Synthesis and Characterization of Betulin-3,28-Diphosphate

Phosphorus oxytrichloride (7.56 mL, 81.6 mmol; Sigma Aldrich, Moscow, Russia, 99%) in dioxane (60 mL) was added dropwise to a solution of betulin (6.0 g, 13.56 mmol) in a mixture of dioxane (120 mL) and pyridine (7.08 mL, 81.6 mmol) at 10–20 °C into a three-necked flask. The reaction mixture was stirred for 24 h at room temperature and then treated by water and ice mixture (1000 g) in the beaker. White precipitate was filtered off and washed by water many times. A wet sediment contained 3–25% of water that corresponds to the hydrate of betulin-3,28-diphosphate × H₂O, where x = 1–9 (7.90 g, 94% as dried product): mp: 147–148 °C (BDP-1), 156–160 °C (BDP-2); UV (EtOH): λ_max (log ε) 256 (2.64) nm; FTIR (KBr): ν_max 3421, 2331, 2342, 1641-1700, 1240, 1031, 973, 501 cm⁻¹; ¹H- NMR (DMSO-d₆, 400 MHz) δ 0.68–1.99 (42H, m, 6CH₃, (CH₂)₁₀, (CH)₄), 2.35–2.42 (1H, m, H-19), 2.97 (0.25H, wide t, α-H-3, J = 7.7 Hz), 3.69 (0.75H, ddd, β-H-3 m, J = 4.6, 7.8, 11.2 Hz), 3.96 (1H, dd, H-28, J = 9.7, 4.5 Hz) and 3.52 (H, dd, H-28′, J = 9.5, 4.5 Hz), 4.55, 4.69 (2H, two s, H-29), 5.69 (protons in the phosphate groups O-P(O)(OH)₂, wide blurred s); ¹³C-NMR (DMSO-d₆, 101 MHz) δ 149.93 (C, C-20), 109.92 (CH₂, C-29), 82.96 (CH, =CHOH), 63.22 (CH₂, CH₂OH), 54.90 (CH, C-5), 49.71 (CH, C-9), 48.11 (CH, C-19), 47.26 (CH, C-18), 46.75 (C, C-17), 42.30 (C, C-14), 40.48 (C, C-8), 38.57 (C, C-4), 38.35 (CH₂, C-1), 37.99 (C, C-10), 37.07 (CH, C-13), 36.57 (CH₂, C-7), 33.79 (CH₂, C-22), 29.11 (CH₂, C-21), 28.96 (CH₂, C-16), 28.17 (CH₃, C-23), 27.90 (CH₂, C-2), 26.57 (CH₂, C-15), 24.83 (CH₂, C-12), 20.43 (CH₂, C-11), 18.84 (CH₃, C-30), 18.01 (CH₂, C-6), 16.14 (CH₃, C-26), 15.91 (CH₃, C-24), 15.70 (CH₃, C-25), 14.56 (CH₃, C-27); ³¹P-NMR (DMSO-d₆, 202.46 MHz) δ −0.4 (d, J = 8.2 Hz, phosphoric acid residue at C-3β), 0.48 ppm (t, J = 4.6 Hz, phosphoric acid residue at C-28).
In the ¹³C-NMR spectrum the signals of the C-28 and C-3 atoms of BDP (60 and 80 ppm, respectively) were shifted by 3 ppm in comparison with botulin, which is characteristic for phosphoric acid esters.
The ³¹P-NMR spectra of BDP in DMSO-d₆ were obtained in the presence of Ph₃P (δ = −6.0 ppm) and H₃PO₄ (δ = −0.14 ppm) which was added just before recording the spectrum at 30 °C. The phosphoric acid residue at C-3β of BDP in the spectrum without decoupling of the protons was recorded as a doublet (δ = −0.4 ppm), while the coupling constant J_H-P ~ 8 Hz is typical for CH-O-P (Figure S1). The phosphoric acid residue at C-28 of BDP is represented as a triplet at δ = +0.48 ppm, with a coupling constant J_H-P ~ 4.6 Hz that is characteristic of the CH₂-O-P fragment of phosphatidic acids (alkylacyl glycerophosphates), where the signal (δ = +0.55 ppm, the coupling constant J_H-P ~ 6.9 Hz) corresponds to the -CH₂-O-P(O)(OH)₂ fragment [29 ].
Phosphorus content determined by spectrophotometric analysis [30 (link)] was equal to 9.59–10.32% (Calc. content for C₃₀H₅₂O₆P₂ is equal to 10.30%).
BDP assay was performed by reversed phase HPLC analysis: 210 nm, 40 °C, mobile phase A30%-B70% v/v (A—acetonitrile (grade 0), B—buffer solution of KH₂PO₄, pH = 6.36), flow 1.0 mL∙min⁻¹. The retention time is 5.19 min.
BDP-1 and BDP-2 separation. The wet BDP crude (9 g) was dissolved in ethanol (600 mL) and the insoluble part (BDP-2) was filtered off. BDP-1 was isolated by precipitation from BDP ethanol solution with acetone, and BDP-1 was recrystallized from ethanol.