Technology Details

• What are the principles of Belyntics PEC-Linker and the Catch-and-Release technology
  Belyntic’s PEC-Linkers are bifunctional, cleavable molecules. Two (bio-) chemical components can be linked via stable covalent bonds (“Catch”): A nucleophile-modified Component A binds with the protected PEC-Linker while exchanging a leaving group. After deprotection of the PEC-Linker, a second aldehyde-modified Component B binds via an oxyamine group at the PEC-Linker by forming an oxime bond. The PEC-Linker can be cleaved with a suitable cleavage protocol. Component A is thereby recovered in its original form (“Release”). A degradation product remains at Component B. The full process is referred to as Catch-and-Release technology.
  
  For Belyntic, the most prominent applications of Belyntic‘s PEC-Linkers is peptide purification and/or peptide modification - we call this Peptide Easy Clean (PEC). The peptide to be purified or modified corresponds to Component A. Activated agarose is used as the Component B. The linking (catch) of the target peptide with the agarose through the PEC-Linker allows the washing out of unbound substances and/or the selective modification of the bound peptide. The subsequent cleavage of the PEC-Linker enables the release of the purified or modified peptide with a free amino group. The degradation product remains on the agarose.
• The PEC-Linker (Basics)

  Belyntic's linker molecules consist of three parts:

   

  

  General PEC-Linker scheme.

  
  
  

  1. A protecting group (PG) modified amino-oxy function for the subsequent catch on the activated filter material

  2. A cleavable unit (LCU)

  3. A leaving group (LG) for coupling to the N-terminus of the peptide. 

  
  

• The PEC-Linker RC+

  The PEC-Linker RC+generation reflects the latest advancements in catch-and-release methodologies. All three construction blocks are optimized and tailored to allow general applicability as well as reliable and highly efficient purification and modification experience for the user.
  

    
  

  
  

  Molecular design of the PEC-Linker RC+

  
  

  1. Remove the Boc protecting group during the acid treatment for cleavage from the SPPS resin. The activated amino-oxy function serves as the anchor to the activated filter material.

  
  2. A Bromo-substituted para azido-benzyl carbamate acts a the cleavable unit and represents the heart of the PEC-Linker RC+. The construction enables a well-balanced stability behavior, depending on the pH of the medium: Reducing the azide to an amine sensitizes the linker to cleavage. However, the fracture does not occur at neutral pH enabling wash out of by-products formed during reduction. Finally, the treatment of the safety-release system with weak acids liberates the peptide through an acid-catalyzed 1,6-elimination.

  
  3. The para-nitrophenol represents an ideal leaving group with precisely tuned reactivity and storage stability. 

• The activated filter material

  Aldehyde-modified agarose reflects the state-of-the-art solid-phase equipment for catch-and-release methodologies. We offer an optimized agarose material (Agarose100) with high stability, and a high loading capacity of 100 µmol per mL settled resin in our current kit products.

  

  Image of the employed Agarose100 as activated filter material.

• Catch-and-release purification with PEC-Linker RC+

  The figure below shows the six steps for peptide purification with Belyntic's PEC-Linker RC+.

  
  

  
  

  
  

  1. The PEC-Linker is coupled with the target peptide at the end of the solid phase peptide synthesis (SPPS). Capping is performed after each amino acid coupling cycle to ensure the selective coupling on the target full-length sequence.

  2. Cleavage of the peptide from the SPPS resin is performed with TFA-cocktails.

  3. Dissolution of the peptide and (4) immobilization through covalent capture („Catch“) on the activated filter material in an oxime ligation.

  5. The covalent capture allows the washing out of unbound substances such as truncated sequences and additionally enables the selective modification of the bound, unprotected peptide.

  6. The subsequent reduction of the PEC-Linker liberates the purified and/or modified peptide via elution ("Release") with a free N-terminal amino group. The degradation product (residue) remains on the filter material.

• What are typical delta masses found during the PEC-process?

