Strecker Synthesis Of 2-amino-2-(Bicyclo[2.2.1] Hept-5-en-2-yl) Acetic Acid And Bioorthogonal Reaction With Tetrazine

Objective:The strained norbornene reacts irreversibly with tetrazine forming dihydropyridazine products and dinitrogen based on inverse electron demand Diels-Alder reaction. The reaction can be carried out in the biological environment with high rates, specificity, minimal byproducts, non-toxicity and no need of any catalyst. It can not only content "click reaction" characteristic, but also meet bioorthogonal reaction criteria. Comparing with former bioorthogonal reactions, tetrazine-based cycloadditions has more features such as higher rates, without need of any catalyst, non-toxicity, with "turn-on" fluorescent probes and readily available starting materials, but it still not emerging in labeling peptides or proteins in vivo/in vitro. If norbornene was incorporated into amino acid, and then integrated into peptides or proteins which then were labeled with tetrazine-fluoresceins. So we could localize target peptides or protein in vivo/in vitro specifically.Methods:5-norbornene-2-glycine was synthesized via Strecker synthesis using 5-norbornene-2-formaldehyde as the substrate; Kidney acylase I was used to isolate L-5-norbornene-2-glycine from DL-5-norbornene-2-glycine; 3,6-dibenzoic acid-1,2,4,5-tetrazine and 3-benzylamino-1,2,4,5-tetrazine were synthesized separately, then study the cycloaddition reactions between 5-norbornene-2-glycine and tetrazine; integrate 5-norbornene-2-glycine (Fmoc protected) into pentapeptide of sperm whale myoglobin and label pentapeptide using tetrazine with additional conjugation handles in PBS buffer.Results and Conclusion:We synthesized 5-norbornene-2-glycine through Strecker reaction with total yield of 41%, separated L-5-norbornene-2-glycine using Kidney acylase with yield of 21%; Then we synthesized 3,6-dibenzoic acid-1,2,4,5-tetrazine and 3-benzylamino-1,2,4,5-tetrazine with yield of 67%and 23%, respectively; We found that 5-norbornene-2-glycine and 3,6-dibenzoic acid-1,2,4,5-tetrazine have poor reactivity in THF and water respectively, but 5-norbornene-2-glycine can quickly react with 3-benzylamino-1,2,4,5-tetrazine in PBS buffer. We used Fmoc solid phase peptide synthesis intergrated Fmoc-5-norbornene-2-glycine into pentapeptide of sperm whale myoglobin and labeled pentapeptide using 3-benzylamino-1,2,4,5-tetrazine in PBS buffer, 3-benzylamino-1,2,4,5-tetrazine could be used as a good exogenous probe for 5-norbornene-2-glycine, because it was electron-deficient, more stable and soluble in aqueous media.