1. Crazy about biomedicine
Designing peptides:
from nature to bench
Crazy about biomedicine
Júlia García Pindado 1

2. Organic chemistry: 5 elements to build us up2

3. A brief reminder about proteins and peptides
What’s an aminoacid?
Chirality
Amino acids (/əˈmiːnoʊ/, /əˈmaɪnoʊ/, or /ˈæmɪnoʊ/) are biologically important organic compounds made from amine (-NH2) and carboxylic acid (-COOH) functional groups, along with a side-chain specific to each amino acid. The key elements of an amino acid are carbon,hydrogen, oxygen, and nitrogen, though other elements are found in the side-chains of certain amino acids. About 500 amino acids are known[1] and can be classified in many ways 3

4. A brief reminder about proteins and peptides4

5. A brief reminder about proteins and peptides5

6. A brief reminder about proteins and peptides
How amino acids form proteins? 6

7. A brief reminder about proteins and peptides7

8. A brief reminder about proteins and peptides
Protein structure 8

9. A brief reminder about proteins and peptides9

10. Peptides as drugs 40 marketed peptides (only 1%)
270 in clinical trials 15% year growth
400 in advanced preclinical trials 10

11. Peptides as drugs Chemically well defined Oral administration
Small molecules
Peptides
Biologics
Chemically well defined
Low production cost
Chemical synthesis
Oral administration
Specificity
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 11

12. Ideal drug candidate
Permeability through biological barriers  reach the target
Stability
Solubility
Active (nM range)
Non toxic 12

13. Which is the appearance of peptides?13

14. Peptide synthesis 14

15. Solid phase peptide synthesis (SPPS)
Developed by Merrifield in 1963
Fast development
Complex peptides can be reached
60-70% efficiency
Microwave assisted SPPS
Manual SPPS 15

16. Solid phase peptide synthesis (SPPS)16

17. Solid phase peptide synthesis (SPPS)
Temporary protecting group
Boc/Bzl Fmoc/tBu
Permanent protecting group 17

18. Solid phase peptide synthesis (SPPS)
Resins
Size of the resin beads ( µm)
Mechanical and thermal stability
Price
Swelling
Types:
Cross-linked polysterene
Polyamide
Tentagel
Soluble 18

19. Solid phase peptide synthesis (SPPS)
Resins
Wang
SWELLING
2-Chlorotrityl chloride
Rink amide Chemmatrix 19

20. Solid phase peptide synthesis (SPPS)
Coupling reagents 20

21. Solid phase peptide synthesis (SPPS)
Protecting groups 21

22. Solid phase peptide synthesis (SPPS)
Protecting groups 22

23. Remove only the desired PG without affecting the others
Solid phase peptide synthesis (SPPS)
The concept of orthogonality
Remove only the desired PG without affecting the others 23

24. Solid phase peptides synthesis (SPPS)
Tests to check coupling reactions
Kaiser
Free amines
Chloranil 24

25. Solid phase peptides synthesis (SPPS)
Side reactions
Diketopiperazine (DKP)
Epimerization: losing the stereochemistry 25

26. Solid phase peptides synthesis (SPPS)
Other inconveniences
Aggregation
Deletion of residues
Problems during the cleavage process 26

27. Solid phase peptides synthesis (SPPS)
Purification
Traditional chromatographic purification 27

28. 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 28

29. Solid phase peptides synthesis (SPPS)
Purification
HPLC can be used to separate the drug from all the other impurities 29

30. How can we improve synthetic peptides?
WHY?
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 30

31. How can we improve synthetic peptides?
Proteases 31

32. N-methylated peptides32

33. Cyclic peptides 33

34. Stapled peptides 34

35. Designing peptides Synthesis of natural peptides
Synthesis of novel peptides 35

36. Mimicking nature
Aromatic AA(Phe)
Variable AA 36

37. Creating new sequences
Envisaging the desired product
Proper selection of the strategy
Resin PG
Final removal of all the PG?
Further reactions and/or deprotections after cleavage
Carrying out the synthesis 37

38. Selecting the resin… 2-Chlorotrityl: 1% TFA in DCM
Protected peptide acid
Wang: 95% TFA
Peptide acid
Rink amide Chemmatrix: 95% TFA
Peptide amide 38

39. Choosing the suitable PG...
Removal in acidic conditions:
pNZ:1-6M SnCl2, 1.6mM HCl (dioxane) in DMF
Boc:25-50% TFA
tBu: 90% TFA
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 39

40. Choosing the suitable PG...40

41. Choosing the suitable PG...41

42. Analysis of the obtained product
High Performance Liquid Chromatography (HPLC) 42

43. Analysis of the obtained product
Mass spectrometry (MS) 43

44. Our lab (Giralt’s group)
Design, synthesis and structure of peptides and proteins 44