Lecture 1: Reasons for synthesizing molecules and types of molecules.
Lecture 2: Med chem and peptide therapeutics, focusing on peptide design.
Current focus: Moving from peptides to proteins.
Solid-phase synthesis (SPPS) for peptides:
Immobilizing amino acids on a polystyrene resin (insoluble bead).
Attaching amino acids via a chemical linker.
Efficient purification by filtering off solvent and reagents.
Using protecting groups to control the directionality of peptide assembly.
Bruce Merrifield: Nobel Prize winner for inventing SPPS.
Limitations of Solid-Phase Synthesis
Smallest proteins: approximately 50 amino acids (e.g., insulin with 51 amino acids).
SPPS limitations:
Aggregation: Growing peptide chains can adopt folded structures, burying the reactive amine terminus.
This reduces the efficiency of elongation and limits the size of peptides that can be synthesized.
Despite SPPS accelerating the production of small peptides, it doesn't extend far into the protein functional domain space.
Protein Therapeutics: Erythropoietin (EPO)
Erythropoietin:
166 amino acids, a human glycoprotein.
Used to treat anemia (low red blood cell count) resulting from chemotherapy or kidney issues.
Produced recombinantly (expressed in cell culture and purified).
Glycosylation:
Attachment of carbohydrates (sugar molecules) to a protein.
Human EPO is highly glycosylated.
Recombinant expression in E. coli lacks the machinery to install complex carbohydrates.
Recombinant proteins may not have the same suite of carbohydrates as endogenously produced proteins.
Synthetic preparation of EPO:
Needed to understand the role of carbohydrates in protein function.
Recombinant expression produces proteoforms or glycoforms, which may not be representative of endogenous forms.
Cost of EPO preparation:
Recombinant expression: Approximately $8,000 per year for treatment.
Total chemical synthesis: Ten years of research.
Post-translational modifications (PTMs):
Modifications to a peptide or protein after ribosome production (e.g., sugars, phosphates, sulfates, methyl groups).
PTMs are important for protein structure and function.
Chemical synthesis is crucial to understanding these modifications.
Historical Perspective: Emile Fischer
Emile Fischer (1902 Nobel Prize in carbohydrate chemistry).
Fischer's statement: The chemical enigma of life won't be solved until organic chemistry masters proteins like carbohydrates.
Fischer determined the polymeric nature of protein structure (repeating amino acid units, polyamides).
Synthesized an 18 amino acid peptide (before protecting groups).
Could not control the sequence (3 leucines and 15 glycines in unknown order).
Fischer's ambition: To synthesize a functional enzyme.
Chemical Ligation: Joining Peptide Fragments
Goal: To synthesize larger proteins by joining synthetic peptide fragments.
Strategy:
Synthesize peptide fragments (e.g., 50 amino acids) using SPPS.
Join the fragments together to reach the protein functional domain space.
Challenge: Selectively creating a bond between two unprotected peptides with various side chains.
Solution: Design mutually reactive functional groups that react specifically with each other.
Chemical ligation: Selectively stitching peptide chains together to form larger molecules.
Peptides should ideally be unprotected to avoid bulky protecting groups and allow for aqueous solutions.
Reaction should occur at physiological pH (7.4) under mild, neutral conditions.
Native Chemical Ligation (NCL)
NCL forms a native amide bond linkage between two peptide chains.
Functional groups involved:
Thioester: Carbonyl group with sulfur replacing the oxygen ($$R-CO-SR'$').
Cysteine: Amino acid with a unique thiol (SH) group.
Reaction:
Thioester reacts with cysteine in the presence of base and water, forming a new linkage and releasing a thiol leaving group.
S to N acyl shift: The acyl group (carbonyl) shifts from the sulfur to the nitrogen, forming an amide bond and freeing the cysteine side chain.
Mechanism (simplified):
Base deprotonates the thiol group of cysteine, forming a thiolate (great nucleophile).
Thiolate reacts as a nucleophile with the electrophilic carbon of the thioester.
The carbonyl group reforms, and a thiophenolate leaves.
Amine acts a nucleophile reacting with carbonyl carbon
The tetrahedral intermediate forms the acyl group reforms and sulfur leaves as a leaving group, finally an H+ from nitrogen is lost and one gained on the sulfur.
Key concept: Proximity effect. Positioning reactants close to each other (like the ribosome) promotes selective amide bond formation.
NCL works with larger peptide chains:
SPPS assembles peptide thioesters and cysteine-containing peptides.
NCL joins them selectively under aqueous conditions with unprotected peptides.
Case Studies: Applications of NCL in Therapeutics
Case Study 1: Cancer Vaccines (MUC1)
MUC1 (mucin-one): A glycoprotein making up the mucus layer on epithelial cells.
Structure:
Extracellular domain with a 20 amino acid tandem repeat sequence.