An improved total synthesis of tunicamycin V

Abstract

The tunicamycins are important biochemical tools to study N-linked glycosylation and protein misfolding in cancer biochemistry fields. We reported a convergent synthesis of tunicamycin V with 21% overall yield from D-galactal. We have further optimized our original synthetic scheme by increasing the selectivity of azidonitration of the galactal derivative and developing a one-pot Büchner-Curtius-Schlotterbeck reaction. An improved synthetic scheme reported here enables the synthesis of tunicamycin V in 33% overall yield. In this article, we describe detailed procedures for a gram-scale synthesis of the key intermediate 12 and synthesizing 100 mg of tunicamycin V (1) from commercially available D-galctal-4,5-acetonide. All chemical steps have been repeated multiple times.

• •Highly selective azidonitration of N-(((3aR,4R,7aR)-2,2-dimethyl-3a,7a-dihydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl)acetamide (D-galctal-4,5-acetonide) to form 2-azido-2-deoxy-α/β-D-galactopyranoside derivatives.
• •Optimized Büchner-Curtius-Schlotterbeck (BCS) reaction procedure for the tunicamycin core structure.
• •Full detail on the 15-chemical step synthesis of tunicamycin V.

Graphical abstract

Specifications table

Subject area:	Chemistry
Morespecific subject area:	A short and high-yielding synthesis of tunicamycin V
Name of your method:	A practical synthesis of tunicamycin V
Name and reference of original method:	K. Mitachi, D. Mingle, W. Effah, A. Sánchez-Ruiz, K.E. Hevener, R. Narayanan, W.M. Clemons, F. Sarabia, M. Kurosu, Concise Synthesis of tunicamycin V and discovery of a cytostatic DPAGT1 Inhibitor, Angew. Chem. Int. Ed. Engl. 61 (2022) e202203225. doi: 10.1002/anie.202203225
Resource availability:	Experimental procedure for 11 via one-pot Büchner–Curtius–Schlotterbeck reaction is available in a video.

Method details

We have reported a total synthesis of tunicamycin V (1, Scheme 1) in 15-chemiacl steps from D-galactal [1]. Our synthesis features Büchner–Curtius–Schlotterbeck (BCS)-type reaction to assemble the complex disaccharide and aldehyde, yielding the tunicamycin core structure V in 90% yield (Fig. 1B). Although our scheme enabled the synthesis of tunicamycin V in 21% overall yield, several transformations are difficult to adapt to a large-scale synthesis. In our program of pharmacokinetic/pharmacodynamic and toxicological studies of tunicamycin analogues, we require to synthesize gram-quantity of the intermediate 12 (Scheme 1). In previous synthetic scheme, azidonitration of I (with DNs protecting group) provided a mixture of the diastereomers (II and III) and uncharacterized by-products (Fig. 1A). Our BCS reaction condition (IV → V) requires extraction of the thermounstable diazo-intermediate with Et₂O and concentration prior to coupling with the aldehyde; these procedures are not feasible for a large-scale synthesis (Fig. 1B). We could solve these drawbacks by 1) introducing the acetoamide group to the galactal 3, and 2) developing a one-pot procedure of BCS reaction (8 → 11 in Scheme 1) with a mixture of solvents (toluene and MeOH) and MgSO₄. Detailed below are the optimized reaction procedures for the 15-step sequence (33.7% overall yield from 2, Scheme 1).

Scheme 1.

Optimized synthesis of tunicamycin V.

Fig. 1.

The chemical steps that require improvement for a large-scale synthesis of tunicamycin analogues in the previousely reported scheme.

