The latest SPPS application data

Transcription

1 The latest SPPS application data -innovative solution for peptide chemistry- Biotage Japan Ltd. Fumio Kumakura Ph,D

2 Biotage With more than 5,000 discovery chemistry systems installed in over 600 facilities worldwide, Biotage automated systems and consumables work together to increase productivity and improve success rates Synthesis Work-up Purification Evaporation Microwave-Assisted Organic Synthesis (MAOS) Solid-Phase Extraction (SPE) Automated Flash Purification Rapid Solvent Evaporation & PEPTIDES

3 Outline Microwave heating Application of Peptide Synthesis -Difficult sequence -N-Methylated amino acids -Selenocysteine -Glycosylated amino acids -ChemMatrix System -Peptide Synthesizer -Comparison: Synthesis method Summary

4 Microwave Heating Gives Faster and more precise heating Faster chemical reactions Greater yields and better purities Novel reactions

5 Advantages of Microwave Heating The rate of heating is generally higher than by conventional means No temperature gradient through the sample The energy transfer is direct to the absorbing reactants Allows reactions to occur in a more controlled manner in a decreased time scal

6 Microwave Assisted Peptide Synthesis Using Biotage Instruments Manual SPPS M. Erdélyi, A. Gogoll, Rapid microwave-assisted solid phase peptide synthesis, Synthesis, 2002, 11, * M. Brandt, S. Gammeltoft, K. J. Jensen, Microwave heating for solidphase peptide synthesis: General evaluation and applications to 15- mer phosphopeptides, International Journal of Peptide Research and Therapeutics, 2006, 12(4), Semi-automated SPPS S. L. Pedersen, K. K. Sørensen, K. J. Jensen, Semi-automated microwave-assisted SPPS: Optimization of protocols and synthesis of difficult sequences, Biopolymers (Peptide Science), 2010, 94, Fully automated SPPS L. Malik, A. P. Tofteng, S. L. Pedersen, K. K. Sørensen and K. J. Jensen, Automated X-Y robot for peptide synthesis with microwave heating: Application to difficult peptide sequences and protein domains, Journal of Peptide Science, 2010, 16,

7 What is a difficult sequence So called difficult sequences are problematic if not impossible to synthesize using standard coupling and deprotection protocols Difficulties are mainly related to: Intra- and/or intermolecular aggregation Secondary structure formation Steric hindrance of protecting groups which can generate premature termination of the sequence

8 Pancreatic Peptide YY3-36 (1-40) analogue H-YLERELKKLERELKKLSPEELNRYYASLRHYLNLVTRQRY-NH 2 The peptide hormone PYY3-36 plays a central role in the regulation of food intake and energy homeostasis. Synthesis is difficult due to reported PYY3-36 analogue consisting helix and loop due to C-terminus.

9 PYY3-36 analogue peptide reagent Resin: Fmoc-TG Rink amide resin 0.24 mmol/g loading Amino Acids: 770-μL of 0.5M Fmoc-AA in NMP with HOBt and HOAt (9:1) Coupling: μl of HBTU/NMP (0.43 M) μl of DIPEA/NMP (2.0 M) De-protect: 2000 μl of 40% piperidine in DMF Wash: NMP Søren L. Pedersen and Knud J. Jensen

10 PYY3-36 analogue method Synthesis Scale: 100 mmol Deprotection: 3 min with 40% piperidine in DMF at RT +10 min with 20% piperidine in DMF at RT Wash: 3 x 45 sec with NMP at RT Coupling: 1 x 45 min for RT or 1 x C (microwave) Wash: 3 x 30 sec with NMP at RT 3 x 30 sec with DCM at RT Søren L. Pedersen and Knud J. Jensen

11 Synthesis of PYY3-36 analogue H-YLERELKKLERELKKLSPEELNRYYASLRHYLNLVTRQRY-NH 2 45 min at RT Product Crude Purity 23% 10 min at 75ºC assisted MW Product Crude Purity 35%

12 Why are we interested in N-Methylated amino acids R N-Methylated amino acid Exist in many biologically-active natural products Help obtain information about backbone conformation Offer improved lipophilicity, proteolytic stability and bioavailability Replacement of natural amino acid for N-methyl amino acid in biologically active peptides has resulted in analogue with improved pharmacological properties

13 Coupling onto N-Methylated amino acids Me ZGYGFL-Resin X Me ZGYGFL-Resin The experiment was to synthesize four different N-methylated peptide sequences. Sequences were Me ZGYGGFL, with Z being Ala, Ile, Phe or Val, these to be some of the most difficult N-methylated amino acids to couple onto.