  Delta Mass	Source	Action required?
  +413.03 (MH2+: +206.52, MH3+: +137.67)	Reductively cleavable linker on peptide (linker on)	No. Your coupling worked well.
  +252.06 (MH2+: +126.03, MH3+: +84.02)	Non-cleaved or re-attached Pbf protecting group	Yes. Treat purified peptide with Reagent K or another thioanisol-containing TFA-cocktail for 4 hours.
  +106.05 (MH2+: +53.03, MH3+: +35.35)	P-hydroxybenzyl cation, generated during TFA cleavage	Yes. Use RAM-linker on the synthesis resin.
  +56.07 (MH2+: +28.04, MH3+: +18.69)	Re-attached tBu	Yes. Use fresh thiols (e.g. EDT) in TFA-cleavage cocktail
  +40.05 (MH2+: +20.03,MH3+: +13.33)	Acetone oxime of peptide-linker conjugate	Yes. Use high purity ether for precipitation after TFA-cleavage and in your solvents for analysis (e.g. MeCN).
  +26.03 (MH2+: +13.02,MH3+: +8.68)	Acetaldehyde oxime of peptide-linker conjugate	Yes. Use high purity ether for precipitation after TFA-cleavage and in your solvents for analysis (e.g. MeCN).
  +12.01 (MH2+: +6.01,MH3+: +4.00)	Formaldehyde oxime of peptide-linker conjugate	Yes. Use high purity ether for precipitation after TFA-cleavageand in your solvents for analysis (e.g. MeCN).

• Are there other applications possible with Belyntic’s PEC-Linkers?
  Yes. The range of possible applications is broad and is not limited to peptide purification or modification. One could consider every application where bifunctional linkers are required to link two components with the option for controlled cleavage or release. The requirements for the binding partners are simple: a suitable nucleophile on Compound A and an aldehyde-modified Component B. We encourage everyone to exploit the potential of our PEC-Linkers and are looking forward to hearing your ideas and thoughts.

Preparations

• Can I reuse the activated Filter Material?
  No. The breakdown species (residues) or the PEC-Linker will remain on the activated filter material, and reuse is not recommended.
• How can I obtain high purities?
  It is essential to consider synthesis aspects before you start:
  1. acetylation after each amino acid coupling (capping) is key to the PEC purification

  • efficient conditions for blocking unreacted amino groups are 2 M acetic anhydride (20 vol%) with either 2 M pyridine (18 vol%) or 2 M 2,6-lutidine (21 vol%) in DMF for 5 min

  2. please pay special attention to possible side reactions during SPPS.

  • minimize by-products during TFA cleavage
  • use Rink Amide (RAM) resins for C-terminal amides
  • avoid amino acid contamination
  • avoid incomplete Fmoc-deprotection, e.g., Ala-, Ile-, Leu-rich sequences

  If purity after PEC is not sufficient for your desired application, chromatography can be applied in a final, orthogonal purification step. This combination allows you to reach a new dimensions of purity because the features of two principles are combined, e.g. critical co-eluting sequence by PEC and polish by HPLC, something that multiple chromatographic runs could only hardly reach.
• Which chemicals do I need additionally to the consumables in the Research Kit?
  Reagents for coupling of the linker:

  • ethyl-2-cyano-2-(hydroxyimino) acetate (Oxyma)
  • scavengers for TFA cleavage cocktail (e.g. TIS, EDT, thioanisol, phenol)

  Salts for buffers:

  • guanidinium chloride (GdmCl, in case of technical grade, filter the solution prior to use)
  • sodium chloride (NaCl)

  Acids / Bases:

  • N,N-diisopropylethylamine (DIEA)
  • trifluoroacetic acid (TFA)
  • acetic acid (AcOH)

  Solvents

  • N,N-dimethylformamide (DMF), ≥ 99.5% (peptide synthesis grade)
  • dimethyl sulfoxide (DMSO), ≥ 99.5% (synthesis grade)
  • dichloromethylene (CH₂Cl₂), ≥ 99.5% (synthesis grade)
  • acetonitrile (MeCN), ≥ 99.5% (synthesis grade & LC-MS grade)
  • deionized water
  • ethanol (EtOH), denatured
  • suitable ether for peptide precipitation (e.g. Et₂O, MTBE, THF/heptane 1:1), ≥ 99.5% (synthesis grade)
• Which chemicals do I need additionally to the consumables in the Developer Kit?
  Reagents for coupling of the linker:

  • ethyl-2-cyano-2-(hydroxyimino) acetate (Oxyma)
  • scavengers for TFA cleavage cocktail (e.g. TIS, EDT, thioanisol, phenol)
  • Cleavage reagent such as Triphenylphosphine or DTT
  • L-Lysine for blocking of the aldehydes on the filter material

  Salts for buffers:

  • guanidinium chloride (GdmCl, in case of technical grade, filter the solution prior to use)
  • sodium chloride (NaCl)
  • citric acid
  • natrium carbonate

  Acids / Bases:

  • N,N-diisopropylethylamine (DIEA)
  • trifluoroacetic acid (TFA)
  • acetic acid (AcOH)

  Solvents

  • N,N-dimethylformamide (DMF), ≥ 99.5% (peptide synthesis grade)
  • dimethyl sulfoxide (DMSO), ≥ 99.5% (synthesis grade)
  • dichloromethylene (CH₂Cl₂), ≥ 99.5% (synthesis grade)
  • acetonitrile (MeCN), ≥ 99.5% (synthesis grade & LC-MS grade)
  • deionized water
  • ethanol (EtOH), denatured
  • suitable ether for peptide precipitation (e.g. Et₂O, MTBE, THF/heptane 1:1), ≥ 99.5% (synthesis grade)
• Which equipment do I need for the purification?
  • shaking device for fritted cartridges
  • centrifuge (recommended) or filtration apparatus to collect peptide precipitates
  • vacuum manifold or vacuum flask for parallel set-up (optional)
• How can I process the cartridges form the Research Kit?
  Single purifications can be processed by hand, using the plunger and push-pull processing. Please make sure your peptide is always fully dissolved. The peptide might otherwise clog in the frit.
  
  We recommend a vacuum manifold or a vacuum flask with a septum for parallel processing.
• How can I run purification with the Developer Kit?
  The Developer Kit comes with the essential materials: PEC-Linker, and activated filter material. Individual cartridges have to be chosen and acquired. We recommend the use of fritted cartridges. For a smaller scale, you can use the SPPS reactors; for larger-scale, we recommend to reach out for solid-phase extraction equipment. Alternatively, you can use fritted glassware such as conventional filter apparatus or SPPS reactors. Please mind, that shaking your reactor for up to 120 minutes is required.

Benefits

• What does parallel purification mean?
  Catch-and-release purification with our novel PEC-Linkers enables the purification of peptides in simple syringe reactors. The protocol relies on covalent and discrete chemical reactions, which proceed independently. This allows the parallel processing of the reactors for example with vacuum flask set-ups or more convenient vacuum manifolds. Having the right equipment in hand, you can therefore readily purify up to 24 Peptides at the time with Belyntics Research Kits.
  
  Higher throughput is possible with customized solutions using the Developer Kit.
• What does “difficult peptide” mean?
  We define all those peptides as “difficult” when their sequence comes with difficulties for conventional purification using chromatography:

  • (very) Hydrophobic Peptides
  • (very) Hydrophilic Peptides
  • long Peptides (>25 AA)
  • aggregating Peptides
• Why does PEC benefits from a low organic solvent consumption?
  The purification process is characterized by low consumption of organic solvents compared to chromatography because no continuous flow of eluents is required. All steps are simple solid-phase reactions, and therefore only little amounts of solution are needed to wash off the excess of reagents.
• Are there limitations in scale?
  In principle no. The PEC-technology can easily be adjusted in scale by the amount of filter material used. For the range between 10 and 100 µmol, you can use the ready-to-use Research Kits, while higher scales can be addressed with the Developer Kit. We showed, for instance, the purification of 1 mmol using a 70 mL fritted cartridge. Please get in touch for production scales. 
• Can I control the quality of PEC?
  PEC offers a high degree of process control due to the serial reaction steps. Mass spectrometry helps to track the impurities in the crude, such as truncated, non-capped sequences and possible side products. This allows a rough prediction of the potential outcome of PEC and gives hint if the synthesis needs to be optimized.
  Also very beneficial is the compatibility of PEC with other chromatographic methods. In case the purity doesn’t meet the requirements, we advise a second purification step with low-, medium, or high-pressure liquid chromatography.
• Why are difficult peptides better to purify with PEC in comparison to chromatography?
  Up to 90% solvents and chaotropic salts can be used to solubilize a variety of hydrophobic or aggregating peptides extending the range of addressable sequences.
• Are there limitations for internal modifications?
  Peptide modification is one of the core strength of the PEC technology. After linker coupling, cleavage from the synthesis resin and immobilization on the activated filter material, one can run several modifications on the unprotected peptide such as conjugation of fatty acids, PEG, dyes as well as cyclization or even a combination of both. Also, the peptide is equally purified when the synthesis requirements (Fmoc SPPS, efficient capping, complete Fmoc-removal, no amino acid contamination) are met.
  
  However, please mind possible side reactions that can occur in reductive conditions when cleaving the peptide after purification. Disulfide bonds are typically not recommended. The effect of solubility is of critical importance, too.
  
  N-terminal modifications are only possible if they allow linker coupling on the side chain, for example, via MTT-protected lysine.

Limitations

• Can I purify peptides with N-terminal modifications?
  No. The PEC-Linker is bound to the N-terminus of the peptide according to the instructions outlined in the manual. Therefore, modifications of the N-terminus are not possible in this version. However, if you have a Lysine with an orthogonal protecting group, you can couple the linker on the backbone and modify your peptide on the N-terminus. Note that specific protocol development is needed.
• What affects the purity?
  In principle, the final product contains every compound that is coupled to the PEC-Linker. Rephrased: Every compound with a free amine during the step of PEC-Linker coupling will bear the PEC-Linker. Potential impurities therefore already arise during synthesis. These may be undesired sequences due to, e.g., amino acid contamination, incomplete Fmoc deprotection, or intra- or intermolecular side reactions (e.g., aspartimides, pyroglutamates) if these are present at the time of linker coupling.
  
  Another essential synthesis-related source of contamination occurs when peptide acetylation is not (or only incompletely) performed after each amino acid coupling. As a result, the PEC-Linker may bind to truncated or undesired sequences, and those will then remain in the product as by-products.
  
  The sequence of the peptide can have a considerable influence on the purification result: Side reactions can play a role during the procedure, or solubility is not always guaranteed. Non-natural or modified peptides may notably be affected. In view of this diversity of peptides, it is unfortunately often not possible to predict in advance whether a satisfactory purification result can be achieved.
  
  Further possible peptide impurities may occur during the cleavage of the peptide from the synthesis resin. These are peptide sequences that already carry the PEC-Linker and then react with reactive species (e.g., cleaved protective groups or degradation products of the cleaved synthesis linker). These usually unwanted sequences are not removed and remain in the final product.
  
  A systemic impurity might be oxidized reducing agent by-products that form in the last step of the reduction-triggered release. The manual suggests a protocol that should remove these impurities. However, traces may still be present in the product.
• What affects the yield?
  At all stages, before the peptide-PEC-Linker-conjugate is bound to the activated filter material, contaminations with aldehydes or ketones in the reaction, precipitation, storage, or analysis media can react with the oxyamine of the PEC-Linker. This side-reaction can reduce the yield and/or distort the analysis. Regarding reduced yield, the purity of solvents used for PEC-Linker coupling or the ether used to precipitate the peptide is of specific importance. During chromatography, the purity of eluents is of particular significance.
  
  Moreover, the adsorption behaviour of the peptide on the activated filter material is of importance, since the peptide needs to be washed off the media after cleavage.
• Can PEC remove unwanted side reactions such as aspartimide formation?
  Unfortunately not. Unwanted side-products formed in the target sequence during synthesis or purification, such as aspartimide or pyroglutamate formation, cannot be removed by PEC.

If you have any further question?

Please contact us directly: support@belyntic.com