A real example:

OHNH

NH

O

O

SH

O

NH2

Leu Cys Ala NH2HO2C

CH2Cl CH2

NH

O

BnS

bocONH

O

NH

O

BnS

ONH

O

O

NH

-Oboc

O

NH3NH

O

SH

OHNH

O

TFA

+

+

DCC

Natural Product Peptides, Peptidomimetics & Peptide Analogues

• “Natural Product” Peptides (nonribosomal peptides)– Product of secondary metabolism– Synthesized on the NRPS– Numerous pharmaceutically relevant peptides:

N

ONH

O

NH

O

NH

O

NH

O

N

ONH

O

NH

OO

NH

NH

O

NH2

NH2

Ph

Ph

N

O

O

NO

NO

NH

O

N

O

O

NO

NO

NH

O

O

N

O

O O

gramicidin S

(antibiotic activity)

Actinomycin C1

(apoptotic activity)

More Nonribosomal Peptides

NON

O

N

OO N

NH

O

NH O

O

NN

N

O

N

O

ONH

OOH

OOH OOH

O

OHOH

OH

NH

ONHO

ONH

ONH

ONH

ONH

H

NH2

O

NH

OOO

OH

Cl

OH

O

OHOHOH

NH2

Cyclosporin A

vancomycin

(immunosuppressive activity)

(antibiotic activity)

• 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

• 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 Mg2+, Li+ & Cu2+

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 + ddH2O

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!

Gly + Tyr

montmorillonite

hectorite

orTyr-Tyr

Gly-Gly

Gly-Gly-Tyr

...etc

60 - 90 oC

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)

OH

O

NH2

R

N C OH

H+

OH

O

NH

R

NH2

O

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

• Step 3:

OH

O

NH

R

NH2

OOH

O

NH

R

NH

O

NO

H+

O

NH

O

O

RN N HOH

NOx

O2

NO

+

Likely present in primitive atmosphere

O

NH

O

O

RN N HOH

-H2O

N N

+

• N is “protected” as a carbamate (recall BOC)

• CO2H activated as an anhydride

Loss of N2 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

ONH

O

OR

NH2 CO2H

R2

NH

CO2H

R2O

NH2

R

CO2

++

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. R1) 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) & R1 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-R2 template hybridizes & peptide bond formation occurs

• Cycle repeats for the third AA (R3) 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