1H, 31P, and 13C NMR spectra were recorded on a 600 MHz spectrometer operating at 600.2, 243.0, and 150.9 MHz, respectively. The residual solvent signals (D2O, δ 4.79 ppm and CDCl3, δ 7.26 ppm) were used as references for 1H NMR measurements [26]. For ApppI(d2), the calibration peak used in the 13C NMR spectrum for characterization is mentioned in the data reported for ApppI(d2) below. The nJCP couplings were calculated from the carbon spectra, with the coupling constant given in parentheses in Hz. The HPCCC purification method has previously been reported elsewhere and was used for the purification of ApppI(d2) [21]. HRMS spectra were recorded on a qTOF mass spectrometer using electrospray ionization (ESI) in negative mode. The purity of the products was determined from the 1H and 31P NMR spectra to be ≥95%, unless stated otherwise.

ApppI(d2)⋅3.25 and ⋅5.25 TBA salts: ATP disodium salt (269 mg, 0.49 mmol) was converted to the corresponding TBA salt by rapid treatment (5 min of stirring) with Dowex H+ cation exchange resin and the addition of 4 equiv of 40% TBAOH in H2O (1260 µL). The mixture was evaporated to dryness in vacuum and kept several days in vacuum to obtain a dry product. This ATP TBA salt was dissolved in dry CH3CN (3 mL), and 3-methylbut-3-en-1-yl-1,1-d2 4-methylbenzenesulfonate (4, 130 mg, 0.54 mmol) was added. The reaction mixture stirred at 45 °C for 55 h before being evaporated to dryness in vacuum. The residue was stirred in diethyl ether (5 mL) for 10 min, and diethyl ether was removed. This was repeated twice. The residue was then dried in vacuum and purified by HPCCC following the method described in Reference [21]. When the appropriate fractions from the HPCCC purification were evaporated, the pure product was obtained in two different portions (70 mg as the ⋅5.25 TBA salt and 24 mg as the ⋅3.25 TBA salt, calculated total yield 11.4%, which was comparable to earlier results [20], see also HPCCC chromatogram in Supporting Information File 1). The product was obtained as very hygroscopic colorless foamy solid. NMR data for ApppI(d2)⋅3.25 TBA salt: 1H NMR (D2O) δ 8.52 (s, 1H), 8.21 (s, 1H), 6.08 (d, J = 6.1, 1H), 4.76–4.73 (m, 1H), 4.69–4.67 (m, 1H), 4.67–4.65 (m, 1H), 4.54–4.52 (m, 1H), 4.36–4.33 (m, 1H), 4.26–4.21 (m, 1H), 4.20–4.15 (m, 1H), 3.17–3.09 (m, 26H, from TBA salt), 2.25–2.18 (m, 2H), 1.61 (s, 3H), 1.63–1.54 (m, 26H, from TBA salt), 1.35–1.25 (m, 26H, from TBA salt), 0.90 (t, J = 7.3, 39H, from TBA salt); 13C NMR (D2O) δ 156.5, 153.7, 150.1, 144.3, 140.9, 119.5, 112.3, 87.6, 85.1 (d, 2JCP = 9.3), 75.4, 71.5, 66.2 (d, 3JCP = 5.4), 65.1 (m, 2H-C-2H, hardly visible), 59.0 (virtual t, from TBA salt), 38.5 (d, 3JCP = 7.5), 24.1 (from TBA salt), 22.6, 20.1 (from TBA salt), 13.8 (from TBA salt, used as calibration peak: value marked same as in the earlier characterization of ApppI [20]); 31P NMR (D2O): δ −11.2 d (2JPP = 19.4), −11.7 d (2JPP = 19.4), −23.4 t (2JPP = 19.4); HRMS–ESI (qTOF, m/z): [M − H]− calcd for C15H212H2N5O13P3, 576.0631; found, 576.0630. All NMR data were comparable to those reported elsewhere for conventional ApppI [19,21].

Preparation of Jones reagent: CrO3 (25 g) was slowly and carefully added to conc H2SO4 (25 mL) with stirring. After complete addition, the formed mixture was further added in very small portions to ice-cold water (75 mL) with vigorous stirring to form the final Jones reagent.

