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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.)

abstract: Conformational changes in biomolecules often take place on longer timescales than are easily accessible with unbiased molecular dynamics simulations, necessitating the use of enhanced sampling techniques, such as adaptive umbrella sampling. In this technique, the conformational free energy is calculated in terms of a designated set of reaction coordinates. At the same time, estimates of this free energy are subtracted from the potential energy in order to remove free energy barriers and cause conformational changes to take place more rapidly. This dissertation presents applications of adaptive umbrella sampling to a variety of biomolecular systems. The first study investigated the effects of glycosylation in GalNAc2-MM1, an analog of glycosylated macrophage activating factor. It was found that glycosylation destabilizes the protein by increasing the solvent exposure of hydrophobic residues. The second study examined the role of bound calcium ions in promoting the isomerization of a cis peptide bond in the collagen-binding domain of Clostridium histolyticum collagenase. This study determined that the bound calcium ions reduced the barrier to the isomerization of this peptide bond as well as stabilizing the cis conformation thermodynamically, and identified some of the reasons for this. The third study represents the application of GAMUS (Gaussian mixture adaptive umbrella sampling) to on the conformational dynamics of the fluorescent dye Cy3 attached to the 5' end of DNA, and made predictions concerning the affinity of Cy3 for different base pairs, which were subsequently verified experimentally. Finally, the adaptive umbrella sampling method is extended to make use of the roll angle between adjacent base pairs as a reaction coordinate in order to examine the bending both of free DNA and of DNA bound to the archaeal protein Sac7d. It is found that when DNA bends significantly, cations from the surrounding solution congregate on the concave side, which increases the flexibility of the DNA by screening the repulsion between phosphate backbones. The flexibility of DNA on short length scales is compared to the worm-like chain model, and the contribution of cooperativity in DNA bending to protein-DNA binding is assessed. / Dissertation/Thesis / Ph.D. Chemistry 2011

Ground state destabilization is a hypothesis to explain enzyme catalysis. The most popular interpretation of it is the entropic effect, which states that enzymes accelerate biochemical reactions by bringing the reactants to a favorable position and orientation and the entropy cost of this is compensated by enthalpy of binding. Once the enzyme-substrate complex is formed, the reaction could proceed with negligible entropy cost. Deamination of cytidine catalyzed by E.coli cytidine deaminase appears to agree with this hypothesis. In this reaction, the chemical transformation occurs with a negligible entropy cost and the initial binding occurs with a large entropy penalty that is comparable to the entropic cost of the uncatalyzed reaction. Our calculations revealed that this reaction occurs with different mechanisms in the cytidine deaminase and water. The uncatalyzed reaction involves a concerted mechanism and the entropy cost of this reaction appears to be dominated by the reacting fragments and first solvation shell. The catalyzed reaction occurs via a stepwise mechanism in which a hydroxide ion acts as the nucleophile. In the active site, the entropy cost of hydroxide ion formation is eliminated due to pre-organization of the active site. Hence, the entropic effect in this reaction is due to a pre-organized active site rather than ground state destabilization. In the second part of this thesis, we investigated peptide bond formation and peptidyl-tRNA hydrolysis at the peptidyl transferase center of the ribosome. Peptidyl-tRNA hydrolysis occurs by nucleophilic attack of a water molecule on the ester carbon of peptidyl-tRNA. Our calculations showed that this reaction proceeds via a base catalyzed mechanism where the A76 O2’ is the general base and activates the nucleophilic water. Peptide bond formation occurs by nucleophilic attack of the α-amino group of aminoacyl-tRNA on the ester carbon of peptidyl-tRNA. For this reaction we investigated two mechanisms: i) the previously proposed proton shuttle mechanism which involves a zwitterionic tetrahedral intermediate, and ii) a general base mechanism that proceeds via a negatively charged tetrahedral intermediate. Although both mechanisms resulted in reasonable activation energies, only the proton shuttle mechanism found to be consistent with the pH dependence of peptide bond formation.

