2 = 0.1587. PPUN: C20H34N4O4. H2O; P21; a = 6.276(1) Å, b = 14.011(2) Å, c = 12.888(1) Å; β = 96.80(1) °; Z = 2; R = 0.0475, wR2 = 0.1322. Chapter 6 describes the pyrrolidine ring puckering states of the Proline residue present in diproline segments in the peptides solved in this thesis, the Cambridge structural database (CSD) [only acyclic diproline containing peptides have been taken into account] and in a non-redundant dataset of proteins in the Protein Data Bank (PDB). The five membered pyrrolidine ring of Proline can be best characterized in terms of the following five endocyclic torsion angles χ1, χ2, χ3,χ4 and θ. Using various values of these endocyclic torsion angles the following puckering states were identified : [1] Cγ-exo (A) [2] Cγ-endo (B) [3] Cβ-exo (C) [4] Cβ-endo (D) [5] Twisted Cγ-exo-Cβ-endo (E) [6] Twisted Cγ-endo-Cβ-exo (F) [7] Planar (G) [8] Cα-distorted (H) [9] Twisted Cβ-exo-Cα-endo (I) [10] Cδ-endo (K) [11] N-distorted (L) [12] Twisted Cδ-endo- Cγ-exo (N). In the case of peptides solved in this thesis for heterochiral diproline segments, the Cγ-exo / Cβ-exo (AC) combination turns out to more preferred than the other combinations. The Cγ-endo / Cγ-endo (BB) state is the second most populated state. The overall investigation of Proline rings in peptides show that the states Cγ-exo (A), Cβ-exo (C) and Twisted Cγ-endo-Cβ-exo (F) are the most preferred states of occurrence of the pyrrolidine ring conformation. In the case of proteins, the overall percentage distribution of various combinations indicates that the AA (Cγ-exo / Cγ-exo), AE (Cγ-exo / Twisted Cγ-exo-Cβ-endo) and FF (Twisted Cγ-endo-Cβ-exo / Twisted Cγ-endo-Cβ-exo) categories are the most preferred combinations. For Proline rings in proteins, the states Cγ-exo (A), Twisted Cγ-exo-Cβ-endo (E) and Twisted Cγ-endo-Cβ-exo (F) are the most preferred states of occurrence of the pyrrolidine ring conformation. Chapter 7 describes the analysis of diproline segments in a non-redundant dataset of proteins In this chapter, the possible conformational states for the diproline segment (LPro-LPro) found in proteins taken from a non-redundant dataset has been investigated an identified with an emphasis on the cis and trans states for the peptide bond between the diproline segment. The occurrence of diproline segments in type VIA1 turns (cis Pro-Pro peptide bond) and other regular secondary structures like type III β-turns and α-helices has been studied. This has been followed up by the amino acid distribution flanking the diproline segment and the conformation adopted by Xaa-Pro and Yaa-Pro segments in proteins. It is observed that for cis Pro-pro peptide bond, the conformation adopted by the first Proline lies in PII region whereas the second Proline inevitably adopts a conformation in the Bridge region, leading to the formation of the type VIA1 β-turn structure. But in the trans case, the conformation adopted by the first Proline is overwhelmingly populated in the PII (Polyproline) and right-handed α-helical region. For position i+2, the major conformation adopted by Proline is P II and α with a substantial amount of occurrences in Bridge and the C7 (γ-turn) region. The analysis also reveals that the cis-cis configuration of the peptide bond is very rare when considering the diproline segment. With a cis-trans peptide linkage, PII-PII conformation is the most stable and favoured conformation for the Pro-Pro segment in proteins. With trans peptide bond linkage between the Proline residues, α- α and PII-Bridge conformations are equally likely for the diproline segment. The population in trans-cis and cis-trans states are comparable indicating that the energy differences between these states is small. However, trans-trans is the most populated state with a percentage occurrence of 85.43%. The analysis and comparison of conformational states for the Xaa-Pro-Yaa sequence reveals that the Xaa-Pro peptide bond exists preferably as the trans conformer rather than the cis conformer. The same is valid for Pro-Yaa segment, with the cis conformer being populated to even lesser extent. The data shows that α- α, PII-α, PII-PII and extended-PII are the most populated states for Xaa-Pro and Pro-Yaa segments as compared to PII-PII and PII-α and states observed for the Pro-Pro segment. Chapter 8 describes the analysis of single and multiple β-turns in a non-redundant dataset of proteins. The analysis on β-turns in proteins has shed a