Academic literature on the topic 'Hydrogen Bond Surrogate'

A novel hydrogen bond surrogate-based (HBS) α-helix mimetic was designed by the combination of covalent H-bond replacement and the use of an ether linkage to substitute an amide bond within a short peptide sequence. The new helix template could be placed in position other than the N-terminus of a short peptide, and the CD studies demonstrate that the template adopts stable conformations in aqueous buffer at exceptionally high temperatures.

This thesis entitled “Design and Synthesis of Peptidomimics Constrained in Helical and Sheet Conformations Using a Novel Covalent Surrogate for the Peptide Main Chain Hydrogen Bond” is divided into six chapters. Chapter 1: Introduction to Ordered Conformations of Peptides and Strategies for Constraining Short Peptides in Ordered Conformations. The first chapter describes the different types of protein secondary structures and introduces the various prominent strategies developed thus far to constrain short peptides in ordered secondary structure-like conformations, with specific emphasis on helical and parallel β-sheet folds. Chapter 2: Design of Structure and General Methodology for the synthesis of Novel H-Bond Surrogate Constrained Cyclic α-Helical Mimics Here we develop the first design of the propyl linker as a covalent surrogate for the peptide H-bond. The first synthetic methodology is described for the synthesis of constraining shortest peptide sequences (tripeptides) in α-helix-like conformations. The Macrolactamization strategy proved to work best as the final step for cyclization. All residues of the turn are completely retained in the constrained sequence, unlike any other earlier method. More importantly, there are no metal involved as catalysts in any of the synthetic transformations, hence removing the problem of metal-bound cyclic structures – which have otherwise rendered these structures non-usable as drug leads in the earlier models. Gly-rich peptides have been constrained as extreme cases of highest chain entropy and least helix propensity. Both secondary and tertiary amide containing peptides have been synthesized using this protocol. Note that the macrolactamization was found to be better than the Fukuyama-Mitsunobu N-alkylation protocol for the final cyclization step. Chapter 3: Synthesis of C-terminal Extended HBS-Constrained Helical Turn Mimics – Validation of the Versatility of Current synthetic protocol The developed cyclization protocol is extended towards the synthesis of C-terminal extended α-helical turn mimics using a solution phase peptide synthesis procedure. Peptides which extend belong the helical turn by a high entropy Gly-residue at the C-terminal are synthesized. The versatility of the synthetic methodology to accommodate sterically constrained amino acid residues – in the form of phenylalanine residue – at any of the positions i+1, i+2 or i+3 of the constrained helical turn is demonstrated. The synthesized are easily isolated without need for column chromatography, in high purity and good yields – this is due to the presence of the N-terminal amino group, salts of which are easily triturated to remove all other organic impurities. Chapter 4: Synthesis and CD conformational analyses of HBS constrained α-Helical turn mimics containing residues with improved helical propensities Alanine residue has the highest helix propensity among all other natural α-amino acid residues. Its enthalpic contribution to the helical conformation is 1 kcal/mol more than that for the Gly residue, which has the least propensity. Incorporation of Ala residue in the Gly-rich cyclic sequences in either the middle of constrained tripeptide or as the C-terminal extended residue has been accomplished. Comparison of the CD spectra of the synthesized cyclic α-helical turn peptides reveals that a tertiary amide linkage is essential for the propyl linker at the C-terminal amino appendage, for helicity to be observed. Helicity improves upon introduction of the first extended residue. The constrained and C-terminal extended α-helical turn mimics show consistently high helicity irrespective of the helix propensities of the component residues showing that the covalent propyl linker surrogate for the H-bond overwhelms the natural propensities of individual amino acid residues towards enabling stabilization of the helical turn and offer far better structural organization to this cause. Chapter 5: Synthesis of shortest HBS-constrained 310 and - helical peptide analogues The unique versatility of the novel covalent propyl linker surrogate for the peptide H-bond is exhibited by its ability to constrain dipeptides in 310-helix like structures. This is the first and the only HBS model that can achieve this synthetic target as the synthetic protocol allows the conservation of both the residues as is in the constrained helical turn. Similarly, the trapping of a pentapeptide in a C-terminal extended rare and unstable -helix like cyclic structure using the current HBS linker is achieved. Considering the high entropic cost for cyclizing such a long 16-membered chain into a constrained structure, this again exhibits the versatility of the currently developed HBS design and the currently developed synthetic methodology. Chapter 6: First design and synthesis of novel H-bond surrogate constrained parallel β-sheet mimics H-bonding interactions stabilize another prevalently observed secondary structure, other than helical structures, namely the -sheets. The parallel -sheets that almost qualify for super secondary structures due to the high contact orders in them are thought to mimic in models, unlike the easier antiparallel -sheets. Here we replace the inter-strand peptide H-bond between parallel -strands to create excised templates as parallel -sheet nucleators. The propyl linker acts as a dynamic linker in these models and the two amino groups are protected with bulky sulphonamides, in order to provide Thorpe-Ingold effect to the peptide chain. The protocol for synthesizing these models has been described and the different analogues that are synthesized thus have been described. This is the first instance of synthesis of parallel -sheet mimics using the covalent surrogates for the peptide H-bond.

