京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18157号 / 医博第3877号 / 新制||医||1003(附属図書館) / 31015 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 清水 章, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-02-16T14:50:28Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Thesis_Gabriel_digital.final.pdf: 10412972 bytes, checksum: ec85b3671bb5bbcd735ea9e83436b08f (MD5) / Made available in DSpace on 2017-02-16T14:50:28Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Thesis_Gabriel_digital.final.pdf: 10412972 bytes, checksum: ec85b3671bb5bbcd735ea9e83436b08f (MD5) Previous issue date: 2016-04-01 / CNPq / Peptídeos antimicrobianos são componentes importantes do sistema de defesa de diversos organismos contra possíveis invasores. Em geral, são pequenos (até 100 aminoácidos), catiônicos e anfipáticos. Eles têm despertado o interesse da comunidade científica por sua capacidade de atuação contra micróbios, que não conseguem desenvolver resistência a esses peptídeos. Ou seja, eles emergem como complemento e/ou alternativa ao uso dos antibióticos convencionais. Este trabalho desenvolveu um modelo computacional de um peptídeo híbrido de pediocina A (N-terminal) e plantaricina 149a (C-terminal), dois peptídeos bactericidas. Dados experimentais obtidos pelo grupo da prof. Dra. Rosângela Itri do IFUSP foram utilizados para modelagem e comparação dos resultados. Foram feitas simulações de MD do peptídeo interagindo com membranas puras e mistas de POPC e POPG utilizando os parâmetros do campo de força GROMOS 53A6 e 54A7. As simulações com uma unidade do peptídeo revelaram a atualização 54A7 era a mais adequado para modelagem desses sistemas. Os mapas de estrutura secundária mostraram que o peptídeo adquire configuração mais ordenada quando interage com membranas com maior quantidade de POPG em sua composição. As simulações com duas unidades do peptídeo sugeririam que o peptídeo interage e penetra na camada de POPG através da região Cterminal. Na simulação com membrana de POPC, nenhuma das porções terminais ficou estável no interior da membrana. O efeito do aumento da concentração de peptídeos foi examinado colocando cinco e dez unidades do peptídeo para interagir com as membranas. Na membrana de POPC, os peptídeos não formam um único aglomerado e causam pouca perturbação na bicamada. Já na membrana de POPG, o efeito da interação do aglomerado de peptídeos é acentuado, provocando grandes deformações na bicamada lipídica, praticamente a destruindo. Esse fenômeno sugere um possível mecanismo carpete para ação do peptídeo sobre a membrana fosfolipídica de bactérias. / Antimicrobial peptides are important components of defense system in various organizations against possible invaders. They are generally small (100 aminoacids), cationic and amphipathic. They have stimulated the interest of the scientific community for its ability to act against microbes that cannot develop resistance to these peptides. That is, they emerge as complement and/or alternative to the use of conventional antibiotics. This study developed a computational model of a hybrid peptide pediocin A (Nterminal) and plantaricin 149a (C-terminal), two bactericidal peptides. Experimental data obtained by the group of prof. Dr. Rosângela Itri (IFUSP) were used for modeling and compare the results. MD simulations were made of the peptide interacting with pure and mixed POPC and POPG membranes. These simulations were performed using the parameters of the force field GROMOS 53A6 and 54A7. Simulations with a single copy of the peptide revealed that the force field 54A7 was the most appropriate for modeling these systems. The secondary structure maps showed that the peptide acquires a more ordered configuration when interacting with membranes with higher amounts of POPG in its composition. The simulations with two copies of the peptide suggest that the peptide interacts and penetrates the POPG layer via the C-terminal part. In the simulation with POPC membrane, none of the end portions remained stable within the membrane. The effect of increasing the peptide concentration of was examined by placing five and ten copies of the peptide to interact with the membranes. In the POPC membrane, the peptides do not form a single cluster and they cause little disturbance in the bilayer. In the POPG membrane, the interaction of peptides cluster is enhanced, causing large deformation and practically destroying the lipid bilayer. This phenomenon suggests a possible carpet mechanism of action of the peptide on the phospholipid membrane of bacteria.

Structural studies of peptides are of great importance in developing novel and effective biomaterials ranging from drugs and vaccines to nano materials with industrial applications. In addition, they provide model systems to study and mimic the protein conformations. The ability to generate folded intramolecularly hydrogen bonded structures in short peptides is essential for peptide design strategies, which rely on the use of folding nuclei in the construction of secondary structure modules like helices and β-hairpins. In these approaches, conformational choices at selected positions are biased, using local stereochemical constraints, that limit the range of accessible backbone torsion angles. X-ray crystallographic studies of designed peptides provide definitive proof of the success of a design strategy, and provide essential structural information that can be utilized in the future design of biologically and structurally important polypeptides. Recent trends in peptide research focus on the incorporation of β-, γ- and higher homologs of the α-amino acid residues in designed peptides as they confer more proteolytic stability to the polypeptides. X-ray crystallographic studies of such modified peptides containing non-protein residues are essential, since information on the geometric and stereochemical properties of modified amino acids can only be gathered from the systematic structural studies of synthetic peptides incorporating them. This thesis reports a systematic study of the structures and conformations of amino acid derivatives and designed peptides containing stereochemically constrained α-, β- and γ-amino acid residues and the structural studies of polymorphic peptide helices. The structures described in thesis contain the Cα,α-dialkyalted α-residues α-aminoisobutyric acid (Aib) and 1-aminocyclohexane-1-carboxylic acid (Ac6c), the β-amino acid residue 1-aminocyclohexane acetic acid (β3,3Ac6c) and the γ-amino acid residue 1-aminomethylcyclohexaneacetic acid (gabapentin, Gpn). The crystal structure determination of peptides incorporating conformationally constrained α-, β- and γ- amino acid residues permitted the characterization of new types of hydrogen bonded turns and polymorphs. The studies enabled the precise determination of conformational and geometric parameters of two ω-amino acid residues, gabapentin and β 3,3Ac6c and provided detailed information about the conformational excursions possible for peptide molecules. This thesis is divided into 10 chapters. Chapter 1 gives a general introduction to the stereochemistry of the polypeptide chain, description of backbone torsion angles of α- and ω- amino acid residues and the major secondary structures of α-peptides, β-peptides, γ-peptides and hybrid peptides. A brief introduction to polymorphism and weak interactions, in particular aromatic interactions, is also provided, followed by a discussion on X-ray diffraction and solution to the phase problem. Chapter 2 describes the crystal structures of gabapentin zwitterion and its eight derivatives (Ananda, Aravinda, Vasudev et al., 2003). The crystal structure of the gabapentin zwitterions determined in this study is identical to that previously reported (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643). Eight of the nine achiral compounds crystallized in centrosymmetric space groups P21/c, C2/c or Pbca, while one derivative (Tos-Gpn-OH) crystallized in non-centrosymmetric space group Pna21 with four independent molecules in the asymmetric unit.The structural studies presented in this chapter reveal that the geminal substituents on the Cβ atom limits the values of dihedral angles θ1 and θ2 to ±60°, resulting in folded backbone conformations in all the examples. Intramolecular hydrogen bonds with 7-atoms in the hydrogen bond turn (C7) are observed in three derivatives, gabapentin hydrochloride (GPNCL), Boc-Gpn-OH (BGPNH) and Piv-Gpn-OH (PIVGPN), while a 9-atom hydrogen bonded turn (C9) is observed in Ac-Gpn-OH (ACGPH). Unique structural features, such as an unusual anti conformation of the COOH group (in ACGPH) and positional disorder of the cyclohexane ring (in BGPNN), indicating the co-existence of both the interconvertible chair conformations, are revealed by the crystal structure analyses. Chapter 3 describes the structural characterization of novel hydrogen bonded conformations of homo oligomers of Gpn. The crystal structures of three peptides, Boc-Gpn-Gpn-NHMe (GPN2), Boc-Gpn-Gpn-Leu-OMe (GPN2L) and Boc-Gpn-Gpn-Gpn-Gpn-NHMe (GPN4) provide the first crystallographic characterization of two new families of polypeptide structures, the C9 helices and C9 ribbons (Vasudev et al., 2005, 2007), in which the molecular conformations are stabilized by contiguous C9 turns formed by the hydrogen bonding between the CO group of residue (i) and the NH group of residue (i+2). The C9 hydrogen bond is characterized by a specific combination of the four torsion angles for the Gpn backbone, with the torsion angles θ1 and θ2 adopting g+/g+ or g /g- conformations. The structural analysis also permits precise determination of hydrogen bond geometry for the C9 structures, which is highly linear in contrast to the analogous γ-turn hydrogen bonds in α-peptides. A comparison of the backbone conformations in the three peptides reveals two classes of C9 hydrogen bonded secondary structures, namely C9 helices and C9 ribbons. The packing arrangement in these γ-peptides follows the same patterns as the helix packing in crystals of α-peptides. Chapter 4 describes ten crystal structures of short hybrid peptides containing the Gpn residue (Vasudev et al., 2007). In addition to the C7 and C9 hydrogen bonded turns which are defined by the backbone conformations at the Gpn residue, hybrid turns defined by a combination of backbone conformations at the α and γ-residues or at the β and γ-residues have been determined. Peptides Boc-Ac6c-Gpn-OH (ACGPH), Piv-Pro-Gpn-Val-OMe (PPGPV) and Boc-Val-Pro-Gpn-OH (VPGPH) reveal molecular conformation stabilized by intramolecular C9 hydrogen bonds, while Boc-Ac6c-Gpn-OMe (ACGPO) and Boc-Gpn-Aib-OH (GPUH) are stabilized by a C7 hydrogen bonded turn at the Gpn residue. An αγ hybrid turn with 12 atoms in the intramolecular hydrogen bonded rings (C12 turns) has been observed in the tripeptide Boc-Ac6c-Gpn-Ac6c-OMe (ACGP3), while βγ hybrid turns with 13 atoms in the hydrogen bonded ring (C13 turns) have been characterized in the tripeptides Boc-βLeu-Gpn-Val-OMe (BLGPV) and Boc- βPhe-Gpn-Phe-OMe (BFGPF). The two βγ C13 turns belong to two different categories and are characterized by different sets of backbone torsion angles for the β and γ residues. A γα C10 hydrogen bond, which is formed in the N→C direction (NHi ••• COi+2), as opposed to the regular hydrogen bonded helices of α-peptides, has also been observed in BFGPF. The Chapter provides a comparison of the backbone torsion angles of the Gpn residue in various hydrogen bonded turns and a brief comparison of the observed hydrogen bonded turns with those of the α-peptides. Chapter 5 describes the crystal structures of three αγ hybrid peptides which show C12/C10 mixed hydrogen bond patterns (Vasudev et al., 2007, 2008a; Chatterjee, Vasudev et al.,2008a). The insertion of gabapentin in the predominantly α-amino acid sequences in Boc-Ala-Aib-Gpn-Aib-Ala-OMe (AUGP5) and Boc-Leu-Gpn-Aib-Leu-Gpn-Aib-OMe results in the observation of helices stabilized by αα C10 (310-turn) and αγ C12 turns. The tetrapeptide Boc-Leu-Gpn-Leu-Aib-OMe reveals a novel conformation, stabilized by C12 (αγ) and C10 (γα) hydrogen bonds of opposite hydrogen bond directionalities. The conformations observed in crystals have been extended to generate C12 helix and C12/C10 helix with alternating hydrogen bond polarities in ( αγ)n sequences. The structure determination of three crystals, providing five molecular conformations, presented in this chapter provides the first crystallographic characterization of two types of helices predicted for the regular αγ hybrid peptides from theoretical calculations. The crystal structure of Boc-Ala-Aib-Gpn-Aib-Ala-OMe also provides an example for the co-existence of left-handed and right-handed helix in the asymmetric unit. Chapter 6 describes the structural studies of αγ hybrid peptides containing Aib and Gpn residues, and is divided into two parts. The first part presents the crystal structure analysis of peptides of sequence length 2 to 4, with alternating Aib and Gpn residues, and illustrates the conformational variability in αγ hybrid sequences as evidenced