new light into the propensity values for amino acid residues at various positions of β-turns which in certain cases have undergone appreciable change in values than previously observed. One of the other notable feature of the analysis is the fact that the data displays a higher occurrence of unprimed β-turns of type I and type II as compared to their primed counterparts of type I’ and type II’ as previously observed. In fact, the results show that type I β-turn is the highest occurring turn both in isolated as well as in consecutive β-turn examples. The analysis of multiple β-turns in proteins has revealed many new categories like the (I,I+1,I+3), (I,I+2,I+3) and combination of turns which can be used for the design of the loops, especially in the case of β-hairpins. Among the multiple turns, double turns occur more frequently than the other consecutive turns like triple and quadruple turns. It is also important to note that the number of examples of a hydrogen bonded turn being followed by a hydrogen bonded turn is very less with type IV turn preceding a primed turn in most of the cases. Thus, the data available from consecutive β-turn analysis and the type-dependent amino acid positional preferences and propensities derived from the present study may be useful for modeling various single and consecutive turns, especially in designing loop regions of β-hairpins.

<p>Enzymes are useful tools as specific analytical reagents. Two different analysis methods were developed for use in the separate fields of protein science and organic synthesis. Both methods rely on the substrate specificity of enzymes. Enzyme catalysis and substrate specificity is described and put in context with each of the two developed methods.</p><p>In <strong>paper I </strong>a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage.</p><p>In paper<strong> II </strong>and <strong>III</strong>, three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase,<em> Candida antarctica</em> lipase<strong> </strong>B<strong> </strong>and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD⁺. The conversion and enantiomeric excess of the sample could be calculated from the relative differences in absorbance.</p>

La compréhension des mécanismes de transport et de dégradation des pesticides émergents est primordiale pour prédire leur devenir dans l’environnement. Les zones humides peuvent intercepter des eaux de ruissellement ou des souterraines contaminées par les pesticides et les traiter par le biais de processus de rétention et de dégradation, encore peu connus. Dans une approche multi-échelles, trois zones humides recevant des eaux polluées par les pesticides ont été utilisées comme des « laboratoires naturels » pour étudier le devenir de pesticides couramment utilisés. Cette thèse souligne l’influence des conditions hydrologiques et redox sur la distribution des pesticides au sein des différents compartiments des zones humides ainsi que sur leur potentiel de dégradation. Alors que les études à grande échelle fournissent des informations intégratives sur la dissipation et la rétention des pesticides en lien avec le développement de la végétation, les études à petite échelle utilisant des techniques innovantes telles que les analyses isotopiques et énantiomériques permettent l’exploration des processus moléculaires de dégradation des pesticides. / A mechanistic understanding of transport and degradation processes of modern agricultural pesticides, including chiral pesticides, is critical for predicting their fate in the environment. In agricultural landscapes, wetlands can intercept pesticide-contaminated runoff or groundwater and improve water quality through various retention and degradation processes, which remain unknown. In a downscaling approach, three different wetlands receiving agricultural runoff were used as ‘natural laboratories’ to investigate the fate of widely used pesticides. Overall, our results showed that dynamics of hydrological and redox conditions largely influenced pesticide sorption mechanisms and their distribution over time within wetland compartments, thereby controlling degradation processes. While large-scale studies provide integrative information on pesticide dissipation and distribution patterns with respect to wetland functioning, small-scale investigations using novel methods such as isotope and enantiomer analyses characterize underlying molecular processes governing pesticide degradation.

