Designing peptides:
from nature to bench
Crazy about biomedicine
Júlia García Pindado
Organic chemistry: 5 elements to build us up
A brief reminder about proteins and peptides
What’s an aminoacid?
Chirality
A brief reminder about proteins and peptides
A brief reminder about proteins and peptides
A brief reminder about proteins and peptides
How amino acids form proteins?
A brief reminder about proteins and peptides
Proteins
A brief reminder about proteins and peptides
Protein structure
A brief reminder about proteins and peptides
Peptides
Peptides as drugs
40 marketed peptides (only 1%)
270 in clinical trials
400 in advanced preclinical trials
15% year growth
Peptides as drugs
Small molecules
Peptides
Chemically well defined
Low production cost
Chemical synthesis
Oral administration
Specificity
Biologics
Specificity
High production cost
Non chemical synthesis
Immunogenicity
No oral administration
Chemically well defined
Oral administration
Low cost of production
Permeability through
biological barriers
No immunogenicity
Specificity
Ideal drug candidate
 Permeability through biological barriers  reach the target
 Stability
 Solubility
 Active (nM range)
 Non toxic
Which is the appearance of peptides?
Peptide synthesis
Solid phase peptide synthesis (SPPS)
Developed by Merrifield in 1963
o Fast development
o Complex peptides can be reached
o 60-70% efficiency
Manual SPPS
Microwave assisted
SPPS
Solid phase peptide synthesis (SPPS)
Solid phase peptide synthesis (SPPS)
Temporary protecting group
Permanent protecting group
Boc/Bzl
Fmoc/tBu
Solid phase peptide synthesis (SPPS)
Resins
Size of the resin beads (80-200 µm)
Mechanical and thermal stability
Price
Swelling
Types:
 Cross-linked polysterene
 Polyamide
 Tentagel
 Soluble
Solid phase peptide synthesis (SPPS)
Resins
Wang
SWELLING
2-Chlorotrityl chloride
Rink amide Chemmatrix
Solid phase peptide synthesis (SPPS)
Coupling reagents
Name
DIPCDI
HOAt
PyBOP
TBTU
DIEA
Structure
Solid phase peptide synthesis (SPPS)
Protecting groups
Name
Structure
Removal conditions
Stable to the removal of
(SPPS)
Fmoc
20% piperidine in DMF
Boc, Trt, Alloc, pNZ
Boc
20-50% TFA in DCM
Fmoc, Trt, Alloc, pNZ
pNZ
1-6M SnCl2, 1.6mM HCl
Boc, Fmoc, Trt, Alloc
(dioxane) in DMF
Trt
Alloc
1% TFA in DCM
Fmoc, Alloc
Pd(PPh)3 cat., scavengers
Boc, Fmoc, Trt, pNZ
PhSiH3 in organic solvents
iVDde
2% N2H4·H2O - DMF
Boc, Fmoc, Trt, Alloc
(dioxane) in DMF
Trt
Solid phase peptide synthesis (SPPS)
TFA:H2O:EDT (90:5:5)
Fmoc, Alloc
Protecting groups
Alloc
Name
Pd(PPh)3 cat., scavengers
Removal conditions
PhSiH3 in organic solvents
(SPPS)
2% N2H4·H2O - DMF
20% piperidine
in DMF
Boc, Fmoc, Trt, pNZ
Stable to the removal of
20-50% TFA in DCM
PMe3 in dioxane
Fmoc, Trt, Alloc, pNZ
-
pNB
SnCl2 in DMF
Boc, Fmoc, Trt, Alloc
pNZ
1-6M SnCl2, 1.6mM HCl
Boc, Fmoc, Trt, Alloc
Acm
(dioxane)
in DMF
I2 (S-S)
Trt
Trt
Bzl
Hg(II) (SH)
TFA:H2O:EDT (90:5:5)
NaOH in organic solvents
Fmoc, Alloc
Boc, Fmoc, pNZ, Trt,
Structure
iVDde
Fmoc
Boc
Azide
N3
Boc, Fmoc, Trt, Alloc
Boc, Trt, Alloc, pNZ
Alloc
Al
0.1 equiv Pd(PPh)3, 10
Boc, Fmoc, pNZ, Trt
equiv PhSiH3
Alloc
Pd(PPh)3 cat., scavengers
Boc, Fmoc, Trt, pNZ
PhSiH3 in organic solvents
iVDde
2% N2H4·H2O - DMF
Boc, Fmoc, Trt, Alloc
Solid phase peptide synthesis (SPPS)
The concept of orthogonality
Remove only the
desired PG without
affecting the others
Solid phase peptides synthesis (SPPS)
Tests to check coupling reactions
Kaiser
Free amines
Chloranil
Solid phase peptides synthesis (SPPS)
Side reactions
Diketopiperazine (DKP)
Epimerization: losing the stereochemistry
Solid phase peptides synthesis (SPPS)
Other inconveniences
Aggregation
Deletion of residues
Problems during the cleavage process
Solid phase peptides synthesis (SPPS)
Purification
Traditional chromatographic purification
Solid phase peptides synthesis (SPPS)
Purification
SPPS enables us to avoid performing several purification steps
Reagents
Solvent
Impurities
Beads with
the peptide
Beads of resin
wash
Reagents
and
byproducts
Solid phase peptides synthesis (SPPS)
Purification
HPLC can be used to separate the drug from all the other impurities
How can we improve synthetic peptides?
To…
- Increase stability
- Enhance cell uptake and permeability through biological barriers
- Improve resistance against proteases




N-methylated peptides
Non natural aminoacids
Cyclic peptides
Stapled peptides
How can we improve synthetic peptides?
Proteases
N-methylated peptides
Cyclic peptides
Stapled peptides
Designing peptides
 Synthesis of natural peptides
 Synthesis of novel peptides
Mimicking nature
Aromatic AA(Phe)
Variable AA
Creating new sequences
1) Envisaging the desired product
2) Proper selection of the strategy
1) Resin
2) PG
3) Final removal of all the PG?
4) Further reactions and/or deprotections after cleavage
3) Carrying out the synthesis
Selecting the resin…
2-Chlorotrityl: 1% TFA in DCM
Protected peptide acid
Wang: 95% TFA
Peptide acid
Rink amide Chemmatrix: 95% TFA
Peptide amide
Choosing the suitable PG...
Removal in acidic conditions:
Boc:25-50% TFA
tBu: 90% TFA
pNZ:1-6M SnCl2, 1.6mM
HCl (dioxane) in DMF
Trt: 1% TFA in DCM
Removal in basic conditions:
Fmoc:20% piperidine/DMF
Bzl: NaOH in organic solvents
Removal in other conditions:
N3
Alloc: Pd(PPh)3
cat., scavengers
PhSiH3 in
organic solvents
Al: Pd(PPh)3
cat., scavengers
PhSiH3 in
organic solvents
Acm: I2 (S-S)
Hg(II) (SH)
Azide: PMe3 in dioxane
Choosing the suitable PG...
Choosing the suitable PG...
Analysis of the obtained product
High Performance Liquid Chromatography (HPLC)
Analysis of the obtained product
Mass spectrometry (MS)
Our lab (Giralt’s group)
Design, synthesis and structure of peptides and proteins