by the observation of conformational polymorphs (Chatterjee, Vasudev et al., 2008b; Vasudev et al., 2007; Ananda, Vasudev et al., 2005). The peptide Boc-Gpn-Aib-NHMe (GUN), Boc-Aib-Gpn-Aib-OMe (UGU), Boc-Gpn-Aib-Gpn-Aib-OMe (GU4O), Boc-Aib-Gpn-Aib-Gpn-OMe (UG4O) and Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), all of which are potential candidates for exhibiting αγ C12 hydrogen bonds, reveal molecular conformations stabilized by diverse hydrogen bonded turns such as C7, C9, C12 and C17 in crystals. The conformational heterogeneity in this class of hybrid peptides is further evidenced by the observation of three polymorphs in the monoclinic space group P21/c for the tetrapeptide Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), providing four independent peptide molecules adopting two distinct backbone conformations. In one polymorph, C12 helices terminated with an unusual three residue ( γαγ) C17 turn is observed, while the unfolding of helical conformation by solvent insertion into the backbone is observed in the other two polymorphs. The studies indicate the possible utility of Gpn residue in stabilizing locally folded conformations in the folding pathway, thus permitting their crystallographic characterization in multiple crystal forms. A discussion of the structural and conformational features of Gpn residues determined from all the crystal structures is presented in the Chapter, along with a φ-ψ plot for the Gpn residue. Part 2 of Chapter 6 describes the crystal structures of two octapeptides, Boc-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-OMe (GU8) and Boc-Leu-Phe-Val-Aib-Gpn-Leu-Phe-Val-OMe (LFVUG8), featuring C12 turns at the Aib-Gpn segments (Chatterjee, Vasudev et al., 2009). GU8 folds into a C12 helix flanked by C9 hydrogen bonds at both the termini, while LFVUG8 adopts β-hairpin conformation with a chain-reversing C12 turn at the central Aib-Gpn segment. A remarkable feature of the Aib-Gpn turn in the β-hairpin structure is the anti conformation about the Cβ-Cα (θ2) bond, which is the only example of a Gpn residue not adopting gauche conformation for both θ1 and θ2. The crystal structures of the two peptides, mimicking the two major secondary structural elements of α-peptides in hybrid polypeptides, permits a comparative study of the mode of molecular packing in crystals of α-peptides and hybrid peptides. The chapter also discusses theoretical calculations on αγ hybrid sequences, which reveal new types of C12 hydrogen bonded turns. Chapter 7 describes the crystal structures of conformationally biased tert-butyl derivatives of Gpn. The crystallographic characterization of the E (trans) and Z (cis) isomers of the residue,three protected derivatives and a tripeptide provides examples of C7 and C9 hydrogen bonded conformations, suggesting that the C7 and C9 hydrogen bonds can be formed by Gpn residues with both the chair conformations of the cyclohexane ring. Chapter 8 describes the systematic structural studies of the derivatives and peptides of the stereochemically constrained β- amino acid residue, β3,3Ac6c (Vasudev et al., 2008c). The backbone torsion angles φ and θ adopt gauche conformation in majority of the examples, owing to the presence of a cyclohexane ring on the Cβ atom. In contrast to Gpn, β3,3Ac6c does not show strong preference for adopting intramolecularly hydrogen bonded conformations. Of the 16 crystal structures determined, intramolecular hydrogen bonds involving the β-residue are observed only in 4 cases. The amino acid zwitterion (BAC6C), the hydrochloride (BACHCL) and the dipeptide Boc-β3,3Ac6c-β3,3Ac6c-NHMe (BAC62N) form N-H•••O hydrogen bonds with 6-atoms in the hydrogen bond ring (C6 turns). An αβ hybrid C11 hydrogen bonded turn is characterized in the dipeptide Piv-Pro-β3,3Ac6c-NHMe, which is distinctly different from the C11 hydrogen bonds observed in αβ hybrid peptide helices. Several unique structural features such as a dynamic disorder of the hydrogen atom of the carboxylic acid group (in BBAC) and cis geometry of the urethane bond (in BBAC, BAC62N and BPBAC) have been observed in this study. A comparison of the backbone conformations of β3,3Ac6c with other β- amino acid residues is also provided. Chapter 9 describes the crystallographic characterization of a new polymorph of gabapentin monohydrate and crystal structures of the zwitterions of E and Z isomers of tert-butylgabapentin and its hydrochloride and hydrobromide (Vasudev et al., 2009). A comparison of the crystal structures of the monoclinic form (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643) of gabapentin monohydrate and the newly characterized orthorhombic form reveals identical molecular conformations and intermolecular hydrogen bond patterns in both the polymorphs. The two polymorphs show differences in the orientation of molecules constituting a layer of hydrophobic interactions between the cyclohexyl side chains. A comparison of the packing arrangements of the zwitterionic amino acid molecules in the crystal structures of gabapentin monohydrate, the tert-butyl derivatives and other co-crystals of gabapentin that had been characterized so far, is provided which would facilitate prediction of new polymorphs of the widely used drug molecule, Gpn. Chapter 10 describes the crystallization of α-peptide helices in multiple crystal forms (Vasudev et al., 2008b). Crystal structures of two peptides, Boc-Leu-Aib-Phe-Phe-Leu-Aib-Ala-Ala-Leu-Aib-OMe (LFF), Boc-Leu-Aib-Phe-Ala-Leu-Ala-Leu-Aib-OMe (D1) in two crystal forms and the crystal structure of a related sequence, Boc-Leu-Aib-Phe-Ala-Phe-Aib-Leu-Ala-Leu-Aib-OMe (D10) permit an analysis of the molecular conformation and packing patterns of peptide helices in crystals. The two polymorphs of LFF, crystallized in the space groups P21 and P22121, reveal very similar molecular conformation (α/310-helix) in both the polymorphic crystals; the two forms differ significantly in the pattern of solvation. The crystal structure determination of a monoclinic (P21) and an orthorhombic polymorph (P21212) of D1 provides five different peptide conformations, four of which are α-helical and one is a mixed 310/α-helix. The crystal structure determination of the three peptides provide an opportunity to compare the nature and role of aromatic interactions in stabilizing molecular conformation and packing and its significance in the observation of polymorphism. An analysis of the Cambridge Structural Database and a model for nucleation of crystals in hydrophobic peptide helices are also discussed.