General

All chemicals were purchased from commercial sources and used without further purification unless otherwise noted. THF, CH₂Cl₂, and DMF were purified via Innovative Technology's Pure-Solve System. All reactions were performed under a nitrogen atmosphere. All stirring was performed with an internal magnetic stirrer. Reactions were monitored by TLC using 0.25 mm coated commercial silica gel plates (EMD, Silica Gel 60F₂₅₄). TLC spots were visualized by UV light at 254 nm, or developed with ceric ammonium molybdate or anisaldehyde or copper sulfate or ninhydrin solutions by heating on a hot plate. Reactions were also monitored by using SHIMADZU LCMS-2020 with solvents: A: 0.1% formic acid in water, B: acetonitrile. Flash chromatography was performed with SiliCycle silica gel (Purasil 60 Å, 230-400 Mesh). Proton magnetic resonance (¹H-NMR) spectral data were recorded on 400, and 500 MHz instruments. Carbon magnetic resonance (¹³C-NMR) spectral data were recorded on 100 and 125 MHz instruments. For all NMR spectra, chemical shifts (δH, δC) were quoted in parts per million (ppm), and J values were quoted in Hz. ¹H and ¹³C NMR spectra were calibrated with residual undeuterated solvent (CDCl₃: δH = 7.26 ppm, δC = 77.16 ppm; CD₃CN: δH = 1.94 ppm, δC = 1.32 ppm; CD₃OD: δH =3.31 ppm, δC = 49.00 ppm; DMSO-d₆: δH = 2.50 ppm, δC = 39.52 ppm; D₂O: δH = 4.79 ppm) as an internal reference. The following abbreviations were used to designate the multiplicities: s = singlet, d = doublet, dd = double doublets, t = triplet, q = quartet, quin = quintet, hept = heptet, m = multiplet, br = broad. Infrared (IR) spectra were recorded on a Perkin-Elmer FT1600 spectrometer. HPLC analyses were performed with a Shimadzu LC-20AD HPLC system. HR-MS data were obtained from a Waters Synapt G2-Si (ion mobility mass spectrometer with nanoelectrospray ionization).

Synthesis of ((3aR,4R,7aR)-2,2-dimethyl-3a,7a-dihydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl 4-methylbenzenesulfonate (P1)

((3aR,4R,7aR)-2,2-Dimethyl-3a,7a-dihydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methanol (2, 3.95 g, 21.2 mmol) was dissolved in CH₂Cl₂ (42.4 mL). Into the reaction mixture at 0 °C, pyridine (8.54 mL, 106.0 mmol) and TsCl (12.10 g, 63.6 mmol) were added. The reaction mixture was warmed to r.t., and was stirred for 8 h. The reaction mixture was quenched with 1N HCl, and extracted with EtOAc. The combined organic extracts were washed with aq. NaHCO₃, dried over Na₂SO₄, and evaporated. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 90/10 - 80/20) to afford ((3aR,4R,7aR)-2,2-dimethyl-3a,7a-dihydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl 4-methylbenzenesulfonate (7.13 g, 99%): [α]²²_D + 0.315 (c = 1.12, CHCl₃); IR (thin film) ν_max = 3067, 2986, 2956, 2932, 1776, 1718, 1650, 1598, 1455, 1368, 1237, 1190, 1177, 1146, 1126, 1096, 1069, 1034, 1023, 983, 889, 866, 828, 816, 790, 761, 725, 679, 660 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.82 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 6.30 (d, J = 6.3 Hz, 1H), 4.81 (ddd, J = 6.3, 3.0, 1.4 Hz, 1H), 4.63 (dd, J = 6.2, 3.0 Hz, 1H), 4.28 (d, J = 5.0 Hz, 1H), 4.26 (d, J = 2.5 Hz, 1H), 4.23 (dt, J = 6.1, 1.7 Hz, 1H), 4.15 (ddd, J = 7.1, 4.9, 1.6 Hz, 1H), 2.45 (s, 3H), 1.39 (s, 3H), 1.29 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 145.02, 144.22, 132.59, 129.89 (2C), 128.01 (2C), 110.78, 102.66, 72.30, 71.77, 68.89, 68.17, 27.83, 26.74, 21.66; HRMS (ESI+) m/z calcd for C₁₆H₂₀NaO₆S [M + Na⁺] 363.0878, found: 363.0883.

