CEM liberty User manual

Liberty BlueTM
Automated Microwave Peptide Synthesizer
User Guide
WARNING
Handle all chemicals under a fume hood, and wear suitable protective clothing such as safety glasses, chemical
resistant gloves, and a laboratory coat. Dispose of all waste in accordance with all applicable local, state, and
federal health and safety recommendations. For detailed information on the safety requirements for the chemicals
used on the Liberty Blue, refer to the appropriate SDS documents.
This User Guide 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, and chemical reagents
• Applications Support: contact CEM at [phone] (US/Canada), [phone], or by email at
peptide.suppor[email protected].
• Technical Support: Contact CEM Service at [phone] (US/Canada) or [phone]
Part Number 600329
November 11, 2018
Rev. 6
Copyright CEM Corporation 2018

Reagents and Concentrations
Reagent Quality
The quality of reagents used for synthesis, including the age of reagents, will have a signicant impact on the quality of
peptides produced. Solvents should be higher than ACS grade. DMF should not be used if it is more than six months old.
TFA that has discolored (is no longer clear) should not be used; this can result in incomplete side chain deprotection.
Solid/undissolved amino acids should be stored at room temperature for no longer than six months, or in a freezer for no
longer than a year.
Reagents and Stability
Reagent Reagent Stability
Amino Acids in solution Two weeks*
Activators: DIC One month
Activator and Activator Bases: HBTU, 6-Cl HBTU, HATU, HOBt, 6-Cl HOBt, HOAt and Oxyma Pure, DIEA in NMP Two weeks
Deprotection solution One month
Cleavage Cocktail 1 day
* Notably, His is only stable for one week. Val, Ile, and Leu will begin to precipitate sooner than other amino acids. The reagent dip tube
lter(s) will need to be replaced if amino acids begin to crystallize.
The Liberty Blue uses stock solutions of all reagents. The table below details the concentrations used on the Liberty Blue
and/or CEM’s preferred reagents.
Reagents 0.005 mmol 0.01 mmol 0.025 mmol 0.05 mmol 0.1 - 0.5 mmol 1.0 - 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% Piperazine
(w/v) in
EtOH:NMP
(10:90)
Amino Acids 0.04 M in DMF 0.08 M in DMF 0.2 M in DMF 0.2 M in DMF 0.2 M in DMF 0.4 M in DMF
Activator** 0.05 M DIC in DMF 0.10 M DIC in
DMF
0.25 M DIC in DMF 0.50 M DIC in
DMF
1.0 M DIC in
DMF
1.0 M DIC in
DMF
Base 0.05 M Oxyma in
DMF
0.1 M Oxyma in DMF 0.25 M Oxyma in
DMF
0.5 M Oxyma in DMF 1.0 M Oxyma in DMF 1.0 M Oxyma
in DMF
*NMP is suitable as a wash solvent or for dissolution/dilution of reagents, but requires 75°C coupling cycles to reduce the risk of insertions.
**View the CarboMAX™application note, ap0124, at www.cem.com for further details.
Alternative Reagents
Piperidine/Piperazine
Piperazine is recommended as an alternative to piperidine. Piperazine has limited solubility in DMF and NMP but shows
improved solubility and deprotection activity in a solution of 10% ethanol in NMP by volume.
HOBt/HOBt • H2O/Oxyma
Anhydrous HOBt is considered explosive. The HOBt monohydrate can be used as a direct substitute for anhydrous HOBt
and is stable to ship. Oxyma can also be used as a direct substitute for HOBt.

