Engineering Enzyme‐Cleavable Oligonucleotides by Automated Solid‐Phase Incorporation of Cathepsin B Sensitive Dipeptide Linkers

Abstract Oligonucleotides containing cleavable linkers have emerged as versatile tools to achieve stimulus‐responsive and site‐specific cleavage of DNA. However, the limitations of previously reported cleavable linkers including photolabile and disulfide linkers have restricted their applications in vivo. Inspired by the cathepsin B‐sensitive dipeptide linkers in antibody–drug conjugates (ADCs) such as Adcetris, we have developed Val‐Ala‐02 and Val‐Ala‐Chalcone phosphoramidites for the automated synthesis of enzyme‐cleavable oligonucleotides. Cathepsin B digests Val‐Ala‐02 and Val‐Ala‐Chalcone linkers efficiently, enabling cleavage of oligonucleotides into two components or release of small‐molecule payloads. Based on the prior success of dipeptide linkers in ADCs, we believe that these dipeptide linker phosphoramidites will promote new clinical applications of therapeutic oligonucleotides.

Note: a After deprotection by 50 mM K2CO3 in methanol (2 mL), 12 µL of acetic acid were added to neutralize the solution. Then, the solvent was removed under high vacuum and the oligonucleotides were subjected to HPLC purification.       (2 µM) + ODN15 (4 µM) groups were incubated with 0.5 U/mL cathepsin B in buffer B for one or three hours at 37 °C, then, subjected to a 12% native PAGE gel electrophoresis assay, and the percentage cleavage was analyzed. The gel was run at 4 °C for one hour. The power was set as 20 W. The error bars indicate the mean ± SD values; n = 3.

Synthesis of Fmoc-Val-Ala-OH
Fmoc-Val-OH (4.0 g, 11.8 mmol), NHS (1.44 g, 12.36 mmol) and DCC (2.56 g, 12.36 mmol) were dissolved in THF (100 mL), and the mixture was stirred at room temperature overnight. Then, the mixture was filtered and the precipitates were washed with THF. The filtrate was collected, and the Fmoc-Val-NHS (4.69 g, 10.7 mmol) was dissolved in 30 mL THF. Then, a solution of 1.0 g L-alanine (11.3 mmol) and 1.0 g NaHCO3 (11.3 mmol) in 30 mL H2O was added to the above THF solution.

Synthesis of Val-Ala-02 phosphoramidite
HDA-Val-Ala-PAP-DMT (0.75 g, 0.96 mmol) was dissolved in 30 mL anhydrous dichloromethane under argon gas protection. Then, 0.8 mL N,N-diisopropylethylamine were added. The mixture was allowed to cool down in an ice bath. Then, 0.5 mL 2-cyanoethyl N,N-diisopropylchlorophosphoramidite were added. The reaction was monitored by thin-layer chromatography. When the reaction was complete, the mixture was washed with saturated NaHCO3 and brine. The organic layer was collected and dried with anhydrous MgSO4. After purification by silica gel column chromatography

Synthesis of Val-Ala-01 phosphoramidite
HDA-Val-Ala-PAB-DMT (0.65 g, 0.84 mmol) was dissolved in 30 mL anhydrous dichloromethane under the protection of argon gas. Then, 1.0 mL DIPEA was added. The mixture was allowed to cool down in an ice bath and then 0.6 mL 2-cyanoethyl N,N-diisopropylchlorophosphoramidite was added. The reaction was monitored by thin-layer chromatography. When the reaction complete, the mixture was washed by saturated NaHCO3 and brine. The organic layer was collected and dried by anhydrous Na2SO4. After purification by silica gel column chromatography (elution solvent: ethyl acetate (1% TEA)), 0.45 g of Val-Ala-01 phosphoramidite (0.46 mmol) was obtained as a solid foam (54.7% yield). 1 H

Synthesis of Val-Ala-PAB-Coumarin
7-Hydroxycoumarin (0.17 g, 1.04 mmol) was dissolved in 10 mL anhydrous DMF, and 0.14 g anhydrous K2CO3 was added. The mixture was stirred for 30 minutes under argon gas protection. Then, Fmoc-Val-Ala-PAB-Br (0.3 g, 0.52 mmol) in 5 mL anhydrous DMF was added. The mixture was stirred overnight under room temperature. Then, the mixture was poured into saturated NaHCO3 solution and extracted with 100 mL EtOAc. The organic layer was collected and dried by Na2SO4. After

Synthesis of Val-Ala-PAB-Chalcone
Chalcone (1.0 g, 2.90 mmol) was dissolved in 15 mL anhydrous DMF and 0.48 g anhydrous K2CO3 (3.46 mmol) was added. The mixture was stirred for 30 minutes under argon gas protection. Then, 1.12 g Fmoc-Val-Ala-PAB-Br (1.94 mmol) in 10 mL anhydrous DMF was added. The mixture was stirred overnight at room temperature. Then, the organic solvent was removed under high vacuum, and the residue was dissolved in 100 mL EtOAc. The mixture was washed with saturated NaHCO3 and brine, and the organic layer was collected and dried by anhydrous Na2SO4. After purification by silica gel