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Crazy about biomedicine Júlia García Pindado

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.

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Crazy about biomedicine Júlia García Pindado

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

  2. Organic chemistry: 5 elements to build us up

  3. A brief reminder about proteins and peptides What’sanaminoacid? Chirality

  4. A brief reminder about proteins and peptides

  5. A brief reminder about proteins and peptides

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

  7. A brief reminder about proteins and peptides Proteins

  8. A brief reminder about proteins and peptides Protein structure

  9. A brief reminder about proteins and peptides Peptides

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

  11. Peptides as drugs Biologics Small molecules Peptides 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

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

  13. Which is the appearance of peptides?

  14. Peptide synthesis

  15. Solid phase peptide synthesis (SPPS) Developed by Merrifield in 1963 • Fastdevelopment • Complexpeptides can be reached • 60-70% efficiency Microwave assisted SPPS Manual SPPS

  16. Solid phase peptide synthesis (SPPS)

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

  18. Solid phase peptide synthesis (SPPS) • Resins • Size of theresinbeads (80-200 µm) • Mechanical and thermalstability • Price • Swelling • Types: • Cross-linkedpolysterene • Polyamide • Tentagel • Soluble

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

  20. Solid phase peptide synthesis (SPPS) Couplingreagents

  21. Solid phase peptide synthesis (SPPS) Protectinggroups

  22. Solid phase peptide synthesis (SPPS) Protectinggroups

  23. Solid phase peptide synthesis (SPPS) The concept of orthogonality Removeonlythedesired PG withoutaffectingtheothers

  24. Solid phase peptides synthesis (SPPS) Teststocheckcouplingreactions Kaiser Free amines Chloranil

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

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

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

  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

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

  30. How can we improve synthetic peptides? WHY? To… - Increase stability - Enhance cell uptake and permeability through biological barriers - Improve resistance against proteases • N-methylatedpeptides • Non natural aminoacids • Cyclicpeptides • Stapledpeptides

  31. How can we improve synthetic peptides? Proteases

  32. N-methylated peptides

  33. Cyclic peptides

  34. Stapled peptides

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

  36. Mimicking nature Aromatic AA(Phe) Variable AA

  37. Creating new sequences • Envisagingthedesiredproduct • Properselection of thestrategy • Resin • PG • Final removal of allthe PG? • Furtherreactions and/ordeprotectionsaftercleavage • Carryingoutthesynthesis

  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

  39. Choosing the suitable PG... Removal in acidicconditions: pNZ:1-6M SnCl2, 1.6mM HCl (dioxane) in DMF Trt: 1% TFA in DCM Boc:25-50% TFA tBu: 90% TFA Removal in basicconditions: Fmoc:20% piperidine/DMF Bzl: NaOH in organic solvents Removal in otherconditions: N3 Acm: I2 (S-S) Hg(II) (SH) Azide: PMe3 in dioxane Alloc: Pd(PPh)3 cat., scavengers PhSiH3 in organic solvents Al: Pd(PPh)3 cat., scavengers PhSiH3 in organic solvents

  40. Choosing the suitable PG...

  41. Choosing the suitable PG...

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

  43. Analysis of the obtained product Massspectrometry (MS)

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

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