Merrifield Synthesis of Peptides
- 1. Merrifield Synthesis of Peptides ACHARYA INSTITUTE OF TECHNOLOGY Piyush Pal I 1AY16BT013 Genomics & Proteomics
- 2. Core Concepts Amino Acids Peptide Bonds Peptide Synthesis Merrifield Synthesis: Genomics & Proteomics Topics for discussion Principle Methodology Advantages and Disadvantages
- 3. DNA is a code of four letters; proteins are made up of amino acids which come in 20 forms. So the ribosome is a very clever machine that reads one language and operates in another. Ada Yonath Defining Amino Acids
- 4. Structural representation of Peptide Bond formation Peptide Bonds This is a dehydration synthesis reaction (also known as a condensation reaction), and usually occurs between amino acids. The bond formed is non-polar and covalent. Genomics & Proteomics
- 5. Production of peptides, compounds where multiple amino acids are linked Peptide Synthesis Solution-phase synthesis retains its usefulness in large-scale production of peptides for industrial purposes. Genomics & Proteomics Chemical peptide synthesis most commonly starts at the carboxyl end of the peptide (C-terminus), and proceeds toward the amino-terminus (N-terminus). The chemical synthesis of peptides can be carried out using classical solution-phase techniques, although these have been replaced in most research and development settings by solid-phase methods.
- 6. 1. SPPS allows the rapid assembly of a peptide chain through successive reactions of amino acid derivatives on an insoluble porous support. 2. With the growing chain covalently an insoluble matrix at all stages of the synthesis, the peptide would also be completely insoluble and furthermore would be in a suitable physical form to permit rapid filtration and washing after completion of each of the synthetic reaction. 3. The fact that all of the steps just described are heterogeneous reactions between a soluble reagent in the liquid phase and the growing peptide chain in the soluble solid phase led to the introduction of the name solid phase peptide synthesis. Basic principle of Merrifield's Solid Phase Peptide Synthesis (SPPS) Genomics & Proteomics
- 7. Linkers may be used that provide a reversible linkage between the synthetic peptide chain and the solid support. Resin is used as solid support. Dry polystyrene beads have an average diameter of 50 µm. An ideal support should have the required mechanical stability, should be stable under the reaction condition, could be functionalised easily and should have very good swelling characteristics. For effective swelling of the resin and solvation of the peptide, the polymer should have optimum hydrophilic-hydrophobic balance. Structure-reactivity, structure-property correlation in crosslinked polymer systems helped to design new supports with optimum reactivity, mechanical stability and other essential requirements of a polymeric support. Solid Support Genomics & Proteomics
- 8. A few notes on Protecting Groups Genomics & Proteomics The Boc approach needs to use highly toxic liquid HF for cleavage from the resin. Due to the development of strategies based on orthogonal protection, Fmoc has become the most important base-labile N-protecting group. In the last 15 years the Fmoc approach has prevailed over the Merrifield SPPS. Deprotection Fmoc group with piperidine in DMF. After last coupling the resin has to be dried in vacuo before cleavage. 8 Note: Refer to note associated with this slide for further information.
- 9. Stages of isolation and purification Peptide Isolation & Purification Genomics & Proteomics Precipitated by addition of cold diethyl ether. Directly from TFA solution or following evaporation of TFA and volatile scavengers. Centrifuge and wash with Et2O 3 times. Dry the solid in vacuo. Purification by HPLC. Characterisation by MALDI-TOF mass spectrum, NMR spectrometry.
- 10. Advantages of SPPS I It is easy to separate the soluble byproducts by filtration as the peptide remains attached to the insoluble polymer support II Excess reagents can be used to force the reaction to completion. This excess reagent can be removed by filtration III Mechanical loss of the material is avoided because the series of reaction steps are carried out in the same reaction vessel. IV The ability to purify, after each reaction by simple filtration and washing and the fact that all reactions can be conducted within a single reaction vessel, made this method ideal for automation. Genomics & Proteomics (A fast and simple method.)
- 11. Disadvantages of SPPS I Incompatibility of the growing peptide chain and the polymer support. II Reaction can't be monitored by the standard method. III Incomplete reactian may lead to deletion sequences. IV During the deprotection step, at least 1% of the resin-peptide bond is also cleaved, resulting in an overall decrease in yield. Genomics & Proteomics 11
- 12. Practical Applications Applications of synthetic peptides Peptides are developed for arthritis, diabetes, Cardiovascular diseases, Inflammation, Vaccines, Ophtalmology, Central Nervous System diseases, Antiviral, Antibacterial. Therapeutic Application : synthetic antigens; vaccines diagnostic tools immunostimulator peptides; muramyl dipeptide tuftsin derivatives Immune peptides conotoxins spider toxins snake toxins ionchanel blockers Toxins 12 Hormones oxytocin vasopressin insulin somatostatin GnRH etc.
- 13. Abbreviations Boc - tert-butoxycarbonyl DMF- N,N-dimethylformamide Fmoc - 9-fluorenylmethoxycarbonyl HF-Hydrofluoric acid SPPS - solid-phase peptide synthesis PEG - polystyrene glycol tBu -tert-butyl TFA-trifluoroacetic acid 3 Genomics & Proteomics Appendix 13
- 14. Resources Peptide synthesis https://en.wikipedia.org/wiki/Peptide_synthesis Basic principles and procedures of solid phase peptide synthesis http://www.cim.unito.it/website/documenti/IP_Probes_Turin_2011/lectures/Basic_principles_and_p rocedures_of_SPPS_2011.pdf SOLID PHASE PEPTIDE SYNTHESIS https://www.bachem.com/fileadmin/user_upload/pdf/Catalogs_Brochures/Solid_Phase_Peptide_Syn thesis.pdf Genomics & Proteomics Online sources used 14
- 15. Genomics & Proteomics Disclaimer Images used are for educational purpose only. No copyright infringement intended.