Peptides have assumed considerable importance in pharmaceutical industry and vaccine research. Understanding the structural features of these peptide molecules can be effective not only in mimicking natural proteins but also in the design of new biomaterials. Polypeptide sequences consisting of twenty genetically coded amino acids possess structural flexibility, which makes the predictions difficult. However, the introduction of non-protein amino acids into the peptide chain restricts the available range of backbone conformations and acts as stereochemical directors of polypeptide chain folding. Such conformationally rigid residues allow the formation of well defined structures like helices, strands etc, which further assemble into super secondary structural motifs by flexible linkages. Crystal structure determination of the oligopeptides by X-ray diffraction gives insight into the specific conformational states, modes of aggregation, hydrogen bond interactions, solvation of peptides and various weakly polar interactions involving the side chains of aromatic residues (Phe, Trp and Tyr). In β-, γ- and higher ω-amino acids, due to the insertion of one or more methylene groups between the N- and Cα-atoms into the peptide backbone the accessible conformational space is greater than the α-amino acids. The β-, γ-, δ-…. amino acid residues belong to the family of ω-amino acids. Extensive research in the field of β-peptides, which have been experimentally verified or theoretically postulated, has assigned several helices, turns and sheets. The use of ω-amino acid residues in conjunction with α-residues permits systematic exploration of the effects of introducing additional backbone atoms into well-characterized α-peptide structures. The observation of new families of hydrogen bonded motifs in the hybrid peptides containing α- and ω-amino acids are the recent interest in this regard. This thesis reports results of X-ray crystallographic studies of eighteen designed peptides and four protected ω-amino acids listed below. Within brackets are given the abbreviations used for the sequences (Symbol U represents Aib). The ω-amino acids reported in this thesis are: (S)-β3-HAla (β3-homoalanine), (R)-β3-HVal, (S)-β3-HVal (β3-homovaline), (S)-β3-HPhe (β3-homophenylalanine), (S)-β3-HPro (β3-homoproline), βGly (β-homoglycine), γAbu (gamma aminobutyric acid) and δAva (delta aminovaleric acid). 1. Boc-Leu-Trp-Val-OMe (LWV), C28H42N4O6 2. Ac-Leu-Trp-Val-OMe (Space group P21) (LWV1), C25H36N4O5 3. Ac-Leu-Trp-Val-OMe (Space group P212121) (LWV2), C25H36N4O5 4. Boc-Leu-Phe-Val-OMe (LFV), C26H41N3O6 5. Ac-Leu-Phe-Val-OMe (LFV1), C23H35N3O5 6. Boc-Ala-Aib-Leu-Trp-Val-OMe (AULWV), C35H54N6O8 7. Boc-Trp-Trp-OMe (WW), C28H32N4O5 8. Boc-Trp-Aib-Gly-Trp-OMe. (WUGW), C34H42N6O7 9. Boc-Leu-Trp-Val-Ala-Aib-Leu-Trp-Val-OMe (H8AU), C57H84N10O11 10. Boc-(S)-β3-HAla-NHMe (BANH), C10H20N2O3 11. Boc-(R)-β3-HVal-NHMe (BVNH), C12H24N2O3 12. Boc-(S)-β3-HPhe-NHMe (BFNH), C16H24N2O3 13. Boc-(R)-β3-HVal-(R)-β3-HVal-OMe (BVBV), C18H34N2O5 14. Boc-(R)-β3-HVal-(S)-β3-HVal-OMe (LVDV), C18H34N2O5 15. Boc-(S)-β3-HPro-OH (BPOH), C11H19N1O4 16. Boc-Leu-Phe-Val-Aib-(S)-β3-HPhe-Leu-Phe-Val-OMe (UBF8), C60H88N8O11 17. Piv-Pro-Gly-NHMe (PA1), C13H23N3O3 18. Piv-Pro-βGly-NHMe (PB1), C14H25N3O3 19. Piv-Pro-βGly-OMe (PBO), C14H24N2O4 20. Piv-Pro-δAva-OMe (PDAVA), C16H28N2O4 21. Boc-Pro-γAbu-OH (BGABU), C14H24N2O5 22. Boc-Aib-γAbu-OH (UG), C13H24N2O5 23. Boc-Aib-γAbu-Aib-OMe (UGU), C18H33N3O6 The thesis is divided into seven chapters. Chapter 1 gives a general introduction to the stereochemistry of polypeptide chains and the secondary structure classification: helices, β-sheets and β-turns followed by an overview of different types of weakly polar interactions involving the side chains of aromatic amino acid residues. This section also provides a brief overview of the conformational analysis of β-, γ- and higher ω-amino acid residues in oligomeric β-peptides and in α,ω-hybrid peptides. A brief discussion on X-ray diffraction and solution to the phase problem is also presented. Chapter 2 describes the crystal structures of the peptides, Boc-Leu-Trp-Val-OMe (LWV), the two polymorphs of Ac-Leu-Trp-Val-OMe (LWV1 and LWV2), Boc-Leu-Phe-Val-OMe (LFV), Ac-Leu-Phe-Val-OMe (LFV1) and Boc-Ala-Aib-Leu-Trp-Val-OMe (AULWV), in order to explore the nature of interactions between aromatic rings, specifically the indole side chain of Trp residues [1]. Peptide LWV adopts a type I β-turn