The thesis entitled "Pushing the Limits of NMR Sensitivity and Chiral Analysis: Design of New NMR Methods and Bio-molecular Tools" consists of six chapters. The research work reported in this thesis is focused on the development of novel chemical and NMR methodological approaches for enantiomeric analysis and mea- surement of residual dipolar couplings (RDCs), and the development of sensitivity enhanced slice selective NMR experiments for obtaining pure shift 1H spectra and the measurement of scalar couplings. The thesis is divided into two parts. The Part I comprises chapters 2-4, where the enantiomeric analysis is discussed, which includes newly developed chiral reagents, two new weak chiral aligning media and design of novel NMR techniques. Part II comprises chapters 5 and 6, which discusses new sensitivity enhanced slice selective NMR techniques. Chapter 1 gives a general introduction to NMR and the problems investigated in the remaining chapters of the thesis. The chapter starts with a brief discussion on the introduction, advancements and general applications of NMR, discussion is also given on the NMR approaches for enantiomeric analysis both in isotropic and anisotropic phases and the measurement of RDCs, including the benefits and limitations associated with each approach. The chapter sets the tone by discussing limitations of the existed NMR enantiomeric approaches and slice-selective techniques, and builds the bridge for the rest of the chapters by addressing these limitations. The chapter also introduces slice selective experiments, their benefits over other conventional methods and limitations. Additional introductory notes are also given on some related concepts. Part I : NMR Chiral analysis and RDCs measurements Chapter 2 discusses chiral sensing properties of RNA nucleosides and their utility as chiral derivatizing agents for the enantio-discrimination of 1o-amines using one dimensional 1H NMR. A three component protocol has been proposed for the complexation of nucleosides with amines, which is rapid, economical and provides maximum diastereomeric conversion. The chiral differentiating ability of nucleosides are examined for different amines based on the 1H NMR chemical shift differences between the diastereomers (∆δ R, S ). Enantiomeric differentiation has been observed at multiple chemically distinct proton sites. It is observed that adenosine and guanosine exhibit large chiral differentiation (∆δ R, S ) due to the presence of a purine ring. The comparison of the diastereomeric excess (de) measured by NMR with those of the gravimetrically prepared ratios are in excellent agreement with each other confirming the robustness of these RNA nucleosides in discriminating primary amines. Chapter 3 establishes the smooth connectivity with the chapter 2 by discussing the limitations of the enantiomeric discrimination using NMR in isotropic solutions. This chapter discusses two new water compatible aligning media that were developed based on self-assembling strategy of small bio-molecules. The self-assembled folic acid, and the binary mixture of 50-GMP and guanosine are introduced as two novel weak aligning media. The properties of these low ordered media have been systematically studied for their easy preparation, physical parameter dependent tunability of their degree of alignment, mesosphere sustainability over a broad range of temperature and the concentration of the ingredients, and the phase reproducibility. The applications of both these new media are demonstrated for chiral and pro-chiral discrimination and also for the measurement of RDCs. Both these liquid crystalline media could be tuned to very low degree of alignment (order parameter of the order of 10−4), which provides simple first order spectra of molecules aligned in them, the analysis provide order dependent NMR spectral parameters. The 50-GMP:guanosine orienting medium can be prepared in less than 1 hour, and has been demonstrated to be an ideal medium for the determination of RDCs that are used as restraints in the structure calculations of small molecules. Chapter 4 describes 1H NMR spectral complexity in isotropic and anisotropic phases and its consequences on enantiomeric analysis. In circumventing such problems, new NMR techniques have been developed and the spin dynamics involved in the designed sequences are discussed. The newly developed 2D 1H NMR experimental method termed as RES-TOCSY, and its applicability for resolving R and S enantiomeric or diastereomeric peaks of all the coupled proton spins in isotropic phase is discussed. The utility of the developed method is demonstrated in diverse situations, such as, for suppressing impurities peaks, resolving the severely overlapped