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Synthesis Optimization Tips

• Extend the coupling time (up to 4 minutes) and/or repeat coupling cycles for difficult regions and deletions.
• For difficult peptides or sequences longer than 25 residues, use a low-loading, high-swelling resin.
• Double couple pairs of adjacent nonpolar amino acids that have branched side chains (Val, Ile, Leu).
• Double couple hydrophobic regions of the peptide (Ala, Pro, Val, Leu, Ile, Met, Phe, Trp).

Peptide Cleavage and Analysis

Cleaving Peptides

Various scavenger molecules are added to the TFA cocktail to prevent the cleaved protecting groups from reattaching to
the peptide. The particular scavengers used depend on the specific peptide sequence. Common scavengers include water
(scavenges t-butyl cations), triisopropyl silane (TIS, scavenges trityl and Pbf cations), ethane dithiol (EDT, scavenges t-butyl
cations, reduces oxidation of Cys/Met side chains), dioxa-1,8-octane-dithiol (DODT, scavenges t-butyl cations, suppresses
oxidation of Cys/Met side chains), phenol (protects Tyr and Trp side chains from oxidation), and thioanisole (aids in removal
of Pbf protecting groups from Arg(Pbf), suppresses oxidation of Cys/Met side chains).

Cleavage can be performed either at room or elevated temperatures. The table below lists CEM’s recommended
parameters for both elevated and room temperature cleavage.

For sequences with more than 3 arginine residues, extend cleaving time by 5 minutes per Arg after 3 if using heated 
cleavage, or by 30 minutes if using room temperature cleavage. Avoid cleaving for over 2 hours total if heated, and over 4 
hours if at room temperature.

CEM Preference

Cleavage Cocktail

Temp

Time

1

TFA/TIS/H

2

O/DODT

1

 (92.5/2.5/2.5/2.5)

40-42 °C

30 min

2

2

TFA/TIS/H

2

O/DODT

1

 (92.5/2.5/2.5/2.5)

Room Temp

3 hours

2

1

DODT (dioxa-1,8-octane-dithiol) is a less malodorous alternative to EDT (ethane dithiol), and can be directly substituted for EDT without any 

difference in cleavage quality.

2

For sequences with multiple Arg(Pbf) residues, cleavage time may need to be extended to ensure complete removal of the Pbf.

 

NOTE

 

The Liberty Blue is not equipped to perform peptide cleavage. All cleavage must be carried out at either room 
temperature or in the Razor™ parallel cleavage system.

Common Mass Differences

Side Chain Protecting Groups

Incomplete cleavage can result in mass additions, due to incomplete removal of side chain protecting groups.

Mass Difference

Reason

+100

Boc

+56

tBu

+242

Trt

+252

Pbf

Содержание liberty

Страница 1: ...e is designed to highlight the basic chemistry and functions of the Liberty Blue Read and understand the Manual PN 600291 before operating the Liberty Blue For More Information Liberty Blue Operation Manual PN 600291 detailed information about setup and operation of the system CEM website www cem com list of spare parts pictures latest information about CEM s products references application notes ...

Страница 2: ... 5 0 mmol Wash Solvent DMF DMF DMF DMF DMF DMF Deprotection Cocktail 4 Piperidine in DMF or 2 Piperazine w v in EtOH NMP 10 90 4 Piperidine in DMF or 2 Piperazine w v in EtOH NMP 10 90 20 Piperidine in DMF or 10 Piperazine w v in EtOH NMP 10 90 20 Piperidine in DMF or 10 Piperazine w v in EtOH NMP 10 90 20 Piperidine in DMF or 10 Piperazine w v in EtOH NMP 10 90 20 Piperidine in DMF or 10 Piperazi...

Страница 3: ...re cleavage of the peptide from the resin low yields Activation methods containing DIEA are more basic and thus preferred to DIC Oxyma for these resins at high temperatures Since lower temperatures should be used the more aggressive HATU activator is recommended CEM Corporation offers Cl TCP Cl ProTide which shows excellent stability to high temperatures and acidic additives such as Oxyma Pure yet...

Страница 4: ...istidine His Boc suppresses epimerization at elevated temperature and by default is coupled using the standard coupling microwave method His Trt is susceptible to epimerization at elevated temperature His Trt should be coupled using the single 50 C 10 min couplpling cycle to minimize epimerization using the parameters in the table below Stage Temp o C Power W Time sec Delta T 1 25 0 120 2 2 50 35 ...

Страница 5: ...eavage of the peptide In these cases the use of Fmoc Asp OMpe OH in place of the more common Asp OtBu can reduce aspartimide formation without the addition of Oxyma Pure HOBt The Asp Gly DG segment is particularly sensitive to base catalyzed aspartimide formation Simple addition of HOBt or Oxyma Pure to the standard deprotection solutions may not be sufficient to prevent this side reaction The use...

Страница 6: ...sses oxidation of Cys Met side chains Cleavage can be performed either at room or elevated temperatures The table below lists CEM s recommended parameters for both elevated and room temperature cleavage For sequences with more than 3 arginine residues extend cleaving time by 5 minutes per Arg after 3 if using heated cleavage or by 30 minutes if using room temperature cleavage Avoid cleaving for ov...

Страница 7: ...positions Reaction Scale for Resin Transfer The resin transfer option is intended for syntheses at 0 1 or 0 25 mmol scale 0 25 1 0 g of resin The resin transfer option should only be used with the 30 mL reaction vessel This option has not been optimized by CEM for use with the 125 mL vessel Transfer Solvent The transfer solvent is used to move the resin into and out of the reaction vessel and to s...

Страница 8: ...ty coated glass 250 mL Bottle used for Activator Base 551325 500 mL Bottle Safety coated glass 500 mL Bottle used for Deprotection 551330 1 L Bottle Safety coated glass 1 L Bottle used for wash solvent 177285 6 L Carboy Plastic 6 L carboy used for waste Temperature Probe Part Number Name Description 314325 Fiber Optic Probe Probe used to measure and control reaction temperature Reaction Vessel Sel...

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