Strategies for Peptide Backbone Modification in Protein Beta-Sheets by George Andrew Lengyel B.S. Chemistry, University of Pittsburgh, 2004 M.A.T. Secondary Science, University of Pittsburgh, 2005 M.S. Chemistry, University of Pittsburgh, 2011 Submitted to the Graduate Faculty of the Kenneth P. Dietrich School of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2014 i UNIVERSITY OF PITTSBURGH DIETRICH SCHOOL OF ARTS AND SCIENCES This dissertation was presented by George Lengyel It was defended on April 16, 2014 and approved by Dr. Dennis Curran, Professor, Department of Chemistry Dr. Steve Weber, Professor, Department of Chemistry Dr. Ron Wetzel, Professor, Department of Structural Biology Committee Chair: Dr. W. Seth Horne, Assistant Professor, Department of Chemistry ii STATEGIES FOR PEPTIDE BACKBONE MODIFICATION IN PROTEIN BETA-SHEETS George Lengyel, PhD University of Pittsburgh, 2014 Copyright © by George Lengyel 2014 iii Strategies for Peptide Backbone Modification in Protein Beta-Sheets George Lengyel, PhD University of Pittsburgh, 2014 Design of foldamers, unnatural backbone oligomers that mimic the structure of proteins, is an important field of research as these species can bind to natural proteins but are resistant to proteolytic degradation. We have focused on developing strategies for the design of unnatural oligomers that adopt β-sheet secondary structures like those commonly found in protein tertiary folds. Our approach is to modify natural peptide sequences that encode for β-sheet folds with various unnatural amino acid building blocks to produce hybrid-backbone peptides that fold like the parent sequence in aqueous solution. Through evaluation of β-hairpin model systems using multidimensional NMR, we have discovered several design strategies that may be applicable to mimicry of sheets found in larger protein tertiary structures and have ranked unnatural monomer types in order of increasing sheet propensity: β- amino acid < N-methyl-α-amino acid ≤ vinylogous γ4-amino acid < cyclic γ-amino acid. These substitutions require a 2:2 or 2:1 α- to β-residue substitution or 1:1 α- to γ- or α- to N-methyl-α-residue substitution to maintain native-like folding behavior. We applied these unnatural backbone substitutions to protein GB1, a 56 residue protein with a complex tertiary fold consisting of a four stranded β-sheet packed against an α-helix. Using thermal denaturation melts and circular dichroism spectroscopy, we have determined that the trend of sheet propensity seen in the hairpin peptide is similar in a tertiary fold with the caveat that the position of the unnatural residues matters greatly. Substitution strategies that lengthen the strands of the β-sheet have varying effects on the stability of the folded structure depending on their placement; substitutions near the center of the strands are significantly more destabilizing than those placed near the termini. Use of N- methylated α-amino acids is not limited in this fashion, but their positioning must be chosen so as to avoid disruption of inter-strand hydrogen bonding. iv Overall, we have determined that several types of unnatural amino acids can be used to promote sheet formation with limited destabilization; these amino acids could potentially be used in other proteins with tertiary folded structures. v TABLE OF CONTENTS 1.0 INTRODUCTION TO BETA-SHEET FOLDAMERS .................................................................. 1 1.1 PEPTIDE THERAPEUTICS .................................................................................................. 1 1.2 FOLDAMERS AND SEQUENCE-BASED DESIGN ........................................................... 3 1.3 PROTEIN BETA-SHEETS ..................................................................................................... 5 1.3.1 Beta-Hairpins .............................................................................................................. 5 1.3.2 Amino Acid Identity and its Impact on Beta-Hairpin Formation .......................... 6 1.4 BETA-SHEET FOLDAMERS ................................................................................................ 8 1.4.1 Beta-Sheet Foldamers Derived from Cyclic Beta-Amino Acids ............................. 8 1.4.2 Beta-Sheet Foldamers Derived from Pyrrolinone-Based Scaffolds ........................ 9 1.4.3 Beta-Sheet Foldamers Templated by Methoxybenzamide .................................... 10 1.4.4 Beta-Sheet Foldamers Containing Alpha-Amino Acid Homologs ........................ 11 1.4.4.1 Beta-Sheet Foldamers Containing Beta-Amino Acids ................................ 11 1.4.4.2 Beta-Sheet Foldamers Containing Gamma-Amino Acids .......................... 12 1.5 SOLVENT CHOICE AND ITS EFFECT ON BETA-SHEET FORMATION................. 