Transcript A real example: - McMaster University

A real example:
HS
O
HO
O
H
N
O
HO2C Leu
NH2
N
H
Cys
Ala
NH2
CH2Cl
CH2
BnS
O
H
N
TFA
N boc
H
O
O
O
DCC
-O
BnS
O
+
O
O
H
N
HO
O
NH
O
HS
O
H
N
N
H
+
NH3
H
N boc
Natural Product Peptides, Peptidomimetics &
Peptide Analogues
• “Natural Product” Peptides (nonribosomal peptides)
– Product of secondary metabolism
– Synthesized on the NRPS
– Numerous pharmaceutically relevant peptides:
O
O
H2N
O
Ph
O
N
H
O
O
N
H
O
HN
H
N
H
N
O
O
N
O
O
O
N
H
NH
O
N
N
N
H
N
O
N
N
O
O
N
O
O
N
O
Ph
NH
HN
NH2
O
O
gramicidin S
(antibiotic activity)
N
O
Actinomycin C1
(apoptotic activity)
O
O
More Nonribosomal Peptides
HO HO
OH
HO
H2N
O
O
O
O
Cl
O
O
HO
O
N H
H
O
O
O
H
N
O
N
H
OH
O
H
N
O
N
H
H
N
O
O
N
N
N
O
O
HN
NH2
HO
N
O
OH
OH
HO
O
HN
O
HN
N
OH
O
vancomycin
(antibiotic activity)
O
N
Cyclosporin A
(immunosuppressive activity)
N
O
HN
N
O
O
• 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
Other Analogues:
NMR structure of magainin 2
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 structureactivity 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
The experiment:
• Uses SFM (scanning force microscopy)
Faults
(cracks)
Apply gly to surface
(at STP)
Hectorite (layered silicate)
containing Mg2+, Li+ & Cu2+
• No visible change in faults or
layers
• HPLC showed no gly peptides
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:
Gly
montmorillonite
or
hectorite
+
Tyr
Tyr-Tyr
Gly-Gly
60 - 90
oC
Gly-Gly-Tyr
Varying the
mineral can give
different
peptides!
...etc
• Another experiment:
– Mixed NaCl + Clay (mineral) + heat
• NaCl alone gave only short peptides
• When clay was added, longer peptides were produced!
Hadean Beach – “the primary pump”
2)
•
This resembles many of the features of chemical peptide
synthesis:
• Step 1: In aqueous phase (i.e., ocean), 25 °C
O
O
NH2
HO
R
•
•
H N C O
+
H
H
N
HO
R
NH2
O
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
NO
O
H
N
HO
NH2
O
NOx
H
N
HO
R
O2
O
HO N N H
R
+
H
N
O
N
O
H
N
R
O
O
O
+
H
H
N
R
O
O
+
HO N N H
O
• N is “protected” as a
carbamate (recall BOC)
• CO2H activated as an
anhydride
-H2O
N N
Loss of N2 is driving force
for rxn
• Step 4 & 5: Condensation
O
HN
O
R2
O
R
+
H2N
+
H2N
CO2H
R2
R
N
H
CO2H
CO2
O
Drives rxn
• Experimentally, this system generates oligo-peptides
with diastereoselection & preferred sequences (?)
• May have given rise to earliest protein catalysts
3)
Nucleic acid templated peptide synthesis:
•
•
Template--
Model for the transfer of RNA world into the protein world?
Basic idea:
• Modify DNA strands with activated amino acids (i.e., DNAlinked 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
• 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
• 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