338 #9

• Peptides


  • Have a variety of biological roles
  • Can be harnessed to create 'tools', such as drugs, or other therapies
• Naturally occurring peptides


  • Often cyclic
  • Feature many disulfide bridges
  • Have other modifications
  • These all help to make them less susceptible to proteolytic digestion
• A challenge for making peptides for in vivo use is designing similar properties
• How to obtain a peptide

  • Purification from tissue
  • Genetic engineering
  • Synthetic chemistry approach

  
• Purification from tissue


  • Often made difficult by the vanishingly low concentrations of some peptides
  • May not be what you want
  • Access to the tissue may be problematic
• Genetic engineering


  • May be incompatible (constituent amino acids, modifications, etc.)
  • Peptides are often produced non-ribosomally
  • These are generally hard!
• If we want a very specific peptide of particular composition and structure a synthetic chemistry approach may work

  
• Advantages of peptide biosynthesis

  • Nature uses enzymes for biosynthesis
  • Regardless of method (ribosomal or non-ribosomal), peptides are built with exquisite control over sequence, stereochemistry, regiochemistry, in presence of other reactive groups, modifications, cyclisation, etc.
  • And quickly!
• Chemical synthesis challenges


  • Reactivity: right bonds forming, wrong bonds not forming?
  • Sequence
  • Speed!
  • Fidelity; want just a single product
  • Purification?
  • Selective modifications?
  • Cyclisation?

  
• Formation of peptide bonds


  1. Linking amino acids together

  2. Not a chemically complex process

  
• Peptide synthesis must proceed in order

  
• The amide bonds must be formed in a specific order
• In protein synthesis, the order of amino acids is specified by the mRNA sequence, and assembly is controlled by the ribosome
• Peptide bond formation


  • Not spontaneous under normal conditions
  • Need a coupling reagent/activator to get an efficient reaction
  • Reactive groups must be activated
  • The -OH of the acid is a poor leaving group, making it difficult to directly displace

  
• Formation of peptide bond

  1. Amides are usually formed by reaction between amines and acid chlorides/reactive esters

  2. Acids are typically converted into an 'activated ester' prior to reaction
• Acyl chlorides


  Have limited value in peptide coupling, because of danger of hydrolysis, racemisation, cleavage of protecting groups, and other side reactions
• DCC-based activation


  Dicyclohexylcarbodiimide is an activator
• Incomplete reaction at one stage can lead to formation of an impurity in the next
• Rapid reaction rates are essential to synthesize long peptides
• Amide bond coupling


  • Competes with side reactions that deplete one or more of the reagents
  • Each coupling step is a race between amide bond formation, and undesired reactions
• Excess reagents at each step is essential for achieving the rapid coupling rates that are necessary to outpace potential side reactions
• The impurities must be laboriously separated during the final purification – this is not easy
• Solid-phase peptide synthesis


  • Synthesis of large peptides chain by sequential addition of amino acids is a long and arduous task
  • Solution is to couple the growing chain to an insoluble resin on the C-terminal end
  • After each new residue is added successively at the free amino-terminus, the elongated product is recovered by filtration and readied for the next synthetic step
  • Wash away excess reactants and contaminants
  • Because the growing chain is coupled to an insoluble resin bead, the method is called solid-phase peptide synthesis

  
• Polymer provides C-term protection

  • Solution-phase: a methyl ester protects the carboxyl group during amide bond formation
  • Solid phase: the polymer particle is the ester protecting group; polystyrene
• Cleavage of peptide from polymer

  1. The peptide-resin linkage must be stable throughout all the peptide elongation steps

  2. But easily cleavable at the end of the synthesis

  3. Cleave under strongly acidic conditions

  
• Side chain protection


  • Reactive side chains (-OH, -NH, -SH, -COOH) must also be protected to prevent side-reactions from occurring
  • Protecting groups must be chosen carefully to be compatible with peptide synthesis strategy

  
• Use protection to control assembly


  1. While the desired coupling reactions proceeds, protect the other functional groups from reaction by blocking

  2. For the peptide Leu-Gly, protect the NH2 group of leucine, and the COOH group of glycine

  
• Requirements for protecting groups

  • The blocking must be removable later under conditions in which the newly formed peptide bonds are stable
  • No point using e.g. an amide to protect the amine, as difficult to hydrolyse that in the presence of the amide bond trying to form
  • Must be stable for the entire synthesis

  
• Orthogonal protection/synthesis


  • A protection strategy allowing the deprotection of functional groups independent of each other
  • Protect NH2 and CO2H differently, so can be removed under different conditions
  • Allows modification of either end of the dipeptide at will

  
• Coupling strategy


  Use protecting groups that are removable only in acid or base to allow independent modification of the N- and C-termini
• SPPS process (using Fmoc protection)

  1. Fmoc-amino acid

  2. e.g. polystyrene ester solid-phase synthesis

  
• Synthetic methods are amendable to incorporation of modifications at time of synthesis
• These methods allow for control of


  • Sequence
  • Stereochemistry and regiochemistry
  • Purification
  • Speed?
  • Incorporation of modifications
• Modifications that could be incorporated

  • Phosphorylation
  • Site-specific incorporation of carbohydrates
• Phosphorylated Ser and Thr

  • Challenging
  • Phosphate group is decomposed by strong acid and lose with base
  • Requires careful selection of protecting groups or, include unprotected Ser and Thr and 'globally' post-assembly phosphorylate
• Phosphorylated Tyr


  Less susceptible to strong acid decomposition and is not at all base labile
• Site-specific incorporation of carbohydrates

  • Require relatively mild conditions for glycopeptide syntheses
  • Repetitive acid treatments can be detrimental to sugar linkages
• General strategies for constructing proteins

  • Stepwise synthesis: synthesise entire protein one amino acid at a time
  • Fragment assembly: individual peptide strands are initially constructed, purified, and linked together to make the protein
  • Directed assembly: get peptide, purify, then non-covalently driven to associate into protein-like structures