Kevin DeSisto’s entire Synthesis, Characterization, and Analysis of Proline Templacted Amino Acids Dissertation is presented on these Thesis pages as a viewable and downloadable PDF.

View and Download (or Print) here> 

The first ten pages of the Thesis are also presented as web text below with accompanying illustrations. The Part 2 and Part 3 sections also have the first ten pages (with illustrations) of the Thesis Chapters 3 and 5 as web text.

Kevin DeSisto’s entire Synthesis, Characterization, and Analysis of Proline Templacted Amino Acids Dissertation is presented on these Thesis pages as a viewable and downloadable PDF.

View and Download (or Print) here>   

The first ten pages of the Thesis are also presented as web text below with accompanying illustrations. The Part 2 and Part 3 sections also have the first ten pages (with illustrations) of the Thesis Chapters 3 and 5 as web text.


A Dissertation Presented
Kevin S. DeSisto
The Faculty of the Graduate College
The University of Vermont
February 2004


The poly-L-proline type II (PPII) secondary structure has emerged as 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 closely related geometries. Proline is often found in peptides as a scaffolding 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 (c1), is important when binding with a substrate. It has been shown c1 of SH3 domain bound peptide residues to favor gauche (-) and trans (-180) dihedral angles. Synthesis of 3-substituted proline analogs was accomplished and the compounds subjected to conformational analysis. Using GMMX (PCModel 7.0) to search for global minima of N-acetyl-3-alkylproline, 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 pucker (f­­max­), and thus the sidechain angle, c1for these systems. The results suggest the ring of the 3-alkylproline analogs exist as a ~50:50 mixture of the two conformations, a North and a South type conformation as described on a pseudorotational wheel. These conformations would allow c1 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 analogs 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 proteinsinteractions. Therefore, it is desirable to generate combinatorial libraries for lead lysine analogs, 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 a PTAA towards lysine libraries has been developed. 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 potential limitless functionality, as a secondary or tertiary amine, without the time of developing a process for each analog.

{T.O.C], {L.O.F.], [L.O.S]


Professor Jose Madalengoitia for his inspiring ideas and the University of Vermont, which supplied fertile grounds to grow these ideas into knowledge.

The Madalengoitia Group (.i.e. Amar, Rui, Floyd, Norm, Steve, Ahmed, Amy, Raksh, Sandy, and others) for listening to me and never being afraid to tell me I was wrong.

Madalengoitia Group meetings, how I will never see “MoM” the same again.

My Family, were always keeping me up-to-date on the progress of my thesis. They probably are as happy and satisfied the work is complete, as I am. Although it wasn’t easy, it’s nice to have support.

John Sharp and the lovely staff (i.e. Judy, Anna, and Andrea) for their dedication and friendly assistance.

To everyone who kept the World from “blowing up” before I could finish this body of work.

Thank you.

Chapter 1 – Introduction- Poly-L-Proline Type II Secondary Structure

Section 1.1 – Significance of PPII Secondary Structure

1.1.1 – Stability of the PPII helix

The poly-L-proline type II (PPII) secondary structure (Figure 1.1) has emerged as a key binding conformation for many protein-protein mediated cellular pathways.­­­­­­(1) The PPII  secondary structure was first identified in collagen (Figure 1.1a), a repeating sequence of (XYGly)n where X is often proline, Y is often hydroxyproline (Hyp), and Gly is glycine. A single triple-helical strand of collagen has been found to exist in a PPII helix. The unusual stability of collagen was once thought to be due to a hydrogen bonding network of water molecules to Hyp residues, which was feignedly confirmed by x-ray crystal structures.