Synthesis of (3aR,4R,7aR)-4-(azidomethyl)-2,2-dimethyl-3a,7a-dihydro-4H-[1,3]dioxolo[4,5-c]pyran (P2)

((3aR,4R,7aR)-2,2-Dimethyl-3a,7a-dihydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl 4-methylbenzenesulfonate (see P1, 7.13 g, 20.9 mmol) was dissolved in DMSO (41.9 mL). Into the reaction mixture, NaN₃ (4.08 g, 62.8 mmol) was added, and the reaction mixture was stirred at 150°C for 1 h. The reaction mixture was quenched with sat. aq. NaHCO₃ and extracted with EtOAc. The combined extract was dried over Na₂SO₄ and evaporated. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 90/10 - 80/20) to afford (3aR,4R,7aR)-4-(azidomethyl)-2,2-dimethyl-3a,7a-dihydro-4H-[1,3]dioxolo[4,5-c]pyran (4.36 g, 99%).%): [α]²¹_D + 0.795 (c = 5.90, CHCl₃); IR (thin film) ν_max = 3068, 2986, 2935, 2889, 1649, 1455, 1380, 1370, 1290, 1238, 1163, 1138, 1114, 1066, 1030, 923, 863, 825, 732 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 6.40 (d, J = 6.2 Hz, 1H), 4.84 (dd, J = 6.2, 2.7 Hz, 1H), 4.67 (dd, J = 6.2, 2.9 Hz, 1H), 4.24 (d, J = 6.3 Hz, 1H), 4.02 (ddd, J = 8.6, 4.9, 1.7 Hz, 1H), 3.74 (dd, J = 12.9, 8.4 Hz, 1H), 3.43 (dd, J = 12.9, 4.9 Hz, 1H), 1.46 (s, 3H), 1.36 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 144.42, 110.76, 102.67, 73.69, 72.57, 68.41, 51.72, 27.95, 26.80; HRMS (ESI+) m/z calcd for C₉H₁₄N₃O₃ [M + H⁺] 212.1035, found: 212.1044.

Synthesis of N-(((3aR,4R,7aR)-2,2-dimethyl-3a,7a-dihydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl)acetamide (3) (P3)

(3aR,4R,7aR)-4-(Azidomethyl)-2,2-dimethyl-3a,7a-dihydro-4H-[1,3]dioxolo[4,5-c]pyran (see P2, 4.36 g, 20.7 mmol) was disolved in pyridine (41.3 mL), and AcSH (7.73 mL, 103.3 mmol) was added at r.t.. The reaction mixture was covered with a tin foil and stirred for 20 h. All volitiles were evaportaed and the residue was attached on a high vacuum (0.1 mmHg) for 6 h. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 20/80 - 0/100) to afford 3 (4.69 g, 100%): [α]²¹_D + 3.772 (c = 8.35, CHCl₃); IR (thin film) ν_max = 3289 (br), 3087, 2985, 2935, 2891, 1646, 1551, 1433, 1370, 1290, 1233, 1164, 1142, 1125, 1105, 1066, 1025, 918, 888, 858, 814, 772, 731 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 6.35 (d, J = 6.2 Hz, 1H), 6.10 (brs, 1H), 4.79 (ddd, J = 6.4, 3.0, 1.4 Hz, 1H), 4.63 (dd, J = 6.1, 2.9 Hz, 1H), 4.24 (d, J = 6.1 Hz, 1H), 3.99 (dd, J = 8.5, 3.5 Hz, 1H), 3.85 (ddd, J = 14.2, 7.7, 3.5 Hz, 1H), 3.35 (ddd, J = 14.2, 8.5, 3.9 Hz, 1H), 2.00 (s, 3H), 1.44 (s, 3H), 1.33 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 170.40, 144.50, 110.63, 102.78, 73.39, 72.95, 68.76, 41.34, 28.01, 26.82, 23.25; HRMS (ESI+) m/z calcd for C₁₁H₁₈NO₄ [M + H⁺] 228.1236, found: 228.1251.

