Investigating the Intercarbonyl X...C' (X=O/S/N) Interactions in Short Peptides and Peptidomimetics. Evidence of charge->II* Interactions. Synthesis and Characterization of Thioimidate Isostere Containing Peptidomimetics

Abstract

This thesis entitled “Investigating the Intercarbonyl X···C′ (X = O/S/N) Interactions in Short Peptides and Peptidomimetics. Evidence of Charge→π* Interactions. Synthesis and Characterization of Thioimidate Isostere Containing Peptidomimetics” is divided into two chapters. First chapter is further subdivided into four sections where investigation of the nature of intercarbonyl X···C′ (X = O/S/N) interactions in short peptides and peptidomimetics has been described. The second chapter also has been subdivided into three parts where the syntheses and characterization of thioimidate (1,3-thiazine) and imidate (1,3-oxazine) isostere containing peptidomimetics have been discussed. Chapter 1: Section A: Revisiting the earlier models for the intercarbonyl O···C′ interactions The proximity between carbonyls is ubiquitous in crystals. Here we review the key reports that have assigned an n→π* nature to interactions between carbonyl oxygen (O) atoms and adjacent carbonyl carbon (C′) atoms (O···C′). Based on earlier hypotheses (by Burgi-Dunitz) that suggest that “the minimum energy trajectory of a nitrogen nucleophile adding to the C′ of carbonyl is at N···C′ distances of ≤ 3.2 Å and along N···C′=O angles of 109±10o”, the optimum trajectory for addition of an O to an adjacent C′ has also been assigned to be the same (O···C′ distance ≤ 3.2 Å and O···C′=O angle is 109±10o). Additionally, all O and C′ atoms within these boundary conditions in crystal structures were assigned a status of interacting and those outside of the same as non-interacting. Based on quantum mechanical models for electronic orbitals that contain the valence electrons of such proximal O and C′ atoms – derived through NBO (Natural Bond Order) calculations (on crystal structures) – it has been proposed that the filled non-bonding lone pair orbital of the O (donor) overlaps with the empty π* orbital of the carbonyl C′ (acceptor), in these O···C′ interactions. Hence, these have been termed as n→π* interactions. Using DFT (Density Functional Theory) calculations energies for these interactions have been predicted to range from 0.5 to 5.0 kcal mol-1, which are similar to those for other strong non-covalent interactions such as H-bonding, weak cation-π, etc. This n→π* interaction model is assumed to prevail between adjacent carbonyls (Oi-1···C′i) at Xaa-Pro dipeptide motifs and to be exclusively responsible for the changes in equilibrium constant values (Kc/t) for the trans to cis isomerisation reaction at Xaa-Pro peptide bond in chosen analogue molecules. Based on this assumption, these Kc/t values have been used as direct experimental equivalents for the energies of these n→π* interactions. Simultaneous to such review of literature, this chapter highlights several anomalies in this n→π* model for the intercarbonyl O···C′ interactions. We discuss the alternate models that also exist for the O···C′ proximities and show that several features – such as improved pyramidalization at the acceptor carbonyl; decrease in Kc/t values at Xaa-Pro peptide bonds; and small changes in 13C NMR chemical shift values for the acceptor carbonyls; etc. – that accompany the shortening of O···C′ distance, can be explained without invoking the n→π* interaction model. Moreover, we discuss key observations such as the presence of near-symmetric antiparallel short contacts between carbonyl groups (C=O) in crystal structures, which cannot be explained by the quantum mechanical n→π* model for the O···C′ interactions. Chapter 1: Section B: Spectroscopic and kinetic investigations into the nature of X···C′ (X = O/S) interactions in N-acyl homoserine lactones (AHLs) In this section the key interactions involving the adjacent carbonyls in model N-acyl homoserine lactones (AHLs) (which are signalling molecules in quorum sensing) in solution, their electronic nature and their influence on solvolysis of the lactone ring have been investigated. Earlier, in the crystal structures of two sterically encumbered synthetic AHL analogues N-trimethyl acetyl