Green Ether Peptide Precipitation
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Green ether for precipitation purposes of peptides after global deprotection
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Green Ether Peptide Precipitation
- 1. Othman Al Musaimi Peptide Science Laboratory School of Health Sciences / School of Chemistry UKZN 1 GREEN ETHER FOR PEPTIDE PRECIPITATION AFTER GLOBAL DEPROTECTION
- 2. 2
- 3. Introduction Diethyl ether (DEE) and methyl tert-butyl ether (MTBE), common ethers of precipitation of peptide after last step of SPPS (global deprotection). 3
- 4. 4 4 DEE *Classified as a highly hazardous chemical. Low flash point - 45⁰C Low boiling point 35⁰C low temperature of auto ignition and it is very prone to form peroxides. MTBE *Classified as a highly hazardous chemical. #Carcinogenic, due carcinogenicity of its two metabolites (formaldehyde and tertiary butanol). Low flash point -28⁰C Low boiling point 55⁰C Unstable under acidic condition (tert butylate the final peptide product).$ Cyclopentyl methyl ether (CPME) Favourable environmental, health and safety (EHS) characteristics: High flash point -1⁰C High boiling point 106⁰C Excellent stability under acidic conditions Low tendency to form peroxides * Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). # World health organization (WHO). $ B.G. de La Torre, D. Andreu, On choosing the right ether for peptide precipitation after acid cleavage, J Pept Sci, 14 (2008) 360-3,10.1002/psc.956.
- 5. 5 TBME M + 56 CPME M + 69 Not Observed Possible Alkylated by product
- 6. 6 6 PEPTIDES AND RESINS USED POLYSTYRENE (PS)-BASED RESIN OR A POLYETHYLENGLYCOL (PEG)-BASED RESIN (CHEMMATRIX, CM).
- 7. Procedure 1. Each peptide resin was divided into two portions. 2. Cleavage mixture TFA/TIS/H2O (95:2.5:2.5) for 1.0 h. 3. Cold DEE / CPME was then added to precipitate the peptide. 4. Mixtures were kept on ice for 30 min. 5. Centrifugation at 5000 rpm for 5 min, and the supernatant was decanted. 6. New ethers added, centrifugation as per step 5. 7. Any remaining ether was dried under N2. 8. Precipitate was dissolved in water. 7
- 8. Procedure 9. Some samples were filtered and injected into HPLC and LCMS systems. 10. Lyophilization, to calculate the amount of the peptide recovered from the resin 8
- 9. Results Leu-enkephalin was not precipitated by CPME! possibly because of the high solubility of this short pentapeptide in the hydrophobic solvent CPME. 9
- 10. 10 GRADIENT ELUTION: 5-95% PHENOMENEX C18 COLUMN (150 × 4.6 MM, 3.6ΜM) BLUE: DEE BROWN: CPME (AFTER EVAPORATION) Enkephalin-OH, CTC-PS
- 11. 11 Entry Resin type Peptide % Yield DEE TBME CPME 1 CTC-PS Leu-Enkephalin 79.2 15.0* 0 2 Wang-PS ACP-OH 58.8 59.4 54.1 3 Rink Amide-PS ACP-NH2 74.9 59.8 89.6 4 Rink Amide-PS ABRF 1992 93.3 94.3 93.1 5 Rink Amide-CM ABRF 1992 31.8 31.7 31.8 6 Rink Amide-CM ABC 26.7 32.8 26.7 7 Rink Amide-PS Thymosin 74.8 71.7 74.5
- 12. 12 ACP-NH2, AM-R-PS GRADIENT ELUTION: 15-25% PHENOMENEX C18 COLUMN (150 × 4.6) mm, 3.6μm BLUE: DEE BROWN: CPME BLACK: MTBE
- 13. 13 ABRF-NH2, AM-R-PS GRADIENT ELUTION: 10-50% PHENOMENEX C18 COLUMN (150 × 4.6) mm, 3.6μm BLUE: DEE BROWN: CPME BLACK: MTBE
- 14. 14 ABC-NH2, AM-CM GRADIENT ELUTION: 20-60% PHENOMENEX C18 COLUMN (150 × 4.6) mm, 3.6μm BLUE: DEE BROWN: CPME BLACK: MTBE
- 15. 15 Thymosin-NH2, AM-CM GRADIENT ELUTION: 15-25% PHENOMENEX C18 COLUMN (150 × 4.6) mm, 3.6μm BLUE: DEE BROWN: CPME BLACK: MTBE
- 16. THANK YOU Refer for our paper for more details: https://doi.org/10.1016/j.cogsc.2018.06.017 16