Studies by R.T. Raines, et al. replaced several of the Hyp residues with 4-fluoroproline (Flp) in a short span of collagen like peptides.(2) The peptides were thermally denatured and the Flp containing helix was found to have higher thermal stability relative to the Hyp and Pro collagen mimics (Figure 1.2). Since F only H-bonds as HF, it was concluded that the collagen’s stability is not due to H-bonding of waterwithin its helix, but also the trend suggested electronegativity was the key factor. An electron withdrawing substituent helps inductively to stabilize the trans amide bond found in the PPII secondary structure. This stabilization is due in part to the gauche effect(3) where C-N and C-F antibonding orbitals (s*) of Flp maximize overlap with adjacent C-H bonding orbital (s) by putting the two vicinal electronegative atoms gauche (~60o). The gauche effect and other stereoelectronic effects (i.e. nàp*) fix the pyrrolidine ring of proline where the f, y, and w angles are found preorganized to values close to that in a collagen triple helix.

1.1.2 – The PPII helix defined

The PPII helix is an extended, left handed helix, which makes a complete turn every 3.3 residues defined by the dihedral angles f = -75o, y  = 145o,w = 180o (Figure 1.3). Minimization of ‘pseudo A(1,3)’ strain constrains the peptide C-C bond or psi angle (y) in an analogous manner similar to allylic strain, A(1,3) strain (Figure 1.3a). The electron resonance of the carbonyl gives the C-N amide bond sp2 character and the resulting geometry is constrained. The pyrrolidine ring of proline sets the phi angle (f) and the s-trans (w = -180o) peptide bond is favored over the s-cis by 2.3-5.0 kcal/mol.(4) A peptide possessing these dihedral angles, whether or not proline is present, is defined as a PPII helix.

1.1.3 – PPII Secondary Structure in biological systems

Recent reports detailing crystal structures,(5) as well as NMR data,(6) have shown many kinases, SH3 and SH2 domains, and other proteins bind ligands in the PPII conformation or closely related geometries. The PPII secondary structure occurs frequently on the surface of globular proteins where binding clefts and other interactions commonly occur. PPII helices are known to be key recognition elements of many protein-protein mediated interactions and other signal transduction pathways. Many native peptides that bind in the PPII conformation are comprised on non-proline amino acids in addition to proline. Furthermore, binding studies(7) have suggested the proline residues are scaffolding and/or hydrophobic contact residues whereas the non-prolyl residues are the AA residues that make contact with the substrate. Non-proline residues generally are not constrained and generally do not stabilize the PPII secondary structure (although Arg, Ala, Ser, Glu are preferred for PPII).(8) Similarly, proline has been found to destabilize other non-PPII secondary structures. A peptide sequence rich in proline, it is theorized, would adopt the PPII secondary structure in preference to a peptide lacking, or with few, proline residues.

Section 1.2 – Proline Templates Amino Acids (PTAAs)

1.2.1 – Developing the Concept of PTAAs

The synthesis of proline analogs that contain non-prolyl side chain functionality (proline templates amino acids, PTAAs) has been envisioned. These amino acid analogs are potentially useful compounds, incorporating any amino acid functionality onto the rigid frame of proline. Coupling of these modified proline residues would generate a contiguous proline backbone with the potential of varying AA functionality (e.g. acidic, basic, and hydrophobic side chains).

Additional interest lies in the potential constraint of the resulting side chain angles (c1, c2, etc.), which have been shown to be critical in many protein-protein binding geometries.(9) PTAA containing oligomers are expected to bind to a receptor with a higher affinity when compared with the same non-PTAA oligomers due to the constrained binding conformations that promote the PPII secondary structure. The net entropic gain relative to native, unconstrained (wild-type) peptides is 1.2-1.6 kcal per constrained bond(10) and consequently +3-4 kcal/mol in binding energy per PTAA.  Flexible installation and position of side chain functionality gives access to a variety of unnatural AA analogs, as well as ability to design specific ligands for potential receptor targets.  The ability to control c1, c2… should enable mapping of a binding cleft with substrate probes. X-ray crystallography is often used for the conformation analysis and provides valuable data, but the need to have substrate and ligand bound within a stable crystal may, in some cases, pose a challenge. Probing with appropriate c1 constrained PTAAs would allow cleft mapping with routine substrate binding studies.