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

Chemical synthesis demonstrated on solid support –Synthesis: weeks (sol n ) → 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 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 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 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

magainin series sites of fluorination: Leu 6, Ala 9, Gly 13, Val 17, and Ile 20 NMR structure of magainin 2 Other Analogues:

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

Examples of Peptidomimetics Mimic  -sheets

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

Hectorite (layered silicate) containing Mg 2+, Li + & Cu 2+ Faults (cracks) Apply gly to surface No visible change in faults or layers HPLC showed no gly peptides (at STP) The experiment: Uses SFM (scanning force microscopy)

Experiment (con’t): Apply gly to surface Alternate cycles of heating to 90 °C + ddH 2 O Small glycine peptides (oligomers) HPLC Gly peptides of up to 6 AAs in length

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!

2)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)

Step 2: –Tide moves out (i.e. AA is now in dry reaction conditions) Step 3: Likely present in primitive atmosphere

N is “protected” as a carbamate (recall BOC) CO 2 H activated as an anhydride Loss of N 2 is driving force for rxn

Step 4 & 5: Condensation Experimentally, this system generates oligo-peptides with diastereoselection & preferred sequences (?) May have given rise to earliest protein catalysts Drives rxn

3)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” Template--

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. R 1 ) has a unique DNA sequence associated with it Nucleic Acid Template Synthesis

Step 2: –Masking of portion of template (i.e., “protect”) –Add other DNA-substrate molecules to the “pot”

Step 3: –Mixture is cooled to 4 °C (for 20 mins) & R 1 template selectively hybridizes –Amine and activated carboxylate are now in close proximity & can undergo “intramolecular” peptide bond formation

Step 4: –Temperature raised, causing dissociation of template –DNA-R 2 template hybridizes & peptide bond formation occurs

Cycle repeats for the third AA (R 3 ) until tripeptide is obtained

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?

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?

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