Synthesis of (3aS,4R,7R,7aR)-4-(acetamidomethyl)-7-azido-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl nitrate (4) (P4)

N-(((3aR,4R,7aR)-2,2-Dimethyl-3a,7a-dihydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl)acetamide (3, 4.69 g, 20.7 mmol) was dissolved in acetone (103.3 mL). Into the reaction mizture, NaN₃ (2.01 g, 31.0 mmol) and [NH₄]₂[Ce(NO₃)₆] (CAN, 33.96 g, 61.9 mmol) were added at -20 °C. The reaction mixture was stirred for 2 h, and quenched with cold H₂O. The partition between H₂O and EtOAc was performed. The combined organic extracts were dried over Na₂SO₄, filtered, and evaporated. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 50/50 - 20/80 - 0/100) to afford 4 (α/β mixture, 6.13 g, 90%): 4α (α-anomer): ¹H NMR (400 MHz, CDCl₃) δ 6.20 (d, J = 4.0 Hz, 1H), 5.81 (brs, 1H), 4.37 (dd, J = 7.7, 5.4 Hz, 1H), 4.29–4.24 (m, 2H), 3.85 (ddd, J = 14.5, 8.1, 3.6 Hz, 1H), 3.83 (dd, J = 7.7, 4.0 Hz, 1H), 3.31 (ddd, J = 13.3, 8.7, 4.4 Hz, 1H), 1.99 (s, 3H), 1.54 (s, 3H), 1.37 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 170.35, 110.67, 96.66, 73.32, 72.78, 69.48, 59.02, 40.00, 27.93, 25.89, 23.25. 4β (β-anomer): ¹H NMR (400 MHz, CDCl₃) δ 6.12 (brs, 1H), 5.43 (d, J = 8.9 Hz, 1H), 4.30–4.23 (m, 1H), 4.20 (d, J = 5.6 Hz, 1H), 4.14 (d, J = 6.5 Hz, 1H), 3.96–3.78 (m, 1H), 3.50 (t, J = 8.0 Hz, 1H), 3.36–3.25 (m, 1H), 2.09 (s, 3H), 1.53 (s, 3H), 1.37 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 172.14, 111.28, 97.86, 77.48, 72.89, 72.34, 61.35, 40.33, 27.97, 25.96, 23.18.

Synthesis of N-(((3aS,4R,7R,7aR)-7-azido-6-hydroxy-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl)acetamide (P5)

(3aS,4R,7R,7aR)-4-(Acetamidomethyl)-7-azido-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl nitrate (4, 6.13 g, 18.5 mmol) was dissolved in DMF (46.3 mL). Into the reaction mixture, H₂NNH₂•AcOH (3.41 g, 37.0 mmol) was added. The reaction mixture was stirred for 4 h, and quenched with brine, and extracted with CHCl₃. The combined organic extracts were dried over Na₂SO₄, filtered, and evaportaed. The crude mixture was purified by silica gel column chromatography (CHCl₃/MeOH = 98/2 - 96/4) to afford N-(((3aS,4R,7R,7aR)-7-azido-6-hydroxy-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl)acetamide (α/β mixture, 4.33 g, 82%): α-anomer: ¹H NMR (400 MHz, CDCl₃) δ 6.23 (dd, J = 7.7, 4.2 Hz, 1H), 5.27 (d, J = 3.3 Hz, 1H), 4.46–4.39 (m, 1H), 4.35 (ddd, J = 7.4, 4.5, 2.5 Hz, 1H), 4.20 (dd, J = 5.4, 2.5 Hz, 1H), 3.85–3.74 (m, 1H), 3.41–3.28 (m, 2H), 2.01 (s, 3H), 1.52 (s, 3H), 1.36 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 171.21, 109.93, 91.72, 73.73, 73.65, 65.39, 61.66, 40.58, 28.15, 26.18, 23.18. β-anomer: ¹H NMR (400 MHz, CDCl₃) δ 6.40 (dd, J = 7.7, 4.4 Hz, 1H), 4.52 (d, J = 8.4 Hz, 1H), 4.09 (dd, J = 5.4, 2.2 Hz, 1H), 3.96 (dd, J = 8.0, 5.3 Hz, 1H), 3.90 (ddd, J = 8.3, 3.8, 2.2 Hz, 1H), 3.85–3.74 (m, 1H), 3.41–3.28 (m, 2H), 2.01 (s, 3H), 1.54 (s, 3H), 1.34 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 171.27, 110.66, 95.67, 77.20, 73.44, 71.04, 66.17, 40.58, 28.12, 26.16, 23.13.

