Proposal of Efficient Peptide Synthesis workflow

Fumio Kumakura1, Cedric Rentier2

1
Peptides Application Engineer, Japan, Biotage Japan Ltd., Japan
2
Peptidesand Biomolecules Application Specialist, Asia Pacific, Biotage Japan Ltd.

Introduction Experiment 2: Purification Concentrate cleavage cocktail and directly obtain crude
peptides prior to purification
Recently, the chemical peptide synthesis have been Biotage Purification System: Isolera
synthesized by full-automated peptide synthesizer. The Pooling and concentration of HPLC or HPFC fractions to obtain
synthesizer are used mainly multiple parallel synthesizer and a pure peptides
microwave heating peptide synthesizer. The crude peptides Remove peptide storage solvents (DMSO) quickly and gently
from removing resin got by HPLC purification and freeze dry Can come handy in organic chemistry as well for high boiling
spending for a lot of time and labor. Here we show efficient points solvents
peptide synthesis workflow obtained crude peptides by flash
rapid evaporation of aqueous and organic solutions
purification and evaporation system in Biotage.
5 mL (95% TFA : 2.5% TIS : 2.5% H2O)
Approx. 5 min to remove cleavage cocktail
Temperature 35 °C
Overview Ready for HPFC

The crude peptide could purify over 100 mg residues by flash

purification once and finish the purification for 30 minutes. The
V10 of evaporation system could evaporate cleavage TFA Figure 6. Freeze Dry peptide by V10 touch
solution and HPLC fraction solution and was complete for 30
minutes. We could get the crude peptide for suitable purity that Experiment 4
decrease a labor time of purification and evaporation after Peptide Synthesis Workflow solution
finishing the peptide synthesizer.

Table 1. HPLC vs HPFC columns purified by both techniques

Purify more peptide in less injections

Similar or better recovery
Reduction in solvent use
Significant reduction in overall purification time

Experimental
Experiment 1: Peptide Synthesis Figure 7. Standard Peptide Workflow Protocol – Room temperature

Biotage Microwave peptide Synthesizer: Initiator+ Alstra

Biotage Parallel peptide Synthesizer: SyroI
Table 2. Different media for different applications

Peptide PYY 3-36(IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-NH2)

5 mL (50% aq. ACN) containing 166 mg crude sample was injected on to a
Biotage® SNAP Bio C18 flash cartridge
Pure peptide was found in fractions 3–6 which were combined and freeze
dried to give 20 mg of pure peptide with >95% purity.

Figure 8. Biotage Workflow – single step purification strategy

Penetratin Synthesis Workflow

Cyclic peptides Synthesis
Peptide: RQIKIWFQNRRMKWKK-NH2
Peptide: CPAYSRRYLDC including Disulfide bond
Scale 0.69 mmol
0.11 mmol scale Figure 3. Chromatogram by HPLC and Purification by Isolera
Peptide cleaved TFA-H2O-TES-EDT (95:2:2:1)
HBTU/HOAt/DIEA, Coupling 75C, 10min Theoretical product weight 1.549 g
Disulfide formation by H2O2, NH3 in DMSO on Resin Reaction TFA cleavage cocktail concentrated on V-10 Touch
HPFC using a Biotage® SNAP Bio C18, 100 g column
Single injection of crude peptide

1 g peptide get for flash purification (65% yield)

Workflow completed within 2 working days
Figure 4. Chromatogram by HPLC after Isoleaa purification
Reference: Prof. Knud J. Jensen, Dr. Kasper K. Sørensen, University of Copenhagen

Next Gen High Performance Flash Chromatography Systems -

Selekt™ and its Sfär columns

Figure 1. Cyclic Petide by Initiator +Alstra

Scheme 1. Cyclic Petide Synthesis scheme
Figure 9. Result of Purification by Isolera and Chromatogram by HPLC
Reference: Prof. Knud J. Jensen, Dr. Kasper K. Sørensen, University of Copenhagen

Conclusion
Figure 5. New purification System: Selekt
Purity 85%

Experiment 3: Evaporation
Biotage evaporation System: V10 Touch

Figure 2.HPLC chart and MS spectra of Cyclic Peptide 83% crude purities
Reference: Prof. Knud J. Jensen, Dr. Kasper K. Sørensen, University of Copenhagen
Biotage Application Note AN93

Table 3.These solvents removed speed

© 2016 Biotage. All rights reserved. All trademarks are the property of their respective companies. This poster was presented at the 56th Japanese Peptide Symposium
www.biotage.com Part Number: P252