Resin Selection
Default cycles are available for both standard resins (such as polystyrene) and high-swelling resins (such as PEG). It is
important to use the appropriate cycles for the resin being used in the synthesis. When the resin type is selected in the
Liberty Method Editor, the appropriate default cycles for that type will automatically be loaded.
Although most resins are compatible with microwave SPPS on the Liberty Blue, CEM recommends the resins shown in the
table below for optimal results.
Peptide Length C-Terminal Acid C-Terminal Amide
Standard (<25 residues) Cl-TCP(Cl) ProTideTM Fmoc Rink Amide ProTideTM
Long (>25 residues) Cl-MPA ProTideTM (LL) Fmoc Rink Amide ProTideTM (LL)
Mesh Size
Only 100-200 mesh (or lower) resin (>75 micron) should be used with the Liberty Blue. The use of higher mesh resins will
result in clogging of the reaction vessel frit and damage to the system.
Special Case: Hyper Acid Sensitive Resins (e.g. 2-chlorotrityl)
Acidic additives (HOBt, HOAt, Oxyma) can lead to premature 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 still exhibits hyper-acid sensitivity. The Cl-TCP(Cl) ProTide resin can be cleaved under 1% TFA/DCM
conditions.
CEM Preference Reagents Resin/Linker Coupling Microwave Method1Exceptions
1 AA/DIC/Oxyma2in DMF Cl-TCP(Cl) ProTide Resin Standard Coupling (90°C 2 min) N/A3
2-Chlorotrityl resin 60°C 5 min Coupling4N/A3
2 AA/HBTU/DIEA5in DMF Cl-TCP(Cl) ProTide Resin 75°C 5 min Coupling Cys/His: 50°C 10 min Coupling
2-Chlorotrityl resin 50°C 10 min Coupling N/A
1Standard deprotection (90°C 1 min) can be performed in all cases.
20.1 equivalents DIEA should be added to Oxyma solution
3Recommended Coupling Microwave Method assumes employment of His(Boc). For other His derivatives, use 50°C 10 min Coupling Micro-
wave Method.
4Coupling time can be extended to 10 min.
5DIEA should be prepared in NMP.

Microwave Method Parameters
Standard Deprotection
Stage Temp (oC) Power (W) Time (sec) Delta T
1 75 155 15 2
2 90 50 50 1
Standard Coupling
Stage Temp (oC) Power (W) Time (sec) Delta T
1 75 170 15 2
2 90 50 110 1
Microwave Power
Specic power settings will vary, depending on scale and the individual microwave. For both the deprotection and coupling
methods the set temperature of 90 °C should be reached within 20-30 seconds and maintained for at least 45 seconds
and 100 seconds respectively. The temperature should not exceed 95 °C. If this occurs, then the power for the stage
where the overage occurred should be decreased.
The power setting can be changed in the Microwave Editor, which can be accessed from the Edit menu. Power settings
can be changed “on-the-y”, meaning that changes made in the Microwave Editor during a run will be applied on the next
microwave step. The Run History le records the temperature during each microwave step and should be reviewed
periodically.
Special Coupling Cycles
DIC/Oxyma (or HOBt) Activation
Histidine
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 (oC) Power (W) Time (sec) Delta T
1 25 0 120 2
2 50 35 480 1
Arginine
Arginine is susceptible to γ-lactam formation, greatly reducing the coupling efciency. By default, Arg is coupled using a
modied double coupling cycle. Both coupling steps use the Standard Coupling microwave method.
Uronium Activation (HBTU/HATU)
Coupling cycles with uronium activation should be performed at 75°C to prevent premature quench of activated amino acid
ester. Below are exceptional cases.
Histidine
His(Trt) is susceptible to epimerization at elevated temperatures. By default, His(Trt) should be coupled using the single
50 ° C 10 min coupling cycle to minimize epimerization using the parameters in the table below. His(Boc) suppresses
epimerization at elevated temperature and can be coupled using the standard coupling microwave method.
Stage Temp (oC) Power (W) Time (sec) Delta T
1 25 0 120 2
2 50 50 480 1

