crazy about biomedicine júlia garcía pindado designing peptides: from nature to bench
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Crazy about biomedicineJúlia García Pindado
Designing peptides: Designing peptides: from nature to benchfrom nature to bench
Organic chemistry: 5 elements to build us up
A brief reminder about proteins and peptides
What’s an aminoacid?What’s an aminoacid?
ChiralityChirality
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?How amino acids form proteins?
A brief reminder about proteins and peptides
ProteinsProteins
A brief reminder about proteins and peptides
Protein structureProtein structure
PeptidesPeptides
A brief reminder about proteins and peptides
Peptides as drugs
40 marketed peptides (only 1%)
270 in clinical trials 15% year growth
400 in advanced preclinical trials
Peptides as drugs
Chemically well defined
Low cost of production
No immunogenicity
Specificity
Oral administration
Permeability through biological barriers
Small moleculesSmall molecules PeptidesPeptides BiologicsBiologics
Chemically well definedLow production costChemical synthesisOral administrationSpecificity
SpecificityHigh production costNon chemical synthesisImmunogenicityNo oral administration
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
Manual SPPSMicrowave assisted
SPPS
o Fast development
o Complex peptides can be reached
o 60-70% efficiency
Solid phase peptide synthesis (SPPS)
Solid phase peptide synthesis (SPPS)
Temporary protecting group
Permanent protecting groupBoc/BzlBoc/Bzl Fmoc/tBuFmoc/tBu
ResinsResins
Size of the resin beads (80-200 µm)
Mechanical and thermal stability
Price
Swelling
Solid phase peptide synthesis (SPPS)
Types: Cross-linked polysterene Polyamide
Tentagel
Soluble
Solid phase peptide synthesis (SPPS)
ResinsResinsWang
2-Chlorotrityl chloride
Rink amide Chemmatrix
SWELLINGSWELLING
Solid phase peptide synthesis (SPPS)
Coupling reagentsCoupling reagents Name Structure
DIPCDI
N
C
N
HOAt
N
N
NN
OH
PyBOP
N
NN
O P
N
N
N
PF6
TBTU
N
N
N
O
NN
BF4
DIEA
N
Solid phase peptide synthesis (SPPS)
Protecting groupsProtecting groupsName Structure Removal conditions
(SPPS)
Stable to the removal of
Fmoc OO
20% piperidine in DMF Boc, Trt, Alloc, pNZ
Boc
O O
20-50% TFA in DCM Fmoc, Trt, Alloc, pNZ
pNZ
O2N
O
O
1-6M SnCl2, 1.6mM HCl
(dioxane) in DMF
Boc, Fmoc, Trt, Alloc
Trt
1% TFA in DCM Fmoc, Alloc
Alloc O
O
Pd(PPh)3 cat., scavengers
PhSiH3 in organic solvents
Boc, Fmoc, Trt, pNZ
Solid phase peptide synthesis (SPPS)
Protecting groupsProtecting groupsAlloc
Pd(PPh)3 cat., scavengers
PhSiH3 in organic solvents
Boc, Fmoc, Trt, pNZ
iVDde
O O
2% N2H4·H2O - DMF Boc, Fmoc, Trt, Alloc
Azide N3 PMe3 in dioxane -
pNB
O2N
SnCl2 in DMF Boc, Fmoc, Trt, Alloc
Acm
NH
O
I2 (S-S)
Hg(II) (SH)
Trt
Bzl
NaOH in organic solvents Boc, Fmoc, pNZ, Trt,
Alloc
Al
0.1 equiv Pd(PPh)3, 10
equiv PhSiH3
Boc, Fmoc, pNZ, Trt
Name Structure Removal conditions
(SPPS)
Stable to the removal of
Solid phase peptide synthesis (SPPS)
The concept of orthogonalityThe concept of orthogonality
Remove only the Remove only the desired PG without desired PG without affecting the othersaffecting the others
Solid phase peptides synthesis (SPPS)
Tests to check coupling reactionsTests to check coupling reactions
Chloranil
Kaiser
Free amines
O
OH
OH
O
+
NH2
R
OH
O
HO
O
N
O
O
Solid phase peptides synthesis (SPPS)
Side reactionsSide reactions
Diketopiperazine (DKP)
Epimerization: losing the stereochemistry
Solid phase peptides synthesis (SPPS)
Other inconveniencesOther inconveniences
Aggregation
Deletion of residues
Problems during the cleavage process
Solid phase peptides synthesis (SPPS)
PurificationPurification
Traditional chromatographic purification
Solid phase peptides synthesis (SPPS)
PurificationPurification
SPPS enables us to avoid performing several purification steps
ReagentsSolventImpuritiesBeads with the peptide wash
Reagents and
byproducts
Beads of resin
Solid phase peptides synthesis (SPPS)
PurificationPurification
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
WHY?WHY?
N-methylated peptides Non natural aminoacids Cyclic peptides Stapled peptides
How can we improve synthetic peptides?
ProteasesProteases
N-methylated peptides
Cyclic peptides
Stapled peptides
Designing peptides
Synthesis of natural peptides
Synthesis of novel peptides
Mimicking nature
Aromatic AA(Phe)
Variable AA
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
Creating new sequences
Selecting the resin…
2-Chlorotrityl: 1% TFA in DCMProtected peptide acid
Wang: 95% TFAPeptide acid
Rink amide Chemmatrix: 95% TFAPeptide amide
Choosing the suitable PG...
Removal in acidic conditions:Removal in acidic conditions:
Boc:25-50% TFA tBu: 90% TFApNZ:1-6M SnCl2, 1.6mM HCl (dioxane) in DMF
Trt: 1% TFA in DCM
O2N
O
O
Removal in basic conditions:Removal in basic conditions:
Fmoc:20% piperidine/DMF Bzl: NaOH in organic solvents
Removal in other conditions:Removal in other conditions:
Alloc: Pd(PPh)3 cat., scavengers PhSiH3 in organic solvents
Al: Pd(PPh)3 cat., scavengers PhSiH3 in organic solvents
NH
O
Acm: I2 (S-S) Hg(II) (SH)
Azide: PMe3 in dioxane
N3
Choosing the suitable PG...
Choosing the suitable PG...
Analysis of the obtained product
High Performance Liquid Chromatography (HPLC)High Performance Liquid Chromatography (HPLC)
Analysis of the obtained product
Mass spectrometry (MS)Mass spectrometry (MS)
Our lab (Giralt’s group)
Design, synthesis and structure of peptides and proteinsDesign, synthesis and structure of peptides and proteins
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