14 Coupling onto N-Methylated amino acids Z Z Coupling condition Coupling condition Coupling onto Me AGYGGFL Coupling onto Me FGYGGFL Coupling condition Coupling condition Coupling onto Me IGYGGFL Coupling onto Me VGYGGFL

15 Synthesis of N-Methylated Peptide Ala- Me Ile-Gly-Tyr-Gly-Gly-Phe-Leu Improved coupling conditions for coupling Fmoc-Ala-OH onto Me IGYGGGFL peptidyl.

16 N-Methylated Trimer Synthesis H- Me Ala- Me Ile- Me Gly-NH 2 Coupling condition for the synthesis of peptide

17 N-Methylated Trimer Synthesis 24 h at RT H- Me Ala- Me Ile- Me Gly-NH mau UV_VIS_1 WVL:215 nm Product Overall synthesis time: ~74 h Crude Purity 39% -200 min x 10 min at 75ºC assisted MW 300 mau Product UV_VIS_1 WVL:215 nm Overall synthesis time: ~3 h Crude Purity 80% -50 min

18 What is a Selenocysteine NH 2 HO 2 C SeH L-Selenocysteine (Sec) The 21st amino acid incorporated in proteins by the genetic codon. The active center of redox selenoenzymes, such as glutathione peroxidase. The application to determination of protein structure.

19 Synthesis of Selenopeptide H-Gly-Gln-Ala-Sec-Ala-Trp-Gly-NH 2 Coupling condition for the synthesis of selenopeptide **Sec protected selenium with p-methoxyphenylmethyl group

20 Synthesis of Selenopeptide H-Gly-Gln-Ala-Sec-Ala-Trp-Gly-NH 2 40 min at RT UV 220 nm Product Overall synthesis time: ~10 h Crude Purity 22% 5 min at 75ºC assisted MW UV 220 nm Product Overall synthesis time: ~5 h Crude Purity 48%

21 Glycosylated amino acids H-TRPAPGST*APPAHGVT*SAPD-NH 2 The 20-mer MUC1 tandem repeat sequence is a large extracellular glycoprotein which exist ubiquitously on the surface of mammalian cell membranes. Glycosylated amino acids use mono-saccharide derivatives of Fmoc-Thr(Ac 4 -β-glc)-oh and Fmoc-Thr(Ac 3 -α-galnac)- OH.

22 Synthesis of Glycopeptide H-TRPAPGST*APPAHGVT*SAPD-NH 2 T* = Thr(Ac 4 -β-glc) or Thr(Ac 3 -α-galnac) Glucose Peptide Galactosamine Peptide

23 Synthesis of GlycoPeptide H-TRPAPGST*APPAHGVT*SAPD-NH 2 20 min at 75 assisted MW Crude Purity 53% 20 min at RT Crude Purity 33% 2 h at RT Crude Purity 38% 20 min at 75 assisted MW +depro 2 min at 60 Crude Purity 15% HPLC chromatograms for the synthesis of the 20 mer peptide 1 using different reaction conditions

24 Synthesis of GlycoPeptide H-TRPAPGST*APPAHGVT*SAPD-NH 2 Entry 5 20 min at 75 assisted MW Crude Purity 64% Entry 6 20 min at 75 assisted MW +depro 2 min at 60 Crude Purity 30% HPLC chromatograms for the synthesis of the 20 mer peptide 2

25 ChemMatrix Resins If your peptide is: long, complex, or hydrophobic: ChemMatrix resin Biotage is now distributing ChemMatrix resins Biotage have selected 5 of the most popular linker chemistries for SPPS (Rink, Wang, HMPB, Trityl, PAL) ChemMatrix is a patented 100% PEG resin from Matrix Innovation that offers substantial advantages over traditional PS & PEG based resins for SPPS Peptides produced with ChemMatrix - higher purity and yields

26 Benefits of ChemMatrix Resin Exceptional stability more stability for the chemistry needed in peptide synthesis No Leaching does not add impurity to the end-users work flow Excellent solvent compatibility organic or aqueous, water or otherwise Many choices of linker and also pre-loaded options available we have access to a wide choice Proven superior performance comparison of synthesis of Selenoglutathione using Rinak amide- ChemMatrix and PS resin shows significant advantages Microwave compatible in peptide synthesizers or manual synthesis

27 Case Study: Selenoglutathione g-glu-sec-gly Se Coupling condition for the synthesis of selenopeptide

28 Case Study: Selenoglutathione MBHA Rink amide resin at RT g-glu-sec-gly UV 220 nm Product Overall synthesis time: ~5 h Crude Purity 34% ChemMatrix Rink amide resin at RT UV 220 nm Product Overall synthesis time: ~5 h Crude Purity 77%