Les cyclodipeptides et leurs dérivés, les dicétopipérazines (DKP), constituent une large classe de métabolites secondaires aux activités biologiques remarquables qui sont essentiellement synthétisés par des microorganismes. Les voies de biosynthèse de certaines DKP contiennent des synthases de cyclodipeptides (CDPS), une famille d’enzymes récemment identifiée. Les CDPS ont la particularité de détourner les ARNt aminoacylés de leur rôle essentiel dans la synthèse protéique ribosomale pour les utiliser comme substrats et ainsi catalyser la formation des deux liaisons peptidiques de différents cyclodipeptides. Le travail de thèse présenté dans ce manuscrit a pour objectif de caractériser la nouvelle famille des CDPS. Dans un premier temps, la caractérisation tant structurale que mécanistique de la première CDPS identifiée, AlbC de Streptomyces noursei, est présentée. Puis, les résultats obtenus avec trois autres CDPS, chacune de ces enzymes ayant des caractéristiques adéquates pour approfondir l’étude de la famille des CDPS, sont décrits. Ainsi, la CDPS Ndas_1148 de Nocardiopsis dassonvillei a permis d’étendre nos connaissances sur les bases moléculaires de la spécificité des CDPS. La CDPS AlbC-IMI de S. sp. IMI 351155 est un bon modèle pour analyser l’interaction de chacun des deux substrats nécessaires à la formation d’un cyclodipeptide. Enfin, la caractérisation de la CDPS Nvec-CDPS2 chez l’animal Nematostella vectensis a permis de fournir le premier exemple d’enzyme d’origine animale impliquée dans la synthèse peptidique non ribosomale. / Cyclodipeptides and their derivatives, the diketopiperazines (DKPs), constitute a large class of secondary metabolites with noteworthy biological activities that are mainly synthesized by microorganisms. The biosynthetic pathways of some DKPs contain cyclodipeptide synthases (CDPSs), a newly defined family of enzymes. CDPSs hijack aminoacyl-tRNAs from their essential role in ribosomal protein synthesis to catalyze the formation of the two peptide bonds of various cyclodipeptides. The aim of the work presented in this thesis manuscript is to characterize the CDPS family. At first, the structural and mechanistic characterization of the first identified CDPS, AlbC of Streptomyces noursei, is presented. Then, the results obtained with three other CDPSs, each of which having suitable properties to increase our understanding of the CDPS family, are described. The CDPS Ndas_1148 of Nocardiopsis dassonvillei extends our knowledge of the molecular bases of the CDPS specificity. The CDPS AlbC-IMI of S. sp. IMI 351155 is a good model to analyze the interaction of each of the two substrates required for the formation of a cyclodipeptide. Finally, the characterization of the CDPS Nvec-CDPS2 from Nematostella vectensis provides the first example of enzymes of animal origin involved in nonribosomal peptide synthesis.

Cyclodipeptides and their derivatives, the diketopiperazines (DKPs), constitute a large class of secondary metabolites with noteworthy biological activities that are mainly synthesized by microorganisms. The biosynthetic pathways of some DKPs contain cyclodipeptide synthases (CDPSs), a newly defined family of enzymes. CDPSs hijack aminoacyl-tRNAs from their essential role in ribosomal protein synthesis to catalyze the formation of the two peptide bonds of various cyclodipeptides. The aim of the work presented in this thesis manuscript is to characterize the CDPS family. At first, the structural and mechanistic characterization of the first identified CDPS, AlbC of Streptomyces noursei, is presented. Then, the results obtained with three other CDPSs, each of which having suitable properties to increase our understanding of the CDPS family, are described. The CDPS Ndas_1148 of Nocardiopsis dassonvillei extends our knowledge of the molecular bases of the CDPS specificity. The CDPS AlbC-IMI of S. sp. IMI 351155 is a good model to analyze the interaction of each of the two substrates required for the formation of a cyclodipeptide. Finally, the characterization of the CDPS Nvec-CDPS2 from Nematostella vectensis provides the first example of enzymes of animal origin involved in nonribosomal peptide synthesis.