peaks and unraveling the peaks masked due to severe line broadening when metal complexes are used as chiral auxiliaries. The advantages and limitations of the method over other methods available in the literature are discussed and the significant advantage of the present method is illustrated by spectral comparison with J-resolved experiment. The appli- cation of the method for the accurate measurement of enantiomeric excess has also been demonstrated. The chapter also introduces another NMR experimental technique developed for resolving enantiomeric peaks and complete unraveling of R and S spectra in anisotropic phase. The developed 2D NMR method is cited in the literature as CH-RES-TOCSY. In addition to spectroscopic visualization of R and S spectra, the method also yields C-H RDCs. The applicability of the new experiment has been demonstrated on a chosen example. The wide utility of the method has also been demonstrated for the assignment of symmetric cis- and trans- isomers. Part II : Sensitivity Enhancement of Slice selective NMR Experiments Chapter 5 describes applications of slice selective NMR experiments over conven tional NMR methods and their limitations as far as the sensitivity of signal detection is concerned, especially in low concentrated samples. The chapter introduces the implementation of Acceleration by Sharing Adjacent Polarization (ASAP) technique in slice selective experiments. It is convincingly demonstrated that ASAP helps in reducing inter scan relaxation delay and consequently permits acquisition of more number of scans in a given time, resulting in the gain in signal enhancement by a factor of two. The pulse sequences have been suitably designed for obtaining the pure shift 1H spectra and in G-SERF experiment for the measurement of 1H-1H couplings, both with significantly enhanced signal intensities. Chapter 6 describes new sensitivity enhanced slice selective NMR methods for mea- surement of scalar couplings. A new experiment has been developed which is named as Quick G-SERF (QG-SERF). It is a 1D NMR slice selective method developed based on real time spin manipulation technique. The method gives multiple scalar couplings of a selected spin with simplified multiplets, which is analogous to the 2D G-SERF but with considerable saving in instrument time by 1-2 orders of magnitude. The rapidness of the experiment arises due to reduced dimensionality. The spin dynamics involved in the pulse sequence and its working principle have been described. The application of the method is illustrated for the measurement of 1H-1H couplings. The sequence has been further improved to obtain the heteronuclear couplings between two abundant spins in an orchestrated manner and has been demonstrated for measurement of 1H-19F couplings. This sequence cited as HF-QG-SERF has been implemented on the molecules containing number of chemically non-equivalent fluorine atoms.

Chirality is the property of objects, including molecules, which are not superimposable on their materialized mirror image. Chiral molecules are omnipresent in living organisms and the constituents of biological macromolecules (proteins and nucleic acids) are chiral. Amino-acids (constituting proteins), ribose and 2-deoxy-ribose (the only chiral constituent of RNA and DNA nucleotides respectively) are furthermore generally present in living organisms only under one of their enantiomeric forms. This is referred to as the homochirality of the living world. The origin of this homochirality is still unexplained, even if many partial scenarios have been proposed in the literature. All scenarios involve the creation of a small enantiomeric excess for certain molecules, amplification of this excess and chirality transfer to other chiral molecules. The origin of homochirality on Earth is closely related to the origin of life, and is currently supposed to have preceded life. As no-one will ever be able to directly observe the phenomena which lead to homochirality, and life, on our planet, the only scientific approach to try and help explain how this occurred is to build scenarios, and test them taking into account all available information on the physical and chemical conditions on the primitive Earth (Earth before life appeared). In our work, we investigated three scenarios related to the origin of homochirality on Earth. One of these scenarios also relates to a very precise step of the origin of life: the selection of beta-d-ribofuranose as component of RNA nucleotides.