13 1.6 GOALS .................................................................................................................................... 14 1.6.1 Model System Selection ............................................................................................ 15 1.6.2 Outline ........................................................................................................................ 17 1.6.2.1 Analysis of α- to β-Residue Substitution ....................................................... 17 1.6.2.2 Analysis of α- to γ-Substitution and N-Methylation .................................... 18 1.6.2.3 Unnatural Residue Substitutions Applied to Protein GB 1 ........................ 18 vi 2.0 IMPACT OF BETA-AMINO ACID INCORPORATION ON FOLDING OF BETA- HAIRPINS IN AQUEOUS SOLUTION ................................................................................................. 19 2.1 1:1 ALPHA- TO BETA-RESIDUE SUBSTITUTION ........................................................ 20 2.1.1 Hairpin Peptide Design ............................................................................................. 20 2.1.2 Qualitative NMR Analysis ........................................................................................ 22 2.1.3 Structural Analysis by NMR .................................................................................... 24 2.1.4 Analysis of 2:1 α- to β-Residue Substitution Strategies ......................................... 30 2.2 2:1 OR 2:2 ALPHA- TO BETA-RESIDUE SUBSTITUTION ........................................... 34 2.2.1 Design of Alpha- to Beta-Residue Substitution Strategies .................................... 34 2.2.2 Selection of Hairpin Model System ......................................................................... 35 2.2.3 Application of Beta-Residue Substitution Strategies to Model Hairpin System . 38 2.2.4 Structural Impact of 2:1 and 2:2 Alpha- to Beta-Residue Substitution ............... 39 2.2.5 Thermodynamic Impact of 2:1 and 2:2 Alpha- to Beta-Residue Substitution .... 43 2.2.6 Conclusions ................................................................................................................ 44 2.3 EXPERIMENTAL .................................................................................................................. 46 2.3.1 Monomer Synthesis ................................................................................................... 46 2.3.1.1 General Information ....................................................................................... 46 2.3.1.2 Synthesis of Fmoc-β2-Monomers ................................................................... 47 2.3.1.3 Synthesis of anti Fmoc-β2,3-Monomers .......................................................... 53 2.3.1.4 Synthesis of syn Fmoc-β2,3-Monomers .......................................................... 57 2.3.1.5 Synthesis of cis Fmoc-ACPC Monomers ...................................................... 68 2.3.1.6 Synthesis of cis Fmoc-ACHC Monomers ..................................................... 71 2.3.2 Peptide Synthesis ....................................................................................................... 74 Table 3. MALDI-TOF data for peptides 1a-17a and 1b-17b. ............................................. 76 2.3.3 NMR Sample Preparation and Data Collection ..................................................... 78 2.3.4 NMR Data Analysis and Structure Determination ................................................ 79 vii 2.3.5 Calculation of Folding Equilbria by NMR ............................................................. 80 3.0 GAMMA RESIDUES AND N-METHYL-ALPHA-RESIDUES IN HETEROGENEOUS BACKBONE HAIRPINS ......................................................................................................................... 82 3.1 1:1 ALPHA- TO GAMMA-RESIDUE SUBSTITUTION................................................... 83 3.1.1 Design of Alpha- to Gamma-Residue Substitution Strategies .............................. 83 3.1.2 Thermodynamic Analysis of 1:1 Alpha- to Gamma-Residue Substitution .......... 84 3.1.3 Structural Analysis of 1:1 Alpha- to Gamma-Residue Substitution .................... 86 3.1.4 Conclusions ................................................................................................................ 91 3.2 N-METHYLATION OF SELECTED ALPHA-RESIDUES .............................................. 92 3.2.1 N-Methylation Strategies Applied to a Model Hairpin Peptide ............................ 92 3.2.2 NMR Analysis of N-Methylated Hairpin Peptides ................................................. 93 3.2.3 Conclusions ...............................................................................................................