Fine-tuning the properties of the thrombin binding aptamer through cyclization: Effect of the 5′-3′ connecting linker on the aptamer stability and anticoagulant activity

• Published in: Bioorganic chemistry 2020-01, Vol.94, p.103379, Article 103379
• Main Authors: Riccardi, Claudia, Meyer, Albert, Vasseur, Jean-Jacques, Russo Krauss, Irene, Paduano, Luigi, Morvan, François, Montesarchio, Daniela
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Anticoagulant activity; Anticoagulants - chemical synthesis; Anticoagulants - chemistry; Anticoagulants - pharmacology; Aptamers, Nucleotide - chemical synthesis; Aptamers, Nucleotide - chemistry; Aptamers, Nucleotide - pharmacology; Biophysical characterization; Blood Coagulation - drug effects; Chemical Sciences; Cyclization; Dose-Response Relationship, Drug; G-quadruplex; Medicinal Chemistry; Molecular Structure; Organic chemistry; Structure-Activity Relationship; Thrombin; Thrombin binding aptamer
• ISSN: 0045-2068; 1090-2120
• DOI: 10.1016/j.bioorg.2019.103379

[Display omitted] •4 novel cyclic TBAs were synthesized by connecting the 5′- and 3′-ends of the sequence.•Their physico-chemical properties, serum resistance and anticoagulant activity were evaluated.•We successfully identify cycTBA II with improved anticoagulant activity. A small library of cyclic TBA analogues (named cycTBA I-IV), obtained by covalently connecting its 5′- and 3′-ends with flexible linkers, has been synthesized with the aim of improving its chemical and enzymatic stability, as well as its anticoagulant properties. Two chemical procedures have been exploited to achieve the desired cyclization, based on the oxime ligation method (providing cycTBA I and II) or on Cu(I)-assisted azide-alkyne cycloaddition (CuAAC) protocols (for cycTBA III and IV), leading to analogues containing circularizing linkers with different chemical nature and length, overall spanning from 22 to 48 atoms. The resulting cyclic TBAs have been characterized using a variety of biophysical methods (UV, CD, gel electrophoresis, SE-HPLC analyses) and then tested for their serum resistance and anticoagulant activity under in vitro experiments. A fine-tuning of the length and flexibility of the linker allowed identifying a cyclic analogue, cycTBA II, with improved anticoagulant activity, associated with a dramatically stabilized G-quadruplex structure (ΔTm = +17 °C) and a 6.6-fold higher enzymatic resistance in serum compared to unmodified TBA.