The synthesis of eGLP1-conjugated ASOs MALAT1 (9), FOXO1 (10), and scrambled control (11) was accomplished, as described in scheme 1 (fig. S1). 5′-Hexylamino ASOs 1 to 3 were synthesized using standard solid-phase oligonucleotide synthesis procedure (31). eGLP1 (8) was generated on Rink Amide-ChemMatrix by microwave-assisted 9-fluorenylmethoxycarbamoyl (FMOC) solid-phase peptide synthesis using a Biotage Initiator+ Alstra synthesizer. All peptide couplings were mediated by ethyl cyanohydroxyiminoacetate (Oxyma Pure) and N,N′-diisopropylcarbodiimide. Compound 4 [propanoic acid, 3-(2-pyridinyldithio)-,2,5-dioxo-1-pyrrolidinyl ester] was obtained from Chem-Impex International Inc. (935 Dillon Drive Wood Dale, IL 60191, USA). A solution of compound 4 (3 μmol) in N,N-dimethylformamide (DMF; 22 μl/μmol) was added dropwise to a solution of ASOs 1 to 3 (1 μmol) in aqueous sodium tetraborate [80 μl/μmol, 0.1 M (pH 8.5)], and the resulting mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with water [fivefold of reaction solution volume (v/v)] and purified by high-performance liquid chromatography (HPLC) (Waters Source 30Q resin; buffer A: 100 mM ammonium acetate in 30% acetonitrile in water; buffer B: 1.5 M sodium bromide in A; 10 to 60% B in 28 column volumes, flow 14 ml min−1, λ 260 nm). Fractions containing full-length ASOs 5 to 7 [assessed by liquid chromatography–mass spectrometry (LC-MS) analysis] were pooled together, diluted to obtain an acetonitrile concentration of 10%, and desalted by HPLC on a reverse-phase column. Product fractions were concentrated to yield 5 to 7 (80 to 83%). Compounds 5 to 7 (1.2 μmol) were dissolved in mixture of degassed water (192 μl/μmol) and 0.1 M sodium bicarbonate solution (192 μl/μmol). eGLP1 peptide 8 (1 μmol) was dissolved in 50% DMF in 0.1 M aqueous sodium bicarbonate solution (215 μl/μmol). eGLP1 peptide solution was added to the above solution of compounds 5 to 7 in small portions (30% of total volume each time) in 5 min intervals. After stirring for 1 hour at room temperature, the reaction mixture was diluted with water [10-fold of reaction solution volume (v/v)], and the product was purified by HPLC (conditions same as above). Product fractions (assessed by LC-MS analysis) were pooled together and desalted by HPLC on a reverse-phase column to yield eGLP1 ASO conjugates 9 to 11 (55 to 65%). LC-MS analysis characterized compounds 9 to 11. 9: ultraviolet (UV) purity, 98.5%; calculated mass, 10766.1; found mass, 10765.1; 10: UV purity, 98.1%; calculated mass, 10838.2; found mass, 10837.8; 11: UV purity, 88.7%; calculated mass, 107780.5; found mass, 107778.5.

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