homoserine lactone and N-tribromoacetyl homosrine lactone the presence of an n→π* orbital overlap type interaction between Oacyl and C′lact had been suggested. Based primarily on this, the operation of similar OacylC′lact interaction was proposed in all AHLs in their solution conformations as well. More intriguingly, the interaction was hypothesized to decrease the rates of lactone hydrolysis, rendering AHLs with longer biological half-life. This is contrary to physical organic understanding of nucleophilic catalysis of addition to carbonyls. Here we synthesize a variety of AHLs and analyze their NMR and FT-IR data in solution. The spectral data reveal that the role of the N-acyl group in AHLs is to withdraw eˉs from lactone C=O inductively and to improve electronic shielding at C′lact. Lack of appreciable changes in C=O stretching frequencies of lactone and 13C NMR chemical shift values of C′lact indicate the absence of electronic perturbation of the π* of the lactone. Similar non-variance of spectral bands with improvement in nucleophilicity of the N-acyl group indicates the absence of any evidence for n→π* nature for the O···C′ interactions (between the lone pair of eˉs from Oacyl to π* at C′lact). Further the spectroscopic data indicate that any change in charge at the acyl O is felt by C′lact and this weak interaction releases energy in the order of ≤ 0.05 kcal mol-1. The combined influence of the electron withdrawing N-acyl group and the weak Oacyl···C′lact interaction in AHLs is that, increasing the charge at Oacyl increases the rate of solvolysis of lactone. Analysis of the conformation of the lactone ring in the LuxR receptor-bound and unbound crystal structure forms reveals the flattening of the puckered ring in the LuxR bound state – facilitated by several interactions with the receptor. Conserved interactions between LuxR and AHLs lock the N-acyl carbonyl motif such that they are orthogonal to the lactone carbonyl and intramolecular interaction between Oacyl and C′lact is precluded. We propose the design of flat cyclic analogues of γ-butyrolactone bearing electron withdrawing side chains as potential molecules for taking advantage of bacterial quorum sensing in environmental applications and biotechnology. Chapter 1: Section C: Spectroscopic investigation into the nature of intercarbonyl X•••C′ (X = O/S/N) interactions: Carbamyl-cisPro model systems In this section we investigate the nature of intercarbonyl X···C′ interactions in carbamyl-Pro model systems using spectroscopic methods like FT-IR and 1D NMR. Further we derive the enthalpic and entropic contributions towards the free energy for trans to cis isomerization (Kc/t) at these model carbamyl-Pro systems. Our results reveal that changes in Kc/t values cannot always be used as proof for the presence or absence of electronic interactions, and hence to unambiguously suggest the nature of these interactions. Cis/trans isomerism exists at Xaa-Pro amide and carbamate motifs, and it was proposed that in acyl-Pro systems the O···C′ interactions are responsible for the stability of either cis or trans depending upon their direction of operation (Forward direction: O of Xaa is the donor of electrons to π* at C′ of Pro; Reverse direction: O of Pro is the donor of electrons to π* at C′ of Xaa). Investigation of the carbamyl-Pro systems can shed further light on this hypothesis. Hence we undertook the first spectroscopic and Van’t Hoff analysis of homologous carbamyl-Pro model systems. The Kc/t of the homologous series surprisingly increased with increase in the bulk at R (R varies from Me to tBu). The spectroscopic data revealed the presence of charge→σ* interactions at carbamyl groups. This interaction locks the carbamyl motif in the s-transoid conformation, along the C′-O σ-bond. Such conformational lock is observed to be greater in carbamyl groups where R has at least one Cα-H bond. Interestingly, we observe the absence of X···C′ electronic interactions that may selectively stabilize the cisPro conformer in these molecules. Van’t Hoff analyses on the other hand showed that as the number of Me substituents in R increases (R = Me to iPr), there is a favorable increase in entropy ( So) associated with the transPro to cisPro