Liberty Blue TM Automated Microwave Peptide Synthesizer. User Guide

Transcription

1 Liberty Blue TM 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 labaty coat. Dispose of all waste in accdance with all applicable local, state, and federal health and safety recommendations. F detailed infmation on the safety requirements f 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 ) befe operating the Liberty Blue. F Me Infmation Liberty Blue Operation Manual (PN ): detailed infmation about setup and operation of the system. CEM website ( list of spare parts, pictures, latest infmation about CEM s products, references, application notes, and chemical reagents Applications Suppt: contact CEM at (800) (US/Canada), (704) , by at peptide.suppt@cem.com. Technical Suppt: Contact CEM Service at (800) (US/Canada) (704) Part Number November 11, 2018 Rev. 6 Copyright CEM Cpation 2018

2 Reagents and Concentrations Reagent Quality The quality of reagents used f synthesis, including the age of reagents, will have a significant impact on the quality of peptides produced. Solvents should be higher than ACS grade. should not be used if it is me than six months old. TFA that has discoled (is no longer clear) should not be used; this can result in incomplete side chain deprotection. Solid/undissolved amino acids should be sted at room temperature f no longer than six months, in a freezer f no longer than a year. Reagents and Stability Reagent Amino Acids in solution Activats: DIC Activat and Activat Bases: HBTU, 6-Cl HBTU, HATU, HOBt, 6-Cl HOBt, HOAt and Oxyma Pure, DIEA in NMP Deprotection solution Cleavage Cocktail Reagent Stability Two weeks* One month Two weeks One month 1 day * Notably, His is only stable f one week. Val, Ile, and Leu will begin to precipitate sooner than other amino acids. The reagent dip tube filter(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/ CEM s preferred reagents. Reagents mmol 0.01 mmol mmol 0.05 mmol mmol mmol Wash Solvent* Deprotection Cocktail 4% Piperidine in 2% Piperazine 4% Piperidine in 2% Piperazine 20% Piperidine in 10% Piperazine 20% Piperidine in 10% Piperazine 20% Piperidine in 10% Piperazine 20% Piperidine in 10% Piperazine Amino Acids 0.04 M in 0.08 M in 0.2 M in 0.2 M in 0.2 M in 0.4 M in Activat** 0.05 M DIC in 0.10 M DIC in Base 0.05 M Oxyma in 0.1 M Oxyma in 0.25 M Oxyma in 0.25 M DIC in 0.50 M DIC in 1.0 M DIC in 1.0 M DIC in 0.5 M Oxyma in 1.0 M Oxyma in 1.0 M Oxyma in *NMP is suitable as a wash solvent f dissolution/dilution of reagents, but requires 75 C coupling cycles to reduce the risk of insertions. **View the CarboMAX application note, ap0124, at f further details. Alternative Reagents Piperidine/Piperazine Piperazine is recommended as an alternative to piperidine. Piperazine has limited solubility in and NMP but shows improved solubility and deprotection activity in a solution of 10% ethanol in NMP by volume. HOBt/HOBt H 2 O/Oxyma Anhydrous HOBt is considered explosive. The HOBt monohydrate can be used as a direct substitute f anhydrous HOBt and is stable to ship. Oxyma can also be used as a direct substitute f HOBt.

3 Resin Selection Default cycles are available f both standard resins (such as polystyrene) and high-swelling resins (such as PEG). It is imptant to use the appropriate cycles f the resin being used in the synthesis. When the resin type is selected in the Liberty Method Edit, the appropriate default cycles f 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 f optimal results. Peptide Length C-Terminal Acid C-Terminal Amide Standard (<25 residues) Cl-TCP(Cl) ProTide TM Fmoc Rink Amide ProTide TM Long (>25 residues) Cl-MPA ProTide TM (LL) Fmoc Rink Amide ProTide TM (LL) Mesh Size Only mesh ( 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-chlotrityl) Acidic additives (HOBt, HOAt, Oxyma) can lead to premature cleavage of the peptide from the resin (low yields). Activation methods containing DIEA are me basic and thus preferred to DIC/Oxyma f these resins at high temperatures. Since lower temperatures should be used, the me aggressive HATU activat is recommended. CEM Cpation 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 Method 1 Exceptions 1 AA/DIC/Oxyma 2 in 2 AA/HBTU/DIEA 5 in 1 Standard deprotection (90 C 1 min) can be perfmed in all cases equivalents DIEA should be added to Oxyma solution Cl-TCP(Cl) ProTide Resin Standard Coupling (90 C 2 min) N/A 3 2-Chlotrityl resin 60 C 5 min Coupling 4 N/A 3 Cl-TCP(Cl) ProTide Resin 75 C 5 min Coupling Cys/His: 50 C 10 min Coupling 2-Chlotrityl resin 50 C 10 min Coupling N/A 3 Recommended Coupling Microwave Method assumes employment of His(Boc). F other His derivatives, use 50 C 10 min Coupling Microwave Method. 4 Coupling time can be extended to 10 min. 5 DIEA should be prepared in NMP.

