Exploration On The Synthesis Of Thrombin-activable Beacon

Photodynamic therapy is a non-invasive or minimally invasive new treatment technique that combines light,photosensitizers,and oxygen molecules in tissues to selectively destroy lesions through photodynamic reactions for therapeutic purposes.According to different target sites,the biological mechanism of PDT can be divided into three different mechanisms: Vascular-targeted Photodynamic Therapy(V-PDT),killing tumor cells and tissues,and inducing immune responses.Since it is difficult to assess tissue damage during the course of treatment,PDT dosimetry has been the subject of significant research efforts.Thrombin is widely considered as an important protein reporter for induction of vascular closure for V-PDT.In order to accurately quantify the amount of drugs administering to patients,this paper initially explored the synthesis of the thrombin activatable beacon(Pyro-thrombin cleavable peptide-ROX)for evaluation of vascular damage during V-PDT treatment.Firstly,Fmoc solid-phase synthesis of thrombin-sensitive peptide was explored.The peptide was synthesized by manual SPPS using Fmoc amine protected amino acids as building blocks and sieber resin as a solid phase.The peptide synthesis cycle consists of three main steps:(1)Fmoc cleavage by 20% piperidine/DMF 3×3 mL×10 min followed by wash with DMF;(2)Amino acid coupling in the presence of activators HOBt ad HBTU.The amino acid/HOBt/HBTU ratio to the resin is 1/2/2:2 and the coupling time is10 min in DIPEA/DMF;(3)Capping by 3×5 mL of 0.3 M acetylimidazole/NMP for 10 min.Secondly,the synthesis of pyropheophorbide-a from silkworm was studied.The synthesis consists of two main steps:(1)a suspension of silkworm in HOAc was refluxing for 4 h to obtain crude pheophorbide-a;(2)crude pheophorbide-a was heated under refluxed temperature in collidine for 4 h.The crude product was chromatographed on a silica column to give pyropheophorbide-a.Thirdly,a three-step for the synthesis of 5-carboxy-X-rhodamine was developed:(1)synthesis of 8-hydroxyjulolidine from 3-aminophenol;(2)synthesis of5-carboxy-phthalaldehydic acid by palladium-catalyzed hydroxycarbonylation of2,5-dibromobenzaldehyde;(3)synthesis of 5-carboxy-X-rhodamine from8-hydroxyjulolidine and 5-carboxy-phthalaldehydic acid using TFE-water as a reaction medium and molecular oxygen as an oxidant.(4)synthesis of 5-carboxyl-X-rhodamine succinimide ester by the reaction of 5-carboxyl-X-rhodamine with2-succinimide-1,1,3,3-tetramethylurea tetrafluoroborate.Fourthly,The final step is to synthesize the beacon: chlorophyll-polypeptide molecule was synthesized.(2)The final product beacon was synthesized by the reaction of 5-carboxyl-X-rhodamine succinimide ester with 2-succinimide-1,1,3,3-tetramethylurea tetrafluoroborate.