This thesis entitled “Design and Synthesis of Peptidomimics Constrained in Helical and Sheet Conformations Using a Novel Covalent Surrogate for the Peptide Main Chain Hydrogen Bond” is divided into six chapters. Chapter 1: Introduction to Ordered Conformations of Peptides and Strategies for Constraining Short Peptides in Ordered Conformations. The first chapter describes the different types of protein secondary structures and introduces the various prominent strategies developed thus far to constrain short peptides in ordered secondary structure-like conformations, with specific emphasis on helical and parallel β-sheet folds. Chapter 2: Design of Structure and General Methodology for the synthesis of Novel H-Bond Surrogate Constrained Cyclic α-Helical Mimics Here we develop the first design of the propyl linker as a covalent surrogate for the peptide H-bond. The first synthetic methodology is described for the synthesis of constraining shortest peptide sequences (tripeptides) in α-helix-like conformations. The Macrolactamization strategy proved to work best as the final step for cyclization. All residues of the turn are completely retained in the constrained sequence, unlike any other earlier method. More importantly, there are no metal involved as catalysts in any of the synthetic transformations, hence removing the problem of metal-bound cyclic structures – which have otherwise rendered these structures non-usable as drug leads in the earlier models. Gly-rich peptides have been constrained as extreme cases of highest chain entropy and least helix propensity. Both secondary and tertiary amide containing peptides have been synthesized using this protocol. Note that the macrolactamization was found to be better than the Fukuyama-Mitsunobu N-alkylation protocol for the final cyclization step. Chapter 3: Synthesis of C-terminal Extended HBS-Constrained Helical Turn Mimics – Validation of the Versatility of Current synthetic protocol The developed cyclization protocol is extended towards the synthesis of C-terminal extended α-helical turn mimics using a solution phase peptide synthesis procedure. Peptides which extend belong the helical turn by a high entropy Gly-residue at the C-terminal are synthesized. The versatility of the synthetic methodology to accommodate sterically constrained amino acid residues – in the form of phenylalanine residue – at any of the positions i+1, i+2 or i+3 of the constrained helical turn is demonstrated. The synthesized are easily isolated without need for column chromatography, in high purity and good yields – this is due to the presence of the N-terminal amino group, salts of which are easily triturated to remove all other organic impurities. Chapter 4: Synthesis and CD conformational analyses of HBS constrained α-Helical turn mimics containing residues with improved helical propensities Alanine residue has the highest helix propensity among all other natural α-amino acid residues. Its enthalpic contribution to the helical conformation is 1 kcal/mol more than that for the Gly residue, which has the least propensity. Incorporation of Ala residue in the Gly-rich cyclic sequences in either the middle of constrained tripeptide or as the C-terminal extended residue has been accomplished. Comparison of the CD spectra of the synthesized cyclic α-helical turn peptides reveals that a tertiary amide linkage is essential for the propyl linker at the C-terminal amino appendage, for helicity to be observed. Helicity improves upon introduction of the first extended residue. The constrained and C-terminal extended α-helical turn mimics show consistently high helicity irrespective of the helix propensities of the component residues showing that the covalent propyl linker surrogate for the H-bond overwhelms the natural propensities of individual amino acid residues towards enabling stabilization of the helical turn and offer far better structural organization to this cause. Chapter 5: Synthesis of shortest HBS-constrained 310 and - helical peptide analogues The unique versatility of the novel covalent propyl linker surrogate for the peptide H-bond is exhibited by its ability to constrain dipeptides in 310-helix like structures. This is the first and the only HBS model that can achieve this synthetic target as the synthetic protocol allows the conservation of both the residues as is in the constrained helical turn. Similarly, the trapping of a pentapeptide in a C-terminal extended rare and unstable -helix like cyclic structure using the current HBS linker is achieved. Considering the high entropic cost for cyclizing such a long 16-membered chain into a constrained structure, this again exhibits the versatility of the currently developed HBS design and the currently developed synthetic methodology. Chapter 6: First design and synthesis of novel H-bond surrogate constrained parallel β-sheet mimics H-bonding interactions stabilize another prevalently observed secondary structure, other than helical structures, namely the -sheets. The parallel -sheets that almost qualify for super secondary structures due to the high contact orders in them are thought to mimic in models, unlike the easier antiparallel -sheets. Here we replace the inter-strand peptide H-bond between parallel -strands to create excised templates as parallel -sheet nucleators. The propyl linker acts as a dynamic linker in these models and the two amino groups are protected with bulky sulphonamides, in order to provide Thorpe-Ingold effect to the peptide chain. The protocol for synthesizing these models has been described and the different analogues that are synthesized thus have been described. This is the first instance of synthesis of parallel -sheet mimics using the covalent surrogates for the peptide H-bond.