conformation, stabilized by an intramolecular 4→1 hydrogen bond. Molecules of LWV pack into helical columns stabilized by two intermolecular hydrogen bonds, Leu(1)NH…O=CTrp(2) and Indole NH…O=CLeu(1). The superhelical columns further pack into the tetragonal space group P43 by means of a continuous network of indole - indole interactions. The peptide Ac-Leu-Trp-Val-OMe crystallized in two polymorphic forms: P21 (LWV1) and P212121 (LWV2). In both forms, the peptide backbone is extended and the crystal packing shows anti-parallel β-sheet arrangement. Similarly, extended strand conformation and anti-parallel β-sheet formation are also observed in the Phe containing analogs, LFV and LFV1. The pentapeptide AULWV adopts a short stretch of 310-helix. Analysis of aromatic - aromatic and aromatic - amide interactions in the structures of peptides LWV, LWV1 and LWV2 are reported along with the examples of 12 Trp containing peptides from the Cambridge Structural Database. The results suggest that there is no dramatic preference for the orientation of two proximal indole rings. In Trp containing peptides specific orientations of the indole ring, with respect to the preceding and succeeding peptide units, appear to be preferred in β-turns and extended structures. Crystal parameters LWV: C28H42N4O6; P43; a = 14.698(1) Å, b = 14.698(1) Å, c = 13.975(2) Å; Z = 4; R = 0.0737, wR2 = 0.1641. LWV1: C25H36N4O5; P21; a =10.966(3) Å, b = 9.509(2) Å; c = 14.130(3) Å, β = 104.94(1)°; Z = 2; R = 0.0650, wR2 = 0.1821. LWV2: C25H36N4O5; P212121; a = 9.533(6) Å, b = 14.148(9) Å, c = 19.53(1) Å, Z = 4; R = 0.0480, wR2 = 0.1365. LFV: C26H41N3O6; C2; a = 31.318(8) Å, b = 10.022(3) Å, c = 9.657(3) Å, β = 107.41(1)°; Z = 4; R = 0.0536, wR2 = 0.1328. LFV1: C23H35N3O5; P212121; a = 9.514(8) Å, b = 13.56(1) Å, c = 20.04(2) Å, Z = 4; R = 0.0897, wR2 = 0.1960. AULWV: C35H54N6O8.2H2O; P21; a = 9.743(3) Å, b = 22.807(7) Å, c = 10.106(3) Å, β = 105.73(2)°; Z = 2; R = 0.0850; wR2 = 0.2061. Chapter 3 describes the crystal structures of three peptides containing Trp residues at both N- and C-termini of the peptide backbone: Boc-Trp-Trp-OMe (WW), Boc-Trp-Aib-Gly-Trp-OMe (WUGW) and Boc-Leu-Trp-Val-Ala-Aib-Leu-Trp-Val-OMe (H8AU). Peptide WW adopts an extended conformation and the molecules pack into an arrangement of parallel β-sheet in crystals, stabilized by three intermolecular N-H…O hydrogen bonds. The potential hydrogen bonding group NE1H of Trp(1), which does not take part in hydrogen bonding interaction with an oxygen acceptor participate in an intermolecular N-H…π interaction. Peptide WUGW adopts a folded structure, stabilized by a consecutive type II-I’ β-turn conformation. The crystal of WUGW contains a stoichiometric amount of chloroform in two distinct sites each with an occupancy factor of 0.5 and the structure provides examples of N-H…π, C-H…π, π…π, N-H…Cl, C-H…Cl and C-H…O interactions [2]. The molecular conformation of H8AU reveals a 310-helix. The crystal structure of H8AU reveals an interesting packing motif in which helical columns are stabilized by side chain - backbone hydrogen bond involving the indole NH of Trp(2) as donor and C=O group of Leu(6) as acceptor of a neighboring molecule, which closely resembles the hydrogen bonding pattern obtained in the tripeptide LWV [1]. Helical columns also associate laterally and strong interactions are observed between the Trp(2) and Trp(7) residues on neighboring molecules [3]. The edge-to-face aromatic interactions between the indoles suggest a potential C-H…π interaction involving the CE3H of Trp (2) Crystal parameters WW: C28H32N4O5; P212121; a = 5.146(1) Å, b = 14.039(2) Å, c = 35.960(5) Å; Z = 4; R = 0.0503, wR2 = 0.1243. WUGW: C34H42N6O7.CHCl3; P21; a = 12.951(5) Å, b = 11.368(4) Å, c = 14.800(5) Å, β = 101.41(2)°; Z = 2; R = 0.1095, wR2 = 0.2706. H8AU: C57H84N10O11; P1; a = 10.494(7) Å, b = 11.989(7) Å, c = 13.834(9) Å, α = 70.10(1)°, β = 82.74(1)°, γ = 78.96(1)°; Z = 1; R = 0.0855, wR2 = 0.1965. Chapter 4 describes the crystal structures of four protected β-amino acid residues, Boc-(S)-β3-HAla-NHMe (BANH); Boc-(R)-β3-HVal-NHMe (BVNH); Boc-(S)-β3-HPhe-NHMe (BFNH); Boc-(S)-β3-HPro-OH (BPOH) and two β-dipeptides, Boc-(R)-β3-HVal-(R)-β3-HVal-OMe (BVBV); Boc-(R)-β3-HVal-(S)-β3-HVal-OMe (LVDV). Gauche conformations about the Cβ-Cα bonds (θ ~ ± 60°) are observed for the β3-HPhe residue in BFNH and all four β3-HVal residues in the dipeptides BVBV and LVDV. Trans conformations (θ ~ 180°) are observed for