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A real example:. Natural Product Peptides, Peptidomimetics & Peptide Analogues . “Natural Product” Peptides (nonribosomal peptides) Product of secondary metabolism Synthesized on the NRPS Numerous pharmaceutically relevant peptides:. More Nonribosomal Peptides.

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natural product peptides peptidomimetics peptide analogues
Natural Product Peptides, Peptidomimetics & Peptide Analogues
  • “Natural Product” Peptides (nonribosomal peptides)
    • Product of secondary metabolism
    • Synthesized on the NRPS
    • Numerous pharmaceutically relevant peptides:
slide6
Chemical synthesis demonstrated on solid support
    • Synthesis: weeks (soln) → days (solid)
    • Employ more and/or different protecting groups
    • Unusual functional groups
    • Cyclization on resin?
    • Other modifications (i.e. sugar moiety)?
  • Solid-supported synthesis has allowed the substitution and/or modification of AAs → analogues
    • AA, functional groups, stereochemistry, substitution, etc
    • Study structure-activity relationships
    • Potential therapeutics
    • Note: Industrial synthesis not performed on solid supported
peptide analogues
Peptide Analogues
  • Recently, there have been developments in the modification of peptides, particularly AMPs
  • AMPs = Antimicrobial Peptides
    • 15-30 AAs in length
    • Produced by all animals (insects to frogs to humans)
    • First line of defense against microbial organisms
    • Answer to antibiotic resistance?
    • Molecular diversity → dependent on structure
amp structure
AMP Structure
  • Large proportion of hydrophobic residues (~ 50 %)
  • Also contain varying amounts of Lys, Arg & His → +vely charged AAs
    • These AAs vary in their arrangement within the peptide
  • This arrangement of AAs allows disruption of bacterial membranes (anionic)
teflon peptide fluorogainin 1
“Teflon” Peptide: Fluorogainin-1
  • Fluorous analogue of the AMP, magainin (isolated from the skin of frogs)
    • Replaced hydrophobic residues (i.e., Val, Leu,etc) with fluorinated versions → “Teflon like”
    • Resulted in more stable peptides:
      • More resistant to unfolding by chemical denaturants & heat
      • NMR also showed higher structural integrity
    • Results also indicated increased antimicrobial activity
      • Likely due to the increased hydrophobicity of peptide
      • This strong hydrophobic interaction may make the peptide less susceptible to proteases
slide10

magainin series sites of fluorination:

Leu 6, Ala 9, Gly 13, Val 17, and Ile 20

NMR structure of magainin 2

Other Analogues:

peptidomimetics
Peptidomimetics
  • Peptide “mimics”
    • Contain non-natural peptidic structural elements (i.e. peptide bonds or unusual functional groups)
    • Molecules vary in size & structure
    • Commonly synthesized using Merrifield resin to study structure-activity relationships
    • Possible drug candidates
peptide synthesis in the prebiotic world
Recall:

Murchison Meteorite

Possible source of AAs (via the Strecker mechanism)

Peptide (oligo) formation ?

Selection of an enantiomer

Selection by crystal faces

Circularly polarized light from stars

Enantioenrichment

Via Serine octamer

Enrichment by sublimation

Peptide Synthesis in the Prebiotic World
peptide synthesis in the prebiotic world14
Peptide Synthesis in the Prebiotic World
  • Also recall: formation of peptides from monomers is energetically unfavorable (i.e., ΔG>0)
    • Modern world  enzymes
    • Chemical synthesis  activation strategies
    • Prebiotic world  some energy input needed?

Possibilities?

  • Synthesis with minerals!
    • Clay has been shown to catalyze the condensation of Gly to peptides up to (Gly)6
slide15

The experiment:

  • Uses SFM (scanning force microscopy)

Apply gly to surface

Faults (cracks)

(at STP)

  • No visible change in faults or layers
  • HPLC showed no gly peptides

Hectorite (layered silicate) containing Mg2+, Li+ & Cu2+

experiment con t
Experiment (con’t):

Small glycine peptides (oligomers)

Apply gly to surface

Alternate cycles of heating to 90 °C + ddH2O

HPLC

Gly peptides of up to 6 AAs in length

other similar experiments
Other Similar Experiments:
  • Another experiment:
    • Mixed NaCl + Clay (mineral) + heat
      • NaCl alone gave only short peptides
      • When clay was added, longer peptides were produced!

Varying the mineral can give different peptides!

slide18
Hadean Beach – “the primary pump”
    • This resembles many of the features of chemical peptide synthesis:
      • Step 1: In aqueous phase (i.e., ocean), 25 °C
      • Similar to Wohler synthesis of urea
      • Amino group is now less reactive (amide-like)
slide20

Likely present in primitive atmosphere

  • Step 2:
    • Tide moves out (i.e. AA is now in dry reaction conditions)
  • Step 3:
slide21

Loss of N2 is driving force for rxn

  • N is “protected” as a carbamate (recall BOC)
  • CO2H activated as an anhydride
slide22
Step 4 & 5: Condensation
  • Experimentally, this system generates oligo-peptides with diastereoselection & preferred sequences (?)
  • May have given rise to earliest protein catalysts

Drives rxn

slide23

Template--

  • Nucleic acid templated peptide synthesis:
    • Model for the transfer of RNA world into the protein world?
    • Basic idea:
      • Modify DNA strands with activated amino acids (i.e., DNA-linked substrate)
      • These DNA strands are specific in sequence in order to “tune” their hybridization abilities
      • DNA acts a template for further reactions, such as peptide bond formation
      • Reactions performed as “one pot”
nucleic acid template synthesis
Nucleic Acid Template Synthesis
  • Step 1:
    • Templates are loaded with an AA
    • Attached to DNA as an N-hydroxysuccinimidyl ester (recall lab 6 → NHS & DCC)
    • Each AA (i.e. R1) has a unique DNA sequence associated with it
slide25
Step 2:
    • Masking of portion of template (i.e., “protect”)
    • Add other DNA-substrate molecules to the “pot”
slide26
Step 3:
    • Mixture is cooled to 4 °C (for 20 mins) & R1 template selectively hybridizes
    • Amine and activated carboxylate are now in close proximity & can undergo “intramolecular” peptide bond formation
slide27
Step 4:
    • Temperature raised, causing dissociation of template
    • DNA-R2 template hybridizes & peptide bond formation occurs
slide29
Model demonstrates that DNA can resemble an enzyme (i.e., ribozyme)
    • Promotes coupling of 2 AAs through non-covalent interactions
    • Specificity (template sequence → one AA selected → tRNA like)
  • Could a similar model or sequence have given rise to peptides in the prebiotic world?
slide30
So far, we have looked at both amino acids & peptides (peptide bond formation) in the prebiotic & modern world
  • Common themes were:
    • Selectivity
      • Regioselectivity
      • Stereoselectivity
      • Protecting groups
    • Overcoming ΔG
      • Activation of carboxylate to make a peptide bond ( E of starting material)
      • Stabilization of TS ( E) (i.e., Lewis acid)
        • What about an active site?
slide31
Peptide → active site?
  • Peptides may fold and/or associate to produce a simple “active site”
  • Proteins/peptides have specific conformations due to intramolecular non-covalent forces:
    • H-bonding
    • salt bridge
    • Ionic
    • Dipole-dipole
    • Van der Waals
  • The sum of many weak forces → strong total binding force to restrict the conformation
    • Folding has a –ve ΔS, but a +ve ΔH
  • Also have covalent bonding: disulphide bridge