Thesis titled, “Design, Synthesis and Conformational Analysis of Hydrogen Bond Surrogate (HBS) Stabilized Helices in Natural Sequences. Helically Constrained Peptides for Potential DNA-Binding”, describes the development of a novel covalent hydrogen bond surrogate (HBS) model and its incorporation in short (4-8 residues) unstructured peptide sequences with coded amino acids, through a facile solution phase synthetic method (SPSM), to constrain them into α- helical conformations with highest known stabilities and helicities. The synthetic protocol was developed for mass scale combinatorial synthesis of helical peptidomimetics. NMR, FT-IR, CD spectra and molecular dynamics simulations of variants of the HBS-constrained helical peptidomimetics were analyzed to determine the optimum number of sp2 atoms and the residue preferences that yield both the α-helical and the 310-helical folds with high structural integrity in the shortest sequences. The HBS-constrained helical peptidomimetics were used to derive experimental evidence that the 2-state Helix-Coil Transition occurs at each residue during helix folding and that this process is entropically driven. Further, the role of temperature on the denaturation of secondary structures was investigated using these HBS-constrained helical models. Helical peptidomimetics of the DNA-binding domain in the zinc-finger human TTK protein have been synthesized, which have proven to be promising mimics for DNA-binding and subsequent transcription regulation.
CSIR

碩士
國立臺灣大學
化學研究所
101
There are many factors that contribute to protein folding and structure stability: intrinsic propensity of amino acids, side chain ion pairing interaction, hydrophobic effect, hydrogen bonding, van der Waals interaction. In this study, we focused on the effect of lysine side chain length on sheet propensity at a non-hydrogen bonded strand position in β-hairpin. The β-hairpin peptides HPTAlaXaa (Xaa= Dap, Dab, Orn, Lys) were designed with the side chain of Lys9 systematically shortened to investigate the effect of Lys side chain length on sheet propensity. The peptides were synthesized by solid phase peptide synthesis using Fmoc-based chemistry. All peptides were purified to 95% purity and were analyzed by 2D NMR experiments. Sequence specific assignment was performed. The hairpin structures were confirmed by chemical shift deviation, 3JHNα coupling constants,and NOE signals.The fraction folded and ΔG of peptides were derived by comparing the chemical shifts with the fully folded and unfolded reference peptides. The percent folding of HPTAlaXaa peptides with Lys analogs at the guest position followed the trend: HPTAlaDap ~ HPTAlaDab < HPTAlaOrn ~ HPTAlaLys, showing that the longer the Lys analogue side chain, the more stable the β-haiprin structure. The HIV Rev protein binds RRE RNA to regulate the transport of unspliced and spliced mRNA from the nucleus to the cytoplasm posttranscriptionally. The Rev peptide is a random-coil. However, the conformation of the Rev peptide changes to an α-helix while binding to RRE RNA. Hydrogen bond surrogate (HBS) is one of the several cross-linking systems for stabilizing an α-helix, using the covalent bond C=C-C-N to substitute the C=O…H-N (i, i+4) hydrogen bond in a short helix. In order to synthesize an HBS peptide, strategy for synthesis of dipeptides that contained an allyl group on the amino group was designed and refined. Two wild type Rev peptides were synthesized by solid phase peptide synthesis using Fmoc-based chemistry. The secondary structure of the two peptides was random-coil analyzed by circular dichroism spectroscopy. The binding specificity of the Rev peptides was determined by gel shift assay. The dissociation constants of the Rev peptides were similar to previous studies.

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.

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.