This thesis titled, “Nature of Local Interactions at cisPro-Aro Peptide Sequences in Proteins: Evidences for van der Waals type Interactions. Design and Synthesis of Novel Covalent Surrogates for the Peptide Hydrogen Bond”, describes two important studies. The first is to gain a thorough understanding of the nature of interactions that govern cisPro stability at Pro-Aro sequences, which described in the first four chapters. The final chapter describes the synthesis of novel 4-carbon covalent surrogates for the peptide H-bonding interaction. Chapter 1: Local Interactions Governing cisPro Stability: Refining the Model Peptides Chapter 1 Section A: Understanding the role of inter-side chain CH•••Aro interaction in cis-trans isomerization at Pro-Aro and Aro-Pro Sequences. This chapter is divided into two sections. In the first section an exhaustive overview of earlier investigations into the nature of local interactions at Xaa-cisPro-Aro and Aro-cisPro-Xaa peptide sequences, by various groups, are discussed. Most studies have found evidence for the close assemblage between side chains of residues flanking cisPro motifs, when at least one of them is an aromatic group. An electronic C-H•••π nature has been proposed for these assemblies and they are proposed to influence the cisPro stability. We highlight those features in these studies that indicate that these interactions are not always electronically tunable, are insensitive to presence of strong chaotropes in the solvent and occur at protein sequences lacking Pro or cisPro; all of which contradict the electronic C-H•••π model for these inter-side chain assemblages and their perceived influence on cisPro stability. Chapter 1 Section B: Investigation of the Nature of H Xaa•••Aro interaction at Xaa-Pro-Pro-Phe-sequences In Section B, we design and synthesize Pro-Aro containing short peptide models to investigate the nature of local C-H•••Aro interactions in them. We synthesize a series of homologous Pro-Pro-Aro containing peptides (modeled based on earlier studies) and investigate the relative populations of its four Xaa-Pro rotamers using extensive 1D and 2D NMR techniques including TOCSY, HSQC and ROESY. We find several drawbacks that make this a relatively deficient model. Firstly, their relative populations of the rotamers (the most important data for current investigation) cannot be determined with high fidelity as they are dependent on the solvent polarity, solute concentration and chemical shift degeneracy of crucial NMR signals for the rotamers. Importantly, the populations of a few rotamers are influenced by strong 13-membered ring backbone H-bonds. Notably, some of the cisPro rotamers do not even contain the inter-side chain assembly, whose nature is under investigation. Design of novel models – unconstrained by H-bonds We design the Acyl-Pro-Pro-Aro-OMe peptides that lack the possibility of forming the 13-membered ring H-bonded structures. Thorough 1D and 2D NMR analyses of these models reveal that strong Type VI β-turn type 10-membered ring H-bonds are formed in the rotamers of these models – hence precluding their applications for current study. Interestingly, the relative rotamer populations are strongly influenced by solvent polarity and are entirely different from those of the corresponding C-terminal amide models. We further discover that the Pro-Pro-Aro motif is not essential to express the inter-side chain interactions – Ala-Pro-Aro are sufficient. Formation of the 10-membered H-bonding interactions, however, are not precluded. Chapter 2: Design and Synthesis of Acyl-Pro-Phe-OMe: Novel models to investigate the role of HαXaa•••Aro interactions on Xaa-cisPro-Aro stability. Chapter 2 Section A: Design, Synthesis and Conformational Analysis of Ibu-Pro-Phe-OMe Chapter 2 is divided into two sections. In Section A, we replace the amino acid at the N-terminal of the putative Pro residue with simple isosteric isobutyryl group, the resulting minimalist dipeptide model shows the exclusive influence of desired inter-side chain interactions in the cisPro rotamer. Solvent polarity and temperature coefficient studies reveal that absence of any intramolecular H-bonding or Oπ* interactions in it. 1D and 2D NMR analyses clearly indicate the close proximity between the side chains of Ibu and Phe exclusively in the cisPro rotamer. The Kc/t value decreases upon mutation of Phe to Ala. All these features favor the