1. Diethyl ether (DEE) and methyl tert-butyl ether (MTBE) are commonly used to precipitate peptides after global deprotection during solid phase peptide synthesis, but have some disadvantages. Cyclopentyl methyl ether (CPME) is proposed as an alternative ether that has more favorable environmental, health and safety properties. 2. Various peptides synthesized on different resin types were cleaved from the resin and precipitated using DEE, CPME or MTBE. CPME successfully precipitated peptides except for the short pentapeptide Leu-enkephalin. HPLC analysis showed comparable purity of peptides precipitated with the different ethers. 3. In conclusion, CPME is a

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).
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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.
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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
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9. Results
 Leu-enkephalin was not precipitated by CPME! possibly because
of the high solubility of this short pentapeptide in the
hydrophobic solvent CPME.
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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

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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

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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

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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
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