The latest SPPS application data

The latest SPPS application data

-innovative solution for peptide chemistry-

Biotage Japan Ltd.

Biotage

Microwave-Assisted Organic Synthesis (MAOS) Rapid Solvent Evaporation Automated Flash Purification

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

Solid-Phase Extraction (SPE)

Synthesis Work-up Purification Evaporation

Outline

• Microwave heating

• Application of Peptide Synthesis

-Difficult sequence

-

N

-Methylated amino acids

-Selenocysteine

-Glycosylated amino acids

-ChemMatrix

• System

-Peptide Synthesizer

-Comparison: Synthesis method

• Faster and more precise heating

• Faster chemical reactions

• Greater yields and better purities

• Novel reactions

• 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

Microwave Assisted Peptide Synthesis

Using Biotage Instruments

Manual SPPS

M. Erdélyi, A. Gogoll, Rapid microwave-assisted solid phase peptide synthesis, Synthesis, 2002, 11, 1592-1596*

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), 349-357

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, 206-212

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, 506-512

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

Pancreatic Peptide YY3-36 (1-40)

analogue

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.

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: + 880 μL of HBTU/NMP (0.43 M) + 380 μL of DIPEA/NMP (2.0 M)

• De-protect: 2000 μL of 40% piperidine in DMF • Wash: NMP

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 10 min @ 75 0_{C (microwave)} • Wash: 3 x 30 sec with NMP at RT

3 x 30 sec with DCM at RT

Synthesis of PYY3-36 analogue

H-YLERELKKLERELKKLSPEELNRYYASLRHYLNLVTRQRY-NH₂ Crude Purity 23% Crude Purity 35% 45 min at RT 10 min at 75ºC assisted MW Product Product

Why are we interested in

N

-Methylated

amino acids

R

• 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

Coupling onto

N

-Methylated amino acids

• 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.

Z Z

Coupling onto

N

-Methylated amino acids

Coupling onto Me_AGYGGFL

Coupling onto Me_FGYGGFL

Coupling onto Me_{IGYGGFL Coupling onto} Me_VGYGGFL

Coupling condition _{Coupling condition}

Synthesis of

N

-Methylated Peptide

Improved coupling conditions for coupling Fmoc-Ala-OH onto Me_{IGYGGGFL peptidyl.}

N

-Methylated Trimer Synthesis

H-

Me

Ala-

Me

Ile-

Me

Gly-NH

₂ Coupling condition for the synthesis of peptide

H-

Me

Ala-

Me

Ile-

Me

Gly-NH

2 24 h at RT 1.00 2.50 3.75 5.00 6.25 7.50 8.75 10.00 -50 100 200 300 UV_VIS_1 mAU min WVL:215 nm 1.00 2.50 3.75 5.00 6.25 7.50 8.75 10.00 -200 0 200 500 UV_VIS_1 mAU min WVL:215 nm 2 x 10 min at 75ºC assisted MW

Overall synthesis time: ~74 h

Overall synthesis time: ~3 h

Product Product Crude Purity 39% Crude Purity 80%

What is a Selenocysteine

• 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.

HO

₂

C

SeH

NH

₂

Synthesis of Selenopeptide

H-Gly-Gln-Ala-Sec-Ala-Trp-Gly-NH

₂

Coupling condition for the synthesis of selenopeptide

Synthesis of Selenopeptide

H-Gly-Gln-Ala-Sec-Ala-Trp-Gly-NH

₂

Overall synthesis time: ~10 h

Overall synthesis time: ~5 h Crude Purity 22% Crude Purity 48% 40 min at RT 5 min at 75ºC assisted MW Product Product UV 220 nm UV 220 nm

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(Ac4_{-β-Glc)-OH and Fmoc-Thr(Ac}3

Synthesis of Glycopeptide

H-TRPAPGST*APPAHGVT*SAPD-NH

₂

T* = Thr(Ac4_{-β-Glc) or Thr(Ac}3_-α-GalNAc)

Glucose Peptide

Synthesis of GlycoPeptide

H-TRPAPGST*APPAHGVT*SAPD-NH

₂

HPLC chromatograms for the synthesis of the 20 mer peptide 1

using different reaction conditions

20 min at 75℃ assisted MW 20 min at RT 2 h at RT 20 min at 75℃ assisted MW +depro 2 min at 60℃ Crude Purity 53% Crude Purity 33% Crude Purity 38% Crude Purity 15%

Synthesis of GlycoPeptide

H-TRPAPGST*APPAHGVT*SAPD-NH

₂

20 min at 75℃ assisted MW _{Crude Purity}

64% Entry 6 Entry 5 Crude Purity 30% 20 min at 75℃ assisted MW +depro 2 min at 60℃

HPLC chromatograms for the synthesis of the 20 mer peptide 2

ChemMatrix Resins

• 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

•

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

Case Study: Selenoglutathione

g

-Glu-Sec-Gly

Se

Case Study: Selenoglutathione

g

-Glu-Sec-Gly

Overall synthesis time: ~5 h

Overall synthesis time: ~5 h Crude Purity

34%

Crude Purity 77%

MBHA Rink amide resin at RT

ChemMatrix Rink amide resin at RT

Product

Product

UV 220 nm

Synthesis of C-terminus of MuLV CTL epitope

H-WFTTLISTIM-NH

₂

Synthesis of Dendrimer Peptide

Different reaction conditions for the synthesis of dendrimer like structure

ChemMatrix Resins

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

Peptide Synthesizer from Biotage

Syrowave (Auto) Syro I (Auto) Syro II (Auto)

Initiator Peptide Workstation

(Manual) Initiator

+ _{SP wave}

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 1-300 µmol scale Includes Dell Desktop PC

Flat Panel Monitor, Printer Syro XP Software

Initiator+ Peptide Workstation: Manual

• Microwave assisted peptide • Manual synthesis

• Disposable 2-5 and 10-20 ml glass reactor vials with HDPE frits

Initiator

+

SP wave: Semi-Automatic

• Microwave assisted peptide and organic synthesis

• Semi-automated

• Disposable 2, 5 and 10 ml PP-reactor vials with PTFE frits

• Synthesis on 5-300 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

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 5-300 µmol scale Includes Dell Desktop PC

Flat Panel Monitor, Printer Syro XP Software

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,

semi-automated and fully semi-automated systems.

• Three different peptides synthesized as test sequences. All the peptides were synthesized on the PEG-based ChemMatrix

Compare to the system

ACP 65-74: H-VQAAIDYING-NH

₂ Crude Purity 97% Product Crude Purity 93% Crude Purity 94% Product

Product _{2 min at 75℃ assisted MW}

2 min at 75℃ assisted MW 2 min at 75℃ assisted MW 1 Syro Wave (Full-Auto) Initiator Peptide Workstation (Manual) 2 _Initiator+_{, SP Wave} (Semi-Auto) 3

Compare the peptide synthesizer

1. ACP 65-74: H-VQAAIDYING-NH

₂

2. LysM: H-LPERVKVVFPL-NH

₂

3. JR: H-WFTTLISTIM-NH

₂

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.

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