β3-HAla residues in both independent molecules in BANH and for the β3-HVal and β3-HPro residues in BVNH and BPOH, respectively. In all these cases except for BPOH, molecules associate in the crystals via intermolecular backbone hydrogen bonds leading to the formation of sheets. The polar strands formed by β3-residues aggregate in both parallel (BANH, BFNH, LVDV) and anti-parallel (BVNH, BVBV) fashion. Sheet formation accommodates both the trans and gauche conformations about the Cβ - Cα bonds [4]. Crystal parameters BANH: C10H20N2O3; P1; a = 5.104(2) Å, b = 9.469(3) Å, c = 13.780(4) Å, α = 80.14(1)°, β = 86.04(1)°, γ = 89.93(1)°; Z =2; R = 0.0489, wR2 = 0.1347. BVNH: C12H24N2O3; P212121; a = 8.730(2) Å, b = 9.741(3) Å, c = 17.509(5) Å; Z = 4; R = 0.0479, wR2 = 0.1301. BFNH: C16H24N2O3; C2; a = 20.54(1) Å, b = 5.165(3) Å, c = 16.87(1) Å, β = 109.82(1)°; Z = 4; R = 0.0909, wR2 = 0.1912. BVBV: C18H34N2O5; P212121; a = 9.385(2) Å, b = 11.899(2) Å, c = 19.199(4) Å; Z = 4; R = 0.0583, wR2 = 0.1589. LVDV: C18H34N2O5; P212121; a = 5.170(4) Å, b = 10.860(8) Å, c = 37.30(3) Å; Z = 4; R = 0.0787, wR2 = 0.1588. BPOH: C11H19N1O4; P1; a = 5.989(2) Å, b = 6.651(2) Å, c = 8.661(3) Å, α = 70.75(1)°, β = 77.42(1)°, γ = 86.98(1)°; Z = 1; R = 0.0562, wR2 = 0.1605. Chapter 5 describes a new class of polypeptide helices in hybrid sequences containing α-, β- and γ-residues. The molecular conformation in crystals determined for the octapeptide Boc-Leu-Phe-Val-Aib-(S)-β3-HPhe-Leu-Phe-Val-OMe (UBF8) reveals an expanded helical turn in the hybrid sequence (ααβ)n. A repetitive helical structure composed of C14 hydrogen bonded units is observed. Using experimentally determined backbone torsion angles for the hydrogen bonded units formed by hybrid sequences, the energetically favorable hybrid helices have been generated. Conformational parameters are provided for C11, C12, C13, C14 and C15 helices in hybrid sequences [5]. Crystal parameters UBF8: C60H88N8O11; P212121; a = 12.365(1) Å, b = 18.940(2) Å, c = 27.123(3) Å; Z = 4; R = 0.0625, wR2 = 0.1274. Chapter 6 describes the crystal structures of five model peptides Piv-Pro-Gly-NHMe (PA1), Piv-Pro-βGly-NHMe (PB1), Piv-Pro-βGly-OMe (PBO), Piv-Pro-δAva-OMe (PDAVA) and Boc-Pro-γAbu-OH (BGABU). A comparison of the structures of peptides PA1 and PB1 illustrates the dramatic consequences upon backbone homologation in short sequences. The molecule PA1 adopts a type II β-turn conformation in the crystal state, while in PB1, the molecule adopts an open conformation with the β-residue being fully extended. The peptide PBO, which differs from PB1 by replacement of the C-terminal NH group by an O-atom, adopts an almost identical molecular conformation and packing arrangement in the crystal state. In peptide PDAVA, the observed conformation resembles that determined for PB1 and PBO, with the δAva residue being fully extended. In peptide BGABU, the molecule undergoes a chain reversal, revealing a β-turn mimetic structure stabilized by a C-H…O hydrogen bond [6]. Crystal parameters PA1: C13H23N3O3; P1; a = 5.843(1) Å, b = 7.966(2) Å, c = 9.173(2) Å, α = 114.83(1)°, β = 97.04(1)°, γ = 99.45(1)°; Z = 1; R = 0.0365, wR2 = 0.0979. PB1: C14H25N3O3.H2O; P212121; a = 6.297(3) Å, b = 11.589(5) Å, c = 22.503(9) Å; Z = 4; R = 0.0439, wR2 = 0.1211. PBO: C14H24N2O4.H2O; P212121; a = 6.157(2) Å, b = 11.547(4) Å, c = 23.408(8) Å; Z = 4; R = 0.050, wR2 = 0.1379. PDAVA: C16H28N2O4.H2O; P21212; a = 11.33(1) Å, b = 25.56(2) Å, c = 6.243(6) Å; Z = 4; R = 0.0919, wR2 = 0.2344. BGABU: C14H24N2O5; P61; a = 9.759(2) Å, b = 9.759(2) Å, c = 29.16(1) Å; Z = 6; R = 0.0773, wR2 = 0.1243. Chapter 7 describes the crystal structures of a dipeptide, Boc-Aib-γAbu-OH (UG) and a tripeptide, Boc-Aib-γAbu-Aib-OMe (UGU) containing a single γAbu residue in each sequence. The structure of UG forms a reverse turn stabilized by a 10-membered intramolecular C-H…O hydrogen bonded ring. The peptide UGU crystallized in the triclinic space group P⎯1 with two molecules in the asymmetric unit resulting in a parallel assembly of sheets in crystals. Notably, the insertion of a single Aib residue at the C-terminus drastically changes the overall conformation of the structures. Crystal parameters UG: C13H24N2O5; P21/c; a = 16.749(3) Å, b = 5.825(1) Å, c = 16.975(3) Å; β = 111.82(1); Z = 4; R = 0.0507; wR2 = 0.1294. UGU: C18H33N3O6; P⎯1; a = 9.576(6) Å, b = 13.98(1) Å, c = 17.83(1); α = 85.31 (1); β = 77.46 (1); γ = 71.39 (1); Z = 4; R = 0.0648; wR2 = 0.1837.