29 Synthesis of C-terminus of MuLV CTL epitope H-WFTTLISTIM-NH 2 Different resins using COMU as coupling reagent

30 Synthesis of Dendrimer Peptide Different reaction conditions for the synthesis of dendrimer like structure

31 ChemMatrix Resins Fmoc-S-RAM-TG 45 min, RT Product Product Fmoc-S-RAM-TG 2 x 120 min, RT Product Fmoc-S-RAM-TG 5 min, 75 ChemMatrix 5 min, 75 Product Fmoc-S-RAM-TG 2 x 10 min, 75 Product

32 Peptide Synthesizer from Biotage Syro I (Auto) Syro II (Auto) Syrowave (Auto) Initiator Peptide Workstation (Manual) Initiator + SP wave (Semi-auto)

33 Syro I:Full-Automatic Single robotic arm 2 x digital syringe pump 1 Vortex mixers: parallel Choice of either: 24 or 48 channel reactor Vacuum Pump Amino Acid Rack: 32 x 50 ml Falcon Tubes Reagent Bottle Rack: 2 x 500 ml, 3 x 200 ml Waste Bottle: 1 x 10 liter Synthesis on µmol scale Includes Dell Desktop PC Flat Panel Monitor, Printer Syro XP Software

34 Initiator+ Peptide Workstation: Manual Microwave assisted peptide Manual synthesis Disposable 2-5 and ml glass reactor vials with HDPE frits Synthesis on µmol scale

35 Initiator + SP wave: Semi-Automatic Microwave assisted peptide and organic synthesis Semi-automated Disposable 2, 5 and 10 ml PPreactor vials with PTFE frits Synthesis on µmol scale Inert gas capability Easy-to use Initiator 4.0 software Use pre-defined templates or define your own Vortex mixing Single robot arm, digital syringe pump

36 Syrowave: Full-Automatic Microwave cavity Single robotic arm 1 x digital syringe pump 2 Vortex mixers: Microwave & parallel Choice of either: 24 or 48 channel reactor Vacuum Pump Amino Acid Rack: 32 x 50 ml Falcon Tubes Reagent Bottle Rack: 2 x 500 ml, 3 x 200 ml Waste Bottle: 1 x 10 liter Synthesis on µmol scale Includes Dell Desktop PC Flat Panel Monitor, Printer Syro XP Software

37 Comparison: Synthesis method 1. ACP65-74: H-VQAAIDYING-NH 2 2. LysM: H-LPERVKVVFPL-NH 2 3. JR: H-WFTTLISTIM-NH 2 Synthesis of a difficult peptide sequences using different levels of automation for microwave assisted SPPS: manual, semiautomated and fully automated systems. Three different peptides synthesized as test sequences. All the peptides were synthesized on the PEG-based ChemMatrix resin.

38 Compare to the system ACP 65-74: H-VQAAIDYING-NH 2 1 Syro Wave (Full-Auto) Product 2 min at 75 assisted MW Crude Purity 97% 2 Initiator+, SP Wave (Semi-Auto) Product 2 min at 75 assisted MW Crude Purity 93% 3 Initiator Peptide Workstation (Manual) Product 2 min at 75 assisted MW Crude Purity 94%

39 Compare the peptide synthesizer 1. ACP 65-74: H-VQAAIDYING-NH 2 2. LysM: H-LPERVKVVFPL-NH 2 3. JR: H-WFTTLISTIM-NH 2

40 Summmary Reduction in synthesis time and an increase in purity using Microwave Irradiation in SPPS -PYY 3-36 of Difficult Sequence -N-Methylated Peptide -Selenopeptide -Glycopeptide Improve the purity using ChemMatrix -MuLV CTL epitope peptide in Difficult Sequence -Selenoglutathione -Dendoric structure peptide Compare to the instruments for microwave assisted SPPS. -The Initiator Peptide Workstation gave the fastest cycle times however, is the most labor intensive. -The Syro Wave system gave excellent purity peptides with the least amount of user input. -The semi-automated Initiator+ SP Wave system required the perfect setup in terms of speed of synthesis, quality of peptides produced and level of user intervention.

41 Conclusion Microwave is powerful technique for accelerating the synthesis of peptides and peptidomimetics. Reduction in synthesis time and an increase in purity. Improve coupling rates and prevent side reactions in SPPS. ChemMatrix is efficient resin for Microwave in SPPS. Three instruments of automation solutions for microwave assisted SPPS provided peptides with high level of purities but differ in cycle times and level of user intervention required.