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Desisto, Kevin S.

Chemistry

2004

Ph. D.

The poly-L-proline type II (PPII) secondary structure has emerged a key binding conformation for many protein-protein mediated cellular pathways. A wealth of NMR and x-ray data have shown short spans of large globular proteins bind in the PPII helix or close related geometries. Proline is often found in peptides as a scoffolding residue with some hydrophobic interactions. Proline helps to stabilize the PPII secondary structure and is the only "constrained" natural amino acid. Constraints include the trans amide bond is favored over the cis in polyproline by 2-3 kcal/mol, minimization of pseudo A(1,3)-strain from the two (2) pyrrolidine rings in a Pro-Pro peptide bond, and the substituents attached to the ring (i.e. C-terminal). Often, non-proline amino acids (i.e. lysine, arginine, etc.) are the critical residues which make contact with the substrate. Ironically, non-prolyl residues have not been found to stabilize a PPII helix.
Therefore it was envisioned, the synthesis of proline analogs that contain functionality of other amino acids. These proline analogs, or Proline_Templated_Amino_Acids (PTAAs), could then be put in place of the natural residue(s) in a short peptide span and would be expected to exist in or form a PPII helix more readily compared to natural "wild type" peptides. The first exocyclic dihedral angle of a side chain, chi angle (Xl), is important when binding with a substrate. It has been shown Xl of SH3 domain bound peptide residues to favor gauche (-) and trans (-180) dihedral angles. Synthesis of 3-substituted proline analogues was accomplished and the compounds subjected to conformational analysis. Using GMMX (PCModel 7.0) to search for global minima of N-acetyl-3alkylproline and two distinct ring puckers were found of virtually equal energy. The spin-spin coupling constants of the endocyclic protons were assigned and subjected to NMR simulation.
The J-values were analyzed using a modified Karplus equation (PSUEROT) to determine the endocyclic torsion angles, the maximum ring pucker (Pmax), and thus the side chain angle, Xl for these systems. The results suggest the ring of the 3-alkylproline analogue(s) exist as ~50:50 mixture of two conformations, a North and a South type conformation as described on a pseudorotational wheel. These conformations would allow Xl of 3-substituted PTAAs access to approximate gauche (-) and trans side chain angles. As a result, these PTAAs could compete with a natural peptide, which binds the substrate with a similar geometry. Several 3-substituted proline analogues have been synthesized and incorporated into synthetic peptides for biological assay. Lysine has been unveiled as a key substrate contact residue in many peptides and proteins interactions. Therefore, it is desirable to generate combinatorial libraries for lead lysine analogues, esp. PTAAs. Rather than synthesizing several different lysine PTAAs individually, a method for rapid diversification from a common intermediate would be more efficient.
Methodology for the solid-phase reductive amination and reductive alkylation of PTAA toward lysine libraries has been developed. A PTAA is attached to a solid support and, in the case of reductive alkylation, a nitrogen side chain is subjected to imine formation by reaction with an aldehyde of desired functionality. Hydride reduction of the imine to the amine follows to furnish the lysine analog upon cleavage from the solid support. Reductive amination is the reaction of an aldehyde side chain with an amine of desired functionality followed by reduction of the imine. These methods allow for the generation of lysine libraries with potentially limitless functionality, as a secondary or tertiary amine, without the time of developing a process for each analog.