4 Microwave Method Parameters Standard Deprotection Standard Coupling Microwave Power Specific power settings will vary, depending on scale and the individual microwave. F both the deprotection and coupling methods the set temperature of 90 C should be reached within seconds and maintained f at least 45 seconds and 100 seconds respectively. The temperature should not exceed 95 C. If this occurs, then the power f the stage where the overage occurred should be decreased. The power setting can be changed in the Microwave Edit, which can be accessed from the Edit menu. Power settings can be changed on-the-fly, meaning that changes made in the Microwave Edit during a run will be applied on the next microwave step. The Run Histy file recds the temperature during each microwave step and should be reviewed periodically. Special Coupling Cycles DIC/Oxyma ( 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 Arginine Arginine is susceptible to γ-lactam fmation, greatly reducing the coupling efficiency. By default, Arg is coupled using a modified double coupling cycle. Both coupling steps use the Standard Coupling microwave method. Uronium Activation (HBTU/HATU) Coupling cycles with uronium activation should be perfmed 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

5 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 Arginine Arginine is susceptible to γ-lactam fmation, greatly reducing the coupling efficiency. F uronium activation, Arg is coupled using an Arg double coupling cycle with the parameters listed in the table below. First Coupling Second Coupling Potential Side Reactions Aspartimide Fmation Aspartimide fmation is a common side reaction that occurs in peptides containing Asp followed by Asn, Asp, Gly, Thr, Ser. The addition of 0.1 M Oxyma Pure 0.1 M HOBt to the deprotection solution will suppress the side reaction. When using the UV moniting option, Oxyma Pure and HOBt show significant UV absbance. 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 me common Asp(OtBu) can reduce aspartimide fmation without the addition of Oxyma Pure/HOBt. The Asp-Gly (DG) segment is particularly sensitive to base-catalyzed aspartimide fmation. Simple addition of HOBt Oxyma Pure to the standard deprotection solutions may not be sufficient to prevent this side reaction. The use of less aggressive deprotecting conditions a Dmb protected dipeptide will prevent aspartimide fmation from occurring. Supression Strategy Microwave Method 5% (w/v) Piperazine w/ 0.1 M Oxyma Pure in 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 f standard peptides Use Fmoc-Asp(OMpe)-OH and any deprotection cocktail applicable f standard peptides. Standard Standard Tetramethyl Guanidinium Capping F extended coupling times (10 minutes longer), tetramethylguanidinium capping of the free amine by HBTU ( other uronium-type activats) is possible. This side reaction is detected as a truncation of target +101 on mass spec. Capping by activat can be minimized by using a slight excess of other reagents (f example, 5 eq AA/4.5 eq HBTU/10 eq DIEA), by using alternate activat strategies (such as DIC/Oxyma Pure DIC/HOBt).

6 Synthesis Optimization Tips Extend the coupling time (up to 4 minutes) and/ repeat coupling cycles f difficult regions and deletions. F difficult peptides 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 perfmed either at room elevated temperatures. The table below lists CEM s recommended parameters f both elevated and room temperature cleavage. F sequences with me than 3 arginine residues, extend cleaving time by 5 minutes per Arg after 3 if using heated cleavage, by 30 minutes if using room temperature cleavage. Avoid cleaving f 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) 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 malodous alternative to EDT (ethane dithiol), and can be directly substituted f EDT without any difference in cleavage quality. 2 F 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 perfm peptide cleavage. All cleavage must be carried out at either room temperature in the Raz 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

7 Amino Acid Deletions Mass Difference Reason Mass Difference Reason Mass Difference Reason -71 -Ala -57 -Gly -97 -Pro Arg His -87 -Ser Asn Ile Thr Asp Leu Trp Cys Lys Tyr Gln Met -99 -Val Glu 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 Fmic salt Liberty Blue Accessies 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 f Resin Transfer The resin transfer option is intended f syntheses at mmol scale ( 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 f 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% /50% DCM by volume. This mixture will ensure optimal suspension of the resin f efficient transfer. It is imptant that approximately 10 ml of transfer solvent is added to the resin tube befe loading resin on the system f 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 f prolonged periods of time. These resins are the only resins that should be loaded onto the HT module without solvent.

8 Spare Parts F additional parts visit our website: Filters Part Number Name Description M Dip Tube Filters Replacement filters f all reagent dip tubes (bag of 30 filters) Vent Line Filter Replacement filter f the vent line (located befe RV1) External Bottle Filter Assembly To be used f main solvent, deprotect, and transfer solvent bottles ONLY Bottles and Caps Part Number Name Description ml Bottle Safety coated glass 250 ml Bottle used f Activat/Base ml Bottle Safety coated glass 500 ml Bottle used f Deprotection L Bottle Safety coated glass 1 L Bottle used f wash solvent L Carboy Plastic 6 L carboy used f waste Temperature Probe Part Number Name Description Fiber Optic Probe Probe used to measure and control reaction temperature Reaction Vessel Selection Part Number Name Description ml Reaction Vessel Standard reaction vessel used f mmol syntheses (up to 1 g of resin) ml Reaction Vessel Optional larger reaction vessel used f mmol syntheses (up to 8 g of resin).