The insertion of ω- amino acid residues as guests into host α-peptide sequences permits expansion of the range of polypeptide secondary structures. The term ω- amino acid is used to refer to the entire family of residues generated by homologation of the backbone of α - amino acid residues. This explores the consequences of insertion of substituted β-residues (β3) , unsubstituted β-residues , unsubstituted γ-residues (gamma aminobutyric acid) and unsubstituted δ-residues (delta aminovaleric acid) into host α -peptide sequences. Chapter 1 provides an introduction to the conformational properties of β-peptides and reviews current literature on the structural features of peptides containing ω-amino acid residues. The available crystallographic information is summarized. The conformational properties of β- residues may be described by three degrees of torsional freedom : φ (N – Cβ) , θ (Cβ -Cα) and ψ (Cα-CO). Similarly, the conformational properties of γ -residues is based on four torsional parameters ( φ , θ1 , θ2, ψ) and the conformational properties of δ - residues is based on five degrees of freedom ( φ , θ1 , θ2, θ3,ψ). The rational use of β -residues in peptide design requires an understanding of the nature of local conformations, which are readily accessible. The conformational space for β -residues can be represented in a three dimensional plot. The observed distribution of φ , θ and ψ values for β -residues in peptide crystal structures presented in this section permits a correlation - between the torsion angle θ and the secondary structure context. The gauche (g+ and g ) conformations induce helical folding and the trans conformation is generally observed in the strands of a hairpin. The most striking feature of hybrid sequences is the observation of novel hydrogen bonded rings in peptide structures. Chapter 2 describes the effects of insertion of β-residues into specific positions in the strand segments of designed peptide hairpins. Insertion of β -residues into the strands of a hairpin changes the orientation of peptide bonds, resulting in a “polar sheet” arrangement. The conformational analysis of three designed peptide hairpins composed of α/β - hybrid segments are described: Boc-Leu-βPhe-Val-DPro-Gly- Leu-βPhe-Val-OMe (BBH8) , Boc-βLeu- Phe-βVal-DPro-Gly-βLeu-Phe-βVal-OMe (BAB8) and CF3COO-H3N+-Leu-Val-Val-βPhe-DPro-Gly-βPhe-Leu-Val-Val-OMe (BHFF10). All the peptides have been characterized by 500 MHz 1H-NMR spectroscopy and several crucial long range NOEs confirm a predominant population of β-hairpin conformations in CD3OH. X-ray diffraction studies on single crystals of peptide BBH8 reveal a β-hairpin conformation, stabilized by three cross-strand hydrogen bonds and a Type II′β-turn at the DPro-Gly turn segment. Designed β-hairpin peptide scaffolds may be used to probe cross-strand sidechain interactions in β-sheet structures. A previously reported peptide β-hairpin, Boc-Leu-Phe-Val-DPro-Gly-Leu-Phe-Val-OMe exhibited an anomalous far UV CD spectrum, which was interpreted in terms of interactions between facing aromatic chromophores, Phe 2 and Phe 7 (Zhao, C.; Polavarapu, P.L.; Das,C. and Balaram, P. J. Am. Chem. Soc., 2000, 122, 8228-8231). In BBH8 and BHFF10 the two cross-strand βPhe residues are at non-hydrogen bonding positions, with the benzyl sidechains pointing on opposite faces of the β- sheet. BBH8 yields a “hairpin –like” CD spectrum, with a minimum at 224 nm. The CD spectrum of BAB8 reveals a negative band at 234 nm and a positive band at 221 nm suggestive of an exciton split doublet. BHFF10 yields a “hairpine-like” CD spectrum, with a negative band at 220 nm. Chapter 3 describes the synthesis and conformational characterization of three hybrid decapeptides : Boc-Leu-Val-βGly-Val-DPro-Gly- Leu-βGly -Val-Val-OMe (BHB10), Boc-Leu-Val-γAbu-Val-DPro-Gly- Leu-γAbu -Val-Val-OMe (BHC10) and Boc- Leu-Val-δAva-Val-DPro-Gly- Leu-δAva -Val-Val-OMe (BHD10). These peptides were designed to systematically investigate the effect of insertion of additional methylene groups into the strands of a hairpin. The incorporation of additional carbon atoms changes the local polarity of the strands. 500 MHz NMR studies establish that BHB10 and BHD10 adopt predominantly β- hairpin conformations in methanol, with interstrand registry established by observation of long range NOEs. The observation of both DPro 4 (CαH) ↔ Gly 5 (NH) and Gly 5 (NH) ↔ Leu 6 (NH) NOEs provides evidence for a Type II ′β - turn for all the hairpins. In BHC10, no long range NOEs were observed. However, X-ray diffraction studies in single crystals reveal a β- hairpin conformation, nucleated by a DPro-Gly Type II′β-turn. Chapter 4 describes an attempt to incorporate one or two ω amino acid residues in the turn region of a potential hairpin, in order to assess the effect of expansion of the nucleating turn. The DPro-LPro segment has been shown to stabilize β-hairpin conformations in both cyclic (Shankaramma,S.C.; Moehle, K. ; James, S.; Vrijbloed, J.W.; Obrecht,D and Robinson, J.A. Chem Commun. 