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+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.
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.
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.
|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).|
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