Ibu-Pro-Phe-OMe as an ideal minimalistic model for investigating the nature of Ibu•••Ph assemblages in the cisPro rotamer. Chapter 2 Section B: Investigation of CH•••Aro /Alp•••Alp interactions in Ibu-cisPro-Xaa-OMe In Section B, the 1D and 2D NMR analyses of the complete set of the aliphatic and aromatic analogues Ibu-Pro-Xaa-OMe were investigated. DMSO-d6 was found to be the best solvent for mimicking both the folded and the unfolded local environments of these short peptide sequences. The HαIbu•••Aro assemblage is observed in Aro analogues, but cannot be electronically tuned. The aliphatic analogues also surprisingly contain the HαIbu•••Alp interactions! The Kc/t values (cisPro %) increase in the aliphatic analogues too, where the aliphatic side chain is long. Increase in cisPro stability is not due to ring current effects or intramolecular H-bonds or Oπ* interactions. It seems to be due to van der Waals type interactions between the involved side chains, either of which need not be aromatic in nature. Chapter 3: Nature of Inter-Side Chain Interactions at Acyl-cisPro-Aro Sequences: Evidence for van der Waals Interactions Chapter 3 Section A: Investigation of nature of inter-side chain interactions in R-CO-cisPro-Phe-OMe Chapter 3 has two sections. Section A describes the systematic design and synthesis of Acyl-Pro-Phe-OMe homologues where first the steric bulk and hence the surface area of the aliphatic side chain of the acyl group is varied. Interaction of the phenyl ring of Phe seems to occur with the Cα-Cβ σ-bond of the acyl group. Branching at either Cα or Cβ seems to destabilize the cisPro rotamer. Aliphatic•••Aromatic interactions overwhelm the cisPro rotamer population to be greater than that of transPro. In the analogues where the acidity of the acyl Cα-H bond is increased, the Kc/t does not increase correspondingly. The Δδ(trans-cis) ppm shifts of HαAcyl protons are dependent exclusively on its acidity rather than on the Kc/t values. In carbamyl-Pro, which entirely lack the HαAcyl proton, the Kc/t values are significantly high and improve as the aliphatic surface on the alkoxy group increases. Introduction of benzyloxy carbamyl group at Pro renders almost the same Kc/t values as that of ethyloxy carbamate. All these data contradict the C-H•••π interaction model and strongly support a van der Waals type interaction between the Acyl (preceding cisPro) group’s Xα-Yβ σ-bond and the Aro or Alp side chains (succeeding cisPro). Chapter 3 Section B: Evidence for the Van der Waals nature of Inter Side Chain (Acyl•••S.C.Aro/Alp) interactions- Determination of Interactions energies In Section B, a thorough investigation of both aliphatic•••aliphatic and aliphatic•••aromatic interactions on the background of homologous Acyl-Pro-Aro/Alp-OMe peptide models is undertaken. These models uniquely allow the delineation of contribution of the van der Waals interactions and the ring current effects to the cis/trans isomerization in these peptides. We see that the energy of the van der Waals component of these aliphatic•••aliphatic and aliphatic…aromatic interactions increase linearly with increase in Kc/t, in both DMSO-d6 and D2O. On other hand, energy from the ring current effects largely remains invariant. The Acyl•••Aro/Alp interactions are not hydrophobic and are facilitated by conformational effects. Chapter 4: Crystallographic evidence for van der Waals interaction-mediated stabilization of cisPro conformers Chapter 4 Section A: Systematic crystallization and crystal structure analyses of homologous Xaa-cisPro-Alp and Xaa-cisPro-Aro rotamers: Evidence for van der Waals interactions Chapter 4 has two sections, both of which present crystallographic evidence for the van der Waals nature of the Xaa•••Aro interactions at Xaa-cisPro-Aro sequences. Section A describes the unique crystal structures of five of the Acyl-Pro-Alp-OMe analogues that have been synthesized in the current study. All of them remarkably crystallize with two features: 1) the Acyl-Pro peptide bond adopts the cisPro rotamer in all; and 2) the aliphatic side chains of the acyl group and the Alp side chain are involved in van der Waals type interactions. The cisPro rotamers of even the bulkiest motifs, namely Ibu-Pro-Val-OMe, Piv-Pro-Ile-OMe and Piv-Pro-Leu-OMe crystallize, stabilized by van der Waals packing between aliphatic groups of the acyl and the Leu/Ile/Val side chains. Where the side chains are not