2003,1842-1843) and acyclic sequences ( Raj Kishore Rai ; S.Raghothama and P. Balaram , unpublished results) . In the present study the following turn segments have been considered: βDPro -αLPro , βLPro -αLPro and βLPro -αDPro. The synthesis and conformational analysis of three octapeptide sequences -Boc-Leu-Phe-Val-βDPro-αLPro-Leu-Phe-Val-OMe (βDPαLP8), Boc-Leu-Phe-Val-βLPro-αLPro-Leu-Phe-Val-OMe (βLPαLP8)and Boc-Leu-Phe-Val-βLPro-αDPro-Leu-Phe-Val-OMe (βLPαDP8) are described. In the βDPro-αLPro peptide, NMR evidence clearly supports a β-hairpin conformation, with a nucleating hybrid βα turn stabilized by a C11 (4 →1) hydrogen bond. In the other two octapeptides, no evidence for folded structures was obtained. These results suggest that nucleating turn formation is facilitated only in the heterochiral βD-αL case. Further expansion of the turn segment in potential hairpins has been investigated by inserting two contiguous β-residues into the center of a host α-peptide sequence. The conformational studies on two synthetic hexapeptides, Boc-Leu-Phe-βDPhe-βLPro-Phe-Leu-OMe (βDFβLP6) and Boc-Leu-Phe-βLPhe-βLPro-Phe-Leu-OMe (βLFβLP6) suggest that the βDPhe-βLPro segment is capable of forming a C12 turn in methanol. Two octapeptide sequences, Boc-Leu-Val-Leu-βDPhe-βLPro-Leu-Phe-Val-OMe (βDFβLP8N) and Boc-Leu-Val-Val-βDPhe-βLPro-Leu-Val-Val-OMe (βDFβLP8V) have also been investigated to probe the possible formation of hairpin structures. In these cases, spectroscopic analysis is hampered by the presence of multiple conformations, because of the tendency of the βDPhe-βLPro bond to exist in both cis and trans conformations. NMR studies on the conformational properties of a hexapeptide Boc-Leu-Val-βDPro-βLPro-Leu-Phe-OMe (βDPβLP6) in CDCl3 reveal that in the major conformer the Val 2(NH) ↔ Leu 5 (NH) NOE is observed, suggesting the presence of a 12-membered hydrogen bonded turn. A ββ - segment can give rise to two types of hydrogen bonded rings , 10 – membered (C10) and 12- membered (C12). In an attempt to generate C10 turns, an N-methylamino acid has been inserted next to a ββ - segment, preventing the formation of the 12 – membered turn. In such a situation formation of a 10-membered turn, with reverse hydrogen bond directionality, may be facilitated. The conformational properties of Boc-Leu-Val-βDPhe-βLPro-(N-Me) Leu- Phe-OMe (βDFβLPNMeL6) has been studied by 500 MHz NMR spectroscopy. The data suggests the formation of a C11 turn at the βLPro- (N-Me) Leu segment in CDCl3-DMSO mixtures, instead of formation of a C10 turn at the βDPhe -βLPro segment. Studies on the peptide Boc-Leu-Phe-Val-βLPro-(N-Me) Leu-Leu-Phe-Val-OMe (βLPNMeL8) also suggest the absence of turn formation and folded structures. In hybrid sequences, an important question to be addressed is whether ω amino acids can be accommodated into helical structures. Two contiguous β- residues have been inserted into a helical sequence. The conformational properties of a 11- residue peptide, Boc-Val-Ala-Phe-Aib-βVal-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA11) are described in Chapter 5. This sequence was based on the parent α- peptide Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe, which adopted a complete helical conformation in crystals (Aravinda, S.; Shamala, N.; Das, C .; Sriranjini, A.; Karle, I.L. and Balaram, P. J. Am. Chem. Soc. 2003, 125, 5308-5315). 500 MHz 1H-NMR studies establish a continuous helix over the entire length of the peptide in CDCl3 solution , as evidenced by diagnostic nuclear Overhauser effects. The molecular conformation in crystals reveals a continuous helical fold, stabilized by seven intramolecular hydrogen bonds. The characterization of two synthetic octapeptides Boc-Val-Ala-βPhe-Aib-Val-Ala-βPhe-Aib-OMe (VAβFU8) (βPhe residues have been incorporated at (i /i+4 positions) and Boc-Val-Ala-βPhe-Aib-βPhe-Ala-Val-Aib-OMe (βFUβF8) (βPhe residues have been incorporated at (i /i+2 positions) is also presented. NMR data suggests the retention of helical conformation in both the peptides. In order to delineate the conformations of hybrid peptides with three contiguous β-residues, two peptides have been synthesized Boc-Phe-Aib-βGly-βLeu-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA10) and Boc-Val-Ala-Phe-Aib-βGly-βLeu-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA12). NMR studies in chloroform support continuous helical conformation in the decapeptide.

This thesis describes NMR studies which probe weak interactions between amino acid side chains in folded peptide structures. Aromatic/aromatic interactions between facing phenylalanine residues have been probed in antiparallel β-sheets, while aromatic/proline interactions have been examined using cyclic peptide disulfides that occur in the venom of marine cone snails. Novel intramolecular hydrogen bonded structures in hybrid peptides containing backbone homologated residues, specifically γ-amino acids, are also described. Chapter 1 provides a brief background to the principles involved in the design of antiparallel β-sheet structures and an introduction to previous studies on aromatic/aromatic and aromatic/proline interactions in influencing peptide conformations. A summary of the NMR methods used is also presented. Chapter 2 discusses the structural characterisation of a designed 14 residue, three stranded β-sheet peptide, Boc-LFVDP-PLFVADP-PLFV-OMe (LFV14). The results described in this Chapter support the presence of multiple conformational states about the χ1 (Cα-Cβ) torsional degree of freedom for the interacting aromatic pairs in solution. Chapter 3 presents the structural characterisation of a designed 19 residue three stranded hybrid β-sheet peptide, Boc-LVβFVDPGLβFVVLDPGLVLβFVV-OMe (BBH19). β-amino acid residues (β-phenylalanine, βPhe) were incorporated at facing positions on antiparallel β-sheets. The BBH19 structure provides an example of interaction between the N and C-terminal strands in a three stranded structure with an α/β hybrid backbone. Chapter 4 focuses on studies of the conformations of the contryphan In936 (GCVDLYPWC*) from Conus inscriptus and the related peptide Lo959 (GCPDWDPWC*) from Conus loroissi. Both peptides possess a macrocyclic 23 membered ring, with multiple accessible conformational states. Chapter 5 describes conformational analysis of a novel 20 membered cyclic peptide disulfide, CIWPWC (Vi804), from Conus virgo. NMR structures were calculated for Vi804 and an analog peptide, CIDWPWC, DW3-Vi804. Chapter 6 explores the solution conformation of hybrid sequences containing α and γ residues. Oligopeptides of the type (αγ)n and (αγγ)n have been studied in solution by NMR methods. Chapter 7 provides a summary of the results described in this thesis and highlights the major conclusions.