Synthesis and characterization of amotosalen HCl

Psoralen is the only class of reversible nucleic acid crosslinkers that can be used in mild physiological conditions, and AMT is the most commonly used one due to its relatively high solubility at 1 mg/ml in aqueous solutions (~3 mM). Nevertheless, crosslinking at 0.5 mg/ml does not approach saturation and therefore the solubility still limits its efficiency¹¹. It is likely that this limited solubility is responsible for the low crosslinking efficiency (0.2–0.5% crosslinked RNA from total RNA)⁹. In a related class of methods that analyzes nucleotide flexibility/accessibility, as exemplified by SHAPE and DMS-seq, the RNA-reactive compounds are typically used at much higher concentrations to merely obtain single hit kinetics (e.g., 100 mM or higher for NAI-N3, and 650 mM for DMS^51,52. In the chemical probing experiments, the reactions would destabilize RNA structures and therefore modifications should be limited to <1 per ~100 nt. However, in the case of crosslinking, RNA structures are stabilized, and therefore higher crosslinking efficiency does not have adverse effects.

One way to improve PARIS is to use psoralen derivatives that are more water soluble. Previous studies have shown that amotosalen (also known as S59 or S-59) is soluble at 50 mg/ml in aqueous solutions^12,53. Amotosalen (compound 2 in patent US5,654,443) was used at 50 µg/ml, irradiated with 3 J/cm² 365 nm UV for inactivation of viruses and bacteria. The activity of amotosalen was slightly better than AMT at the same concentration⁵³. The synthesis of amotosalen was described on page 44 of patent US5,654,443, but the procedure is unnecessarily complex. We synthesized amotosalen from trioxalen using a simplified three-step procedure as follows (see Supplementary Fig. ¹).

Trioxsalen + ClCH₂OCH₃ → CMT + methanol;

CMT + Boc-ethanolamine → Boc-amotosalen → amotosalen + Boc.

All chemicals for synthesis were obtained from commercial sources and used as received unless stated otherwise. Solvents were reagent grade. Thin-layer chromatography was performed using commercial Kieselgel 60, F254 silica gel plates. Flash chromatography was performed on silica gel (40–63 µm, 230–400 mesh). Drying of solutions was performed with MgSO₄ and solvents were removed with a rotary evaporator. Chemical shifts for NMR measurements were determined relative to the residual solvent peaks (δ_H 7.26 for CHCl₃ and 2.50 for DMSO, δ_C 77.0 for CHCl₃, and 40.0 for DMSO). The following abbreviations are used to indicate signal multiplicity: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; brs, broad signal; appt, apparent triplet.

Compound 2 was synthesized as previously reported⁵⁴. Trioxsalen (1.9 g, 4.4 mmol) was dissolved in AcOH by gently heating after which the solution was cooled back to room temperature. Chloromethyl methylether (16.0 g, 200 mmol) was added and the resulting reaction mixture was stirred at room temperature for 24 h. Next, more chloromethyl methylether (16.0 g, 200 mmol) was added and the solution was stirred at 35 °C. for 48 h. The reaction was cooled down to room temperature and allowed to stand for another 24 h. The formed precipitate was filtered off yielding 1.5 g (65%) of a white cotton-like solid. ¹H NMR (400 MHz, CDCl₃) δ 7.60 (s, 1H), 6.27 (s, 1H), 4.74 (s, 2H), 2.58 (s, 3H), 2.54–2.52 (m, 6H).

The conversion of CMT to amotosalen can be accomplished with a Williamson ether synthesis method. Compound 2 (1.5 g, 5.4 mmol) was mixed with N-(2-hydroxyethyl)trifluoroacetamide (3.0 g, 19.1 mmol) and heated for 1 h at 100 °C. The mixture was cooled down to room temperature and recrystallized from methanol yielding an off-white powder. ¹H NMR (400 MHz, DMSO) δ 8.30 (s, 1H), 7.70 (s, 1H), 6.31 (s, 1H), 4.62 (s, 2H), 3.52 (t, J = 5.6 Hz, 2H), 3.35 (t, J = 5.4 Hz, 2H), 2.46 (s, 6H), 2.43 (s, 3H).

Compound 3 was dissolved in 0.5 M Cs₂CO₃ in methanol and stirred at room temperature for 16 h. The mixture was concentrated in vacuo and purified using flash chromatography (DCM:MeOH, 9:1) yielding yellow crystals. The product was dissolved in ethanol and the mixture was cooled on an ice bath. A total of 1 M HCl in diethyl ether was added and the mixture was stirred for 4 h on ice. The white precipitate was collected by filtration yielding Amotosalen HCl. ¹H NMR (400 MHz, DMSO) δ 8.02 (s, 3H), 7.80 (s, 1H), 6.34 (s, 1H), 4.68 (s, 2H), 3.62 (t, J = 5.1 Hz, 2H), 2.97 (d, J = 4.9 Hz, 2H), 2.52–2.44 (m, 9H).