Cysteine
Cysteine is susceptible to epimerization at elevated temperatures in the presence of bases such as DIEA and NMM. If an
uronium activation strategy is being used, Cys should be coupled using the single 50 ° C 10 min coupling cycle with the
parameters listed in the table below.
Stage Temp (oC) Power (W) Time (sec) Delta T
1 25 0 120 2
2 50 50 480 1
Arginine
Arginine is susceptible to γ-lactam formation, greatly reducing the coupling efciency. For uronium activation, Arg is coupled
using an Arg double coupling cycle with the parameters listed in the table below.
First Coupling
Stage Temp (oC) Power (W) Time (sec) Delta T
1 25 0 1500 2
2 75 30 120 1
Second Coupling
Stage Temp (oC) Power (W) Time (sec) Delta T
1 75 30 300 2
Potential Side Reactions
Aspartimide Formation
Aspartimide formation is a common side reaction that occurs in peptides containing Asp followed by Asn, Asp, Gly, Thr,
or Ser. The addition of 0.1 M Oxyma Pure or 0.1 M HOBt to the deprotection solution will suppress the side reaction. When
using the UV monitoring option, Oxyma Pure and HOBt show signicant UV absorbance. When using acid-sensitive linkers
other than Cl-TCP(Cl), Oxyma Pure and HOBt can cause premature cleavage 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 sufcient to prevent this side reaction. The use of less
aggressive deprotecting conditions or a Dmb protected dipeptide will prevent aspartimide formation from occurring.
Supression Strategy Microwave Method
5% (w/v) Piperazine w/ 0.1 M Oxyma Pure in EtOH:NMP (10:90) Two step room temperature deprotection:
1. Conventional initial deprotection
2. Conventional deprotection
Use Fmoc-Asp(OtBu)-(Dmb)Gly-OH dipeptide and any deprotection cocktail applicable for
standard peptides
Standard
Use Fmoc-Asp(OMpe)-OH and any deprotection cocktail applicable for standard peptides. Standard
Tetramethyl Guanidinium Capping
For extended coupling times (10 minutes or longer), tetramethylguanidinium capping of the free amine by HBTU (or other
uronium-type activators) is possible. This side reaction is detected as a truncation of target +101 on mass spec. Capping
by activator can be minimized by using a slight excess of other reagents (for example, 5 eq AA/4.5 eq HBTU/10 eq DIEA),
or by using alternate activator strategies (such as DIC/Oxyma Pure or DIC/HOBt).

Synthesis Optimization Tips
• Extend the coupling time (up to 4 minutes) and/or repeat coupling cycles for difcult regions and deletions.
• For difcult 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 specic 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/H2O/DODT1(92.5/2.5/2.5/2.5) 40-42 °C 30 min2
2 TFA/TIS/H2O/DODT1(92.5/2.5/2.5/2.5) Room Temp 3 hours2
1DODT (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.
2For 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

Amino Acid Deletions
Mass Difference Reason Mass Difference Reason Mass Difference Reason
-71 -Ala -57 -Gly -97 -Pro
-156 -Arg -137 -His -87 -Ser
-114 -Asn -113 -Ile -101 -Thr
-115 -Asp -113 -Leu -186 -Trp
-103 -Cys -128 -Lys -163 -Tyr
-128 -Gln -131 -Met -99 -Val
-129 -Glu -147 -Phe
Side Reactions N-terminal Modifications
Mass Difference Reason Mass Difference Reason
-18 Aspartimide +223 Fmoc
+67 Aspartimide +42 N-Acetyl
+101 Tetramethylguanidium
Salts
Because peptides are charged molecules, the target is often detected as a salt when analyzing peptides by ESI-MS.
Mass Difference Reason
+23 Na+ salt
+39 K+ salt
+114 TFA salt
+46 Formic salt
Liberty Blue Accessories
HT12 and HT24 Resin Loader
The HT12 and HT24 resin loaders increase the throughput of the Liberty Blue system by adding a modular resin loading
option with up to 24 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 swell the resin. The
recommended transfer solvent is 50% DMF/50% DCM by volume. This mixture will ensure optimal suspension of the resin
for efcient transfer.
It is important that approximately 10 mL of transfer solvent is added to the resin tube before loading resin on the system
for swelling, with the exception of Cl-TCP(Cl) ProTide and Cl-MPA ProTide. The Cl-TCP(Cl) ProTide and Cl-MPA ProTide resins
can hydrolyze, reducing yield, and should not sit in the swelling solvent for prolonged periods of time. These resins are the
only resins that should be loaded onto the HT module without solvent.

Spare Parts
For additional parts visit our website: www.cem.com
Filters
Part Number Name Description
551005-M Dip Tube Filters Replacement lters for all reagent dip tubes (bag of 30 lters)
551085 Vent Line Filter Replacement lter for the vent line (located before RV1)
551000 External Bottle Filter Assembly To be used for main solvent, deprotect, and transfer solvent bottles ONLY
Bottles and Caps
Part Number Name Description
551320 250 mL Bottle Safety 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 Selection
Part Number Name Description
167260 30 mL Reaction Vessel Standard reaction vessel used for 0.005 - 0.25 mmol syntheses (up to 1 g of resin).
542415 125 mL Reaction Vessel Optional larger reaction vessel used for 0.5- 5 mmol syntheses (up to 8 g of resin).