Introduction of Stereo Chemical Constraints into β -Amino Acid Residues

Abbreviations

SBT: Seabuckthorn; RVNA: Rabies Virus Neutralizing Antibody; RFFIT: Rapid Fluorescent Focus Inhibition Test; LPS: Lipopolysaccharide; MPL A: Monophosphoryl Lipid A; CTL: Cytotoxic T-lymphocyte; CFTRI: Central Food Technological Research Institute; SC-CO2: Supercritical Carbon Dioxide; SCE: Supercritical Carbon Dioxide Extract; Rb: Inactivated Rabies antigen; QS21: Quillaja saponaria fraction 21; TNF-α: Tumor Necrosis Factor-alpha; IL-1β: InterLeukin-1 beta; CD: Cluster of Differentiation; FBS: Fetal Bovine Serum; PE: Phycoerythrin; FITC: Fluorescein IsoThio Cyanate

Introduction

Over the last 20 years, a large body of work in the literature has focused on the folded structures formed by peptide sequences containing backbone homologated residues. Currently increasing interest in peptide based vaccines for several infectious diseases, and non-infectious diseases. The work of Seebach in Zurich [1] and Gellman in Madison [2], established that oligomers of β amino acid residues can form novel helical structures in solution and in the solid state. Two distinct types of hydrogen bonded helical structures were demonstrated in these studies for oligomeric β peptides. The C₁₂ helix which is an analog of the canonical 3C₁₀ helical structure in “all α” sequences, has the same hydrogen bond directionality (C=O_i…..H-Ni+3). The second helical form, the C₁₄ helix, has the opposite directionality (C=O_i…..H-N_i+4), which is unprecedented in α peptide sequences [3,4]. These reports sparked a flurry of activity on the conformational properties of β peptide oligomers. An early study from Appavu et al. [3-7], had demonstrated that unsubstituted β and γ amino acid residues can be incorporated into oligopeptide helices, without disturbing the overall helical fold [4].

β-amino acid residues

This study suggested that hybrid sequences with expanded hydrogen bonded, rings could indeed be constructed. A very large number of recent studies have greatly expanded our understanding of the conformational properties of substituted β and γ residues, when incorporated into α peptide host sequences [5]. One approach that has been investigated in this laboratory is to examine the role of gem dialkyl substitution on the conformational properties of β and γ residues. The ready availability of the achiral β, β-disubstituted γ amino acid, gabapentin (1-aminomethylcyclohexaneacetic acid, Gpn), has permitted detailed exploration of the structural combinations, (+60°, +60°) and (-60°, -60°) are indicated. (21 structures in (a) and 11 in (b)) (Figure 1).

Figure 1: Chemical structure of Gabapentin (Gpn).

The presence of gem dialkyl substituents at the central β carbon atom, limits the accessible conformations about the C^β-C^γ(θ₁), and C^β-C^α(θ₂) bonds. A large body of crystallographic evidence has been presented, which suggests that in the case of Gpn θ₁, and θ₂, are largely restricted to gauche conformations (θ₁±60, θ₂±60), a property that favors locally folded conformation at this residue. Consequently, a very large number of folded peptides have been characterized, in which diverse hydrogen bonded rings are facilitated by the gabapentin residue [2-7]. Figure 2 provides a summary of the conformational characteristics for the gabapentin residue.

Figure 2: Nine torsion angles θ₁ and θ₂ in gabapentin. (a) Gpn with chair conformation of the cyclohexane ring in which the aminomethyl group is equatorial and carboxymethyl group axial, (b) the inverted chair conformation of the cyclohexane ring with respect to (a), aminomethyl group is axial and carboxymethyl group is equatorial.

The success in generating folded structures in hybrid peptides containing, the gabapentin residue prompted an examination of the related β-amino acid residues 1-aminomethylcyclohexane carboxylic acid (β^2,2Ac₆c), and 1-aminocyclohexane acetic acid (β^3,3A_cc). Figure 3 shows the structures of the four related residues, all of which possess 1,1-disubstituted cyclohexane rings. The parent α amino acid residue 1-aminocyclohexane 1-carboxylic acid (Ac₆c) has been conformational characterized in a number of synthetic peptides [8]. The Ac₆c residue strongly favors helical conformations, with φ60±30, and ψ30±20. Thus, both β-turn and 3C₁₀/α-helical structures can readily accommodate the Ac₆c residue. Two β amino acid homologs may be considered viz β^2,2Ac₆c, and β^3,3A_cc. Earlier studies from this laboratory focused on the more readily synthetically accessible residue β^3,3A_cc.^3,4,5,6,7,8 X-ray crystallographic characterization of a number of small peptides containing the β^3,3A_cc residue revealed that internally hydrogen bonded conformations were rarely observed. The overwhelming majority of the β-amino acid residues (149) adopt gauche conformation (θ=±60°). Out of a total of 210 examples, 61 residues adopt the Trans conformation (φ=-180°). Figure 4 provides a summary of φ,ψ values for all β amino acid residues in which θ values of ±60° have been obtained. Most β^3,3A_cc amino acid residue fall out outside the region, expected for intramolecularly hydrogen bonded structures, which have been characterized for other β amino acid residues. Only one example of a hydrogen bonded hybrid αβ C₁₁ turn has been observed in Piv-Pro-β^3,3A_cc-NHMe. These results suggest that the intrinsic conformational preferences of the β^3,3A_cc residue may not readily facilitate its incorporation into folded, intramolecular hydrogen bonded structures in short peptides. Therefore, an examination of the conformational properties of the isomeric β^2,2Ac₆c residue was undertaken.

Figure 3: Definition of backbone torsion angles in , β and γ amino acid residues.

Figure 4: The scatter plot in 2D space (θ = ±60°) for intramolecular hydrogen bonded β-residues. Regions accommodating C₁₂ and C₁₄ helices (ββ) and C₁₁ helices (αβ) are shaded.

Thus far, relatively few structural reports are available for β^2,2Ac₆c residue. Figure 5 shows a view of the structure of the peptide Boc-β^2,2Ac₆c-β^2,2Ac₆c-β^2,2Ac₆c-OMe which was already reported at the time these studies undertaken. In this structure the unusual ββ C₁₀ hydrogen bond with reverse directionality and a C6 hydrogen bond were [9,10]. Chapter 5 describes hydrogen bonded C₁₁ turns obtained in αβ hybrid sequences incorporating the β^2,2Ac₆c residue.

Figure 5: Molecular conformation of the peptide Boc-β^2,2Ac₆c-β^2,2Ac₆c-β^2,2Ac₆c-OMe, which has a single C6 hydrogen bond at β^2,2Ac₆c(1) and a non-helical ββ C₁₀ hydrogen bond with reversed directionality, at the β^2,2Ac₆c(2)- β^2,2Ac₆c(3) segment. The backbone dihedral angles are marked [10-12].

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