long enough to make sub-van der Waals contacts with each other, their acyl C′-Cα σ-bond rotations are restricted due to Oσ* interactions involving the charge on the acyl carbonyl O. Where this occurs, the short space between the acyl and Alp side chains are filled in by aliphatic groups from neighbouring molecules at sub van der Waals distances. The Pro, Alp and χ1(Alp) dihedral angles are restricted to narrow range of values, irrespective of the length of Alp side chain, indicating that this backbone conformation is a conformational minimum when i+3i backbone H-bond is removed, with Pro at the i+1st position. This is further substantiated in Piv-Pro-Gly-OMe, which crystallizes in trans-Pro form, but still adopts similar backbone dihedral angles in spite of lacking any Alp side chain for interactions with the acyl group. Three of the Acyl-Pro-Aro-OMe models also crystallize in cisPro rotamer forms – both exhibit van der Waals type contacts between the Acyl group and backbone of Phe, rather than the aromatic ring of Phe. The phenyl ring of Phe may or may not form intramolecular Ph•••Pro inter-side chain contacts – which is not a pre-requisite for cisPro stabilization. No C-H••• interactions are observed anywhere in these peptides – van der Waals type contacts alone predominate in all cases. There are no abnormal distortions in bond angles or lengths even in the most sterically hindered cases, signifying that the conformations of these cisPro rotamers involving aliphatic•••aliphatic type contacts are natural minima. Chapter 4 Section B: Mining the PDB for Statistical Evidence of van der Waals interactions Section B of chapter 4 describes the data mining and statistical analyses of Xaa-cisPro-Phe, Xaa-cisPro-Val and Xaa-cisProLeu sequences in the PDB. The PEARL program was used to mine the PDB data. The overall frequency of 5.3% for appearance of cisPro among all Xaa-Pro peptide bonds, improves when Xaa is Phe or Tyr. However, several anomalies highlight the need for refining the analyses set to only those sequences where the side chains of Xaa and Aro/Alp face each other. In this refined set, clearly, inter side chain Xaa•••Alp/Aro contacts take precedence over even Aro•••Pro interactions at Aro-cisPro sequences (where Xaa is Aro). The Phe and the Leu side chains induce similar conformational effects on the preceding Xaa-Pro backbone. So does Val. Strong aliphatic•••aliphatic inter side chain contacts at van der Waals distances are observed to flank cisPro in several proteins. Substitution at the Cα of Xaa governs the proximity of the approaching side chain of Alp / Aro residue. The Cα-H of Xaa steers away from the Aro side chain at Xaa-Pro-Phe sequences, as the Aro group gets closer to it – implying the absence of ordered C-H••• contacts between them. There is consistent parallel alignment between Cα-Cβ -bond of Xaa and the C -C bond of the approaching side chain of Alp or Aro group – clearly highlighting the presence of van der Waals type interactions between them. All these evidences clearly point towards the van der Waals nature of local interactions at cisPro-Aro/Alp peptide sequences. Chapter 5: A novel 4-carbon covalent surrogate model for peptide H-Bond Chapter 5 describes the design and synthesis of novel 4-carbon covalent surrogates for the peptide H-bond (HBS). These surrogates would allow the unique constraining of two peptide strands in their extended conformations. The covalent HBS contain four orthogonal functional groups for independent extension at all of the four ends – similar to an endogenous inter-strand peptide H-bond. The synthesis of the surrogate is achieved by directly using natural chiral amino acid derivatives, beginning from amino alcohols obtained from reduction of desired amino acids. Suitably N-protected alcohols undergo oxidation to aldehyde followed by Grignard addition of allyl magnesium bromide, TBDMS protection of the homoallylic alcohol and reductive ozonolysis of the olefin to get a primary alcohol which is subject to Fukuyama-Mitsunobu reaction with desire protected peptide. The residue preferences that produce strongest inter-strand H-bonds were explored. The designed 4-carbon covalent HBS was incorporated using this methodology in a Gramicidin-S analogue, its first structural mimic containing only a single turn motif. This HBS model will have wide applications for constraining peptides in a number of secondary structures.