<p><p>Enantiomeric excesses (up to 15 %) of alpha-methylated alpha-amino-acids have been detected in meteorites which fell on Earth during the 20th century. No enantiomeric excess is detected for the corresponding alpha-hydroxy-acids in the same meteoritic samples and small (2% at most) or no enantiomeric excesses have been measured for non-methylated alpha-amino-acids. In the first part of our work, we investigated if photolysis by circularly polarized light (CPL) in space could be at the origin of the presence (or absence) of an enantiomeric excess for these compounds. Experiments to reproduce UV-CPL photolysis are difficult to undertake: they require high-energy circularly polarized photons, hence the use of a synchrotron. In our work, we used quantum mechanical calculations to obtain the electronic circular dichroïsm (ECD) spectra of two -methylated -amino-acids, their corresponding alpha-hydroxy-acids and one non-methylated alpha-amino-acid. Differences are observed between these spectra, and we propose a scenario to explain the experimental measurements reported here above: the enantioselective photolysis, in the gas phase at low temperatures (20K at most), of the alpha-amino-acids by UV-CPL with lambda>210 nm. Under these conditions no photolysis of the alpha-hydroxy-acids would occur. This scenario concerns the first step in the origin of homochirality on Earth: the creation of a small enantiomeric excess for some chiral molecules.<p><p>The second scenario that we investigated relates to the enantiomeric amplification step of the origin of homochirality on Earth, for which the role of the alpha-amino-acid serine has been suggested in the literature. Serine clusters have been observed in the gas phase by mass spectrometry. Among these clusters the octamer has been shown to be a magic number cluster and to have a preference for homochirality. An enantiomeric amplification via cycles of formation and dissociation of the octamer has been suggested. No complete scenario has however been proposed in the literature to explain how this could have occurred on the primitive Earth, but any scenario would most probably include an aqueous phase. We aimed at determining if the homochiral preference of serine octamers also exists in solution and therefore we first investigated if serine octamers exist in solution. For this study, we used nuclear magnetic resonance and infrared spectroscopies, which are well-adapted to the study of molecular assemblies in solution. We were able to demonstrate that most probably serine clusters are not present in solution, and if they are it could only be in extremely low concentration. The scenario suggested in the literature is discussed in the light of our results and of literature data on serine clusters.<p><p>As last hypothesis, we investigated a possible scenario for the selection of beta-d-ribofuranose as component of RNA nucleotides. The currently known prebiotic synthesis pathways to ribose also lead to the formation of many other carbohydrates, and ribose is only a minor product of these syntheses. Our hypothesis is that beta-d-ribofuranose could have been selected through favorable interactions with alpha-amino-acids already present on the primitive Earth under one enantiomeric form. Indeed, it is plausible that a peptidic world emerged before the presence of RNA and that homochiral alpha-amino-acids were present on Earth when RNA was synthesized. Under this hypothesis, we investigated the role that alpha-l-amino-acids could have played in the selection of alpha-d-ribofuranose as component of RNA nucleotides. This work is related to the last step of the origin of homochirality: chirality transfer. Our scenario was investigated via nuclear magnetic resonance studies of the interaction between alpha-amino-acids and carbohydrates. We were able to show that, in the systems that we studied, when an interaction occurs it is very weak (affinity constant less than 1M−1) and non enantioselective. Our results most probably discard the role that alpha-amino-acids alone could have played in the selection of beta-d-ribofuranose as component of RNA nucleotides, but does not discard the role that peptides could have played in this selection. / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished

Enzymes are useful tools as specific analytical reagents. Two different analysis methods were developed for use in the separate fields of protein science and organic synthesis. Both methods rely on the substrate specificity of enzymes. Enzyme catalysis and substrate specificity is described and put in context with each of the two developed methods. In paper I a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage. In paper II and III, three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The conversion and enantiomeric excess of the sample could be calculated from the relative differences in absorbance. / QC 20101108

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