Synthesis of 3-methylbut-3-enoic acid (2) [27]: 3-Methylbut-3-en-1-ol (1, 3.0 mL, 2.56 g, 0.030 mol) was dissolved in acetone (150 mL), and the reaction mixture was cooled to 0–3 °C. Then, Jones reagent (15.6 mL) was added, and the reaction mixture was stirred at 0–3 °C for 1 h. The reaction mixture was made basic by adding an appropriate volume of 4 M NaOH with stirring (pH value measured by pH paper), and acetone was removed by evaporation in vacuum. The remaining mixture was acidified by addition of 6 M HCl and extracted with diethyl ether (3 × 15 mL). The combined diethyl ether fractions were dried over MgSO4 and filtered. After evaporation of the solvent, the final product was distilled in vacuum. The product was obtained as colorless liquid (1.5 g, 50%), bp 50–53 °C/2.0 mbar. According to the 1H NMR spectrum, the product purity was about 88%, which was considered to be pure enough to be used in the next step. 1H NMR (CDCl3) δ 4.95 (br, 1H), 4.89 (br, 1H), 3.08 (s, 2H), 1.84 (s, 3H); 13C NMR (CDCl3) δ 177.7, 138.1, 115.5, 43.2, 22.5.

Synthesis of 3-methylbut-3-en-1,1-d2-1-ol (3) [28]: LiAlD4 (320 mg, 7.6 mmol, 0.5 equiv) was added to dry and cold (0–3 °C) diethyl ether (15 mL) in an ice–water bath, and 3-methylbut-3-enoic acid (2, 1.5 g, 15 mmol), dissolved in dry diethyl ether (5 mL), was added in small portions with vigorous stirring. After completed addition, the ice–water bath was removed, and the reaction mixture was stirred for 3 h at room temperature before it was cooled again to 0–3 °C. Then, solid Na2SO4·10 H2O (1.0 g) was added, and the reaction mixture was stirred for 0.5 h at 0–3 °C. All solids were filtered off, and diethyl ether was removed slowly using a rotary evaporator in vacuum. The crude product was obtained as colorless liquid (415 mg, 31%). Most of the product had apparently been concomitantly evaporated with diethyl ether, and the remaining crude product still contained diethyl ether and side products. However, most of the obtained material was the desired product 3-methylbut-3-en-1,1-d2-1-ol (3), which was used directly in the next step. 1H NMR (CDCl3) δ 4.87 (br, 1H), 4.78 (br, 1H), 2.28 (s, 2H), 1.76 (s, 3H).

Synthesis of 3-methylbut-3-en-1-yl-1,1-d2 4-methylbenzenesulfonate (4) [28]: 3-Methylbut-3-en-1,1-d2-1-ol (3, 415 mg, 4.7 mmol) was dissolved in dry acetonitrile (5 mL), and distilled pyridine (375 µL, 368 mg, 4.7 mmol) and tosyl chloride (900 mg, 4.7 mmol) were added. Then, the reaction mixture was stirred for 4 h at room temperature before being evaporated to dryness in vacuum. The residue was dissolved in diethyl ether, filtered, and evaporated to dryness in vacuum. The crude product (635 mg) was purified by preparative TLC using EtOAc/hexane 5:95 as eluent. The pure product was obtained at Rf = 0.11 (139 mg). However, also the fraction at Rf = 0.21 (181 mg) was observed to contain the desired product with some impurities, according to the 1H NMR spectrum. The fraction at Rf = 0.21 was further purified twice by analytical TLC, and the desired product was obtained purely with Rf = 0.27 (140 mg). The total amount of purified 3-methylbut-3-en-1-yl-1,1-d2 4-methylbenzenesulfonate (4) was 279 mg (24%). 1H NMR (CDCl3) 7.79 (d, J = 8.0, 2H), 7.34 (d, J = 8.0, 2H), 4.79 (br, 1H), 4.67 (br, 1H), 2.45 (s, 3H), 2.34 (s, 2H), 1.66 (s, 3H);13C NMR (CDCl3) δ 144.9, 140.2, 133.3, 130.0 (2C), 128.0 (2C), 113.2, 68.0 (m, 2H-C-2H, 1JCD = 22.9, hardly visible), 36.7, 22.5, 21.8.