conformational isomerism. As a result, the population of the cisPro conformer improves significantly as the steric bulk at R increases. We note that the enthalpy of cisPro is however relatively small and remains unfavourable as R-bulk increases (Me to iPr). These data reveal the influence of electrostatic interactions between charged groups, on the change in entropy associated with cis/trans isomerism at carbamayl-Pro motifs. This not only opposes the n→π* model, but also provides an example for the important point that changes in Kc/t can/should not be taken as direct evidences of any single electronic interaction. Importantly, this study provides another example where electronic interactions between charged, polarized carbonyl motif rather than nonbonding lone pair eˉs of carbonyl motifs influence cis/trans isomerism at Xaa-Pro systems. Chapter 1: Section D: Investigation of the stereoelectronic nature of the X···C′ (X = O/S) contacts In this section we provide experimental evidence for the existence of inverse correlation between the charge on the O nucleophile and the O···C′ distances. We show that O and C′ atoms (of adjacent carbonyls), which are separated at distances > 3.20 Å in carefully chosen analogues, come together to σ-bonding distances when the charge on O is increased to -1. Additionally, the influence of backbone steric factors on these charge→π* interactions is investigated. A partial covalent nature was proposed for the O···C′ interactions. Our study showed that the shortest intercarbonyl O···C′ distances between the O of 1°, 2° and 3° amide carbonyls and proximal C′ in molecules found in the Cambridge Structural Database (CSD) (v5.36, November 2014) show an inverse linear correlation with the partial negative charge (δ‾) on the amide carbonyl O rendered by natural amide carbonyl polarization. These data suggest the interaction of charge on the nucleophilic O with π* of the acceptor carbonyl. Further on increasing the charge on nucleophilic carbonyl O to -1 in the model compound, we achieved the formation of σ-bond through non-native (natively disallowed) Oi‾¹→C′i-1 interaction. Here we provide the first experimental evidences that suggest the interaction between charge of O and π* at adjacent C′ (the charge→π* interaction) and the latent covalent nature of the O···C′ interactions. This charge→π* model explains the origins of variations in O···C′ distances (3.20 Å–1.43 Å) in proteins and complexes that occur to suit biological functions; and the mutual interactions between antiparallel carbonyls. Further the effect of 3 key steric factors – namely the allowed τ (N-Cα-C′) angle, entropy and allowed (ϕ,ψ) angles – on the non-native Oi→C′i-1 interactions were investigated in the model compounds. Our kinetic data revealed that, the allowed τ angles have the greatest influence on charge→π* interaction, followed by entropy. Importantly the allowed (ϕ,ψ) torsional angles for residues, that govern protein folding pathways, have little influence on the O···C′ electronic interactions. Chapter 2: Section A: Design and synthesis of novel 1,3-Thiazine containing peptidomimetics This section describes the first synthesis of peptidomimetics containing the 1,3-thiazine isostere (thioimidate isostere for the peptide bond), at the C-terminus and also at the middle of the peptide. The synthesis of the 6-membered heterocycles – 1,3-oxazine (Oxa) – have earlier been reported. Oxa motifs constrain preceding amino acid backbones into natively disallowed conformations. Here we present the first synthesis of peptidomimetics containing the 1,3-thiazine (Thi) (the thioimidate analogue of Oxa) motif, by the treatment of N-(3-hydroxypropyl)thioamides with MsCl/Et3N, which leads to intramolecular S-alkylation / cyclization. When placed at the C-terminus of acyl-Pro motifs the Thi group selectively improves the stability of the rare s-cis conformation of the acyl-Pro peptide bond. Further this method has been used to synthesize peptidomimetics in which an endogenous peptide bond is replaced with the Thi isostere. These Thi analogues are shown to be stable to standard conditions of peptide coupling and N- and C- terminus protection, deprotection and can be extended selectively at their N- or C- termini. Chapter 2: Section B: Epimerization in 1,3-Thiazine containing peptidomimetics The epimerization in 1,3-thiazine containing peptidomimetics and its mechanism has been described in this section. Further the aggregation behaviour of these thiazines, in solution and crystal structures, has been studied. It has been well-documented that epimerization (Racemization) occurs at the chiral centers at the C(2) exo methine of 1,3-thiazolines and 1,3-thiazoles. Similar epimerizations in 1,3-thiazines have however not been explored. Here we report our observation of epimerization in chiral aminoacid (non Pro) containing 1,3-thiazine peptidomimetics. Our studies revealed that, the epimerization happens at C2 positions of chiral (non-Pro) amino acids-derived 1,3-thiazine containing peptiomimetics. And NH of chiral (non-Pro) amino acid fused to Thi ring at C2 position is necessary for the epimerization. Further we investigated the Boc-Xaa*-Thi analogues in solution, which showed two resonances for the carbamate N-H (HN) and the H of Xaa*, irrespective of the side chain in Xaa, in CDCl3 a weakly polar solvent. The integral ratios of the major : minor peak increased with increase in concentration for Boc-Val*-Thi, indicating the formation of H-bonded aggregates. Even in the polar aprotic (DMSO-d6) and polar protic (D2O) solvents the two sets of resonances were observed for Boc-Val*-Thi in 1H NMR. But when the thioimidate N is protonated (N of Thi is no longer a H-bond acceptor), showed only a single set of resonances. Formation of intermolecular H-bonds involving N of Thi in solution is thus evident in the aggregates. This is further suggested by the crystal structures obtained for the peptide mimetics Boc-Val*-Thi, Boc-Leu*-Thi and Boc-Phe*-Thi in which the racemic pair, instead of one enantiomer of it, are present in the unit cell and are locked in a pair of intermolecular 10 membered H-bonding interactions between NThi and HLeu* similar to an antiparallel β-sheet. A mechanism for racemization is proposed, where this strong H-bond assists enamination/racemization process. Chapter 2: Section C: Influence of a disallowed conformation of Aib on the structure of a 310-helical fold In this section, the effect of the presence of a disallowed conformation of Aib at the C-terminus of a 310-helical peptide, on the structure and fold of the rest of the peptide body has been studied in solution. We constrain the C-terminal Aib in the Aib-rich octapeptide (N-tert-butoxycarbonyl-Leu1-Aib2-Ala3-Leu4-Aib5-Ala6-Phe7-Aib8-CO2Me (1), which adopts a complete 310-helical conformation throughout the peptide body in the crystal structure and in solution) in one of its disallowed conformations using a method earlier developed in our group. This involves the synthetic modification of the C-terminal ester (Aib8-CO2Me) in 1 to an Oxa (Aib*8-Oxa) in 2 and the study of its effect on the peptide body. Analyses of the solution FT-IR, CD, ¹H, 2D (TOCSY, HSQC, HMBC and ROESY) and solvent polarity dependent NMR data reveal that 2 adopts a 310-helical conformation similar to that of 1. The C-terminal CO2Me → Oxa (E → O) modified Aib*8-Oxa motif is constrained in a unique conformation where the two Cβ atoms of Aib*8 are staggered with respect to the Aib*8 C=O and are both interacting with the two Hβ of Phe7. Here the Aib* backbone is constrained by a 5-membered ring NOxa∙∙∙HAib* H-bond, in a C5i structure. Solvent polarity dependent ¹H NMR data indicate the formation and persistence of C5i H-bond at the Aib*8-Oxa motif in 2. Analyses of the ROESY, solvent polarity dependent ¹H NMR and CD spectra reveal that four crucial changes in ROESY cross peaks occur at the Phe7-Aib*8 motif of 2, compared to that in 1. From these spectroscopic data it has been confirmed that there is no change in the structure of 2 from Leu1 to Ala6. Whatever the crucial changes happened are at Phe7-Aib*8 motif of 2. Hence our study showed that the significant structural consequences of this disallowed conformation of Aib* are primarily observed to occur in the residue in its immediate vicinity, rather than in the whole peptide body. Presence of a disallowed fold at a residue need not result in disruption of the structure, or the overall fold, in the rest of the peptide body.