Synthesis of N-(((3aS,4R,7R,7aR)-7-(1,3-dioxoisoindolin-2-yl)-6-hydroxy-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl)acetamide (5) (P6)

N-(((3aS,4R,7R,7aR)-7-Azido-6-hydroxy-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl)acetamide (see P5, 4.33 g, 15.1 mmol) was disolved in MeOH (75.6 mL) and 10% Pd/C (0.43 g) was added slowly under N₂. H₂ gas was introduced, and the reaction mixture was stirred under H₂ atmosphere (a double-folded balloon) for 4 h. The reaction mixture was filtered through celite, and evaporated. The crude product was dissolved in in THF (30.2 mL) and PhthCO₂Et (6.63 g, 30.2 mmol) and Et₃N (6.32 mL, 45.3 mmol) were added. The reaction mixture was stirred for 9 h at r.t., and was quenched with brine, and extracted with CHCl₃. The combined CHCl₃ extracts were dried over Na₂SO₄, and evaportaed. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc = 50/50 - 20/80 - 0/100) to afford 5 (α/β mixture, 5.05 g, 86% for 2 steps): 5α (α-anomer): ¹H NMR (400 MHz, CDCl₃) δ 7.87 (dd, J = 5.5, 3.1 Hz, 2H), 7.76 (dd, J = 5.5, 3.1 Hz, 2H), 6.07 (d, J = 8.0 Hz, 0H), 5.30–5.27 (m, 1H), 5.25–5.18 (m, 1H), 5.12 (dd, J = 9.8, 5.0 Hz, 1H), 4.55–4.50 (m, 1H), 4.31 (dd, J = 5.1, 2.5 Hz, 1H), 4.20–4.14 (m, 1H), 4.12–4.08 (m, 1H), 3.98–3.92 (m, 1H), 3.36 (ddd, J = 14.2, 8.6, 3.9 Hz, 1H), 2.01 (s, 3H), 1.56 (s, 3H), 1.32 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 170.77, 134.62 (2C), 131.41 (2C), 123.79, 110.08, 92.76, 69.67, 65.97, 60.41, 54.67, 40.49, 27.89, 23.33, 21.06. 5β (β-anomer): ¹H NMR (400 MHz, CDCl₃) δ 7.83 (dd, J = 5.4, 3.0 Hz, 2H), 7.72 (dd, J = 5.4, 3.1 Hz, 2H), 6.25 (dd, J = 8.1, 4.0 Hz, 1H), 5.29 (d, J = 8.6 Hz, 1H), 4.85 (dd, J = 9.2, 5.0 Hz, 1H), 4.22 (dd, J = 5.0, 2.1 Hz, 1H), 4.20–4.14 (m, 1H), 4.13–4.07 (m, 1H), 3.92 (ddd, J = 14.3, 7.9, 3.5 Hz, 1H), 3.36 (ddd, J = 14.2, 8.6, 3.9 Hz, 1H), 2.01 (s, 3H), 1.62 (s, 3H), 1.32 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 168.37, 134.15 (2C), 131.76 (2C), 123.40, 110.62, 92.58, 74.10, 73.70, 71.60, 56.59, 40.67, 27.96, 26.42, 23.25.

Synthesis of (4aR,6R,7R,8R,8aS)-6-(((3aS,4R,6S,7R,7aR)-4-(acetamidomethyl)-7-(1,3-dioxoisoindolin-2-yl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-7-azido-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-8-yl acetate (7) (P7)

N-(((3aS,4R,7R,7aR)-7-(1,3-Dioxoisoindolin-2-yl)-6-hydroxy-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl)acetamide (5, 2.00 g, 5.12 mmol), 6 (6.05 g, 15.4 mmol), and MS3Å (6.00 g) were suspended in CH₂Cl₂ (25.6 mL) and stiired for 30 min. The reaction mixture was cooled to 0°C, and AgBF₄ (1.00 g, 5.12 mmol) and NBS (2.74 g, 15.4 mmol) were added. After 16 h, the reaction mixture was quenched with Et₃N (5.0 mL) and aq. NaHCO₃ (10.0 mL). The reaction mixture was passed through a pad of celite, and eluted with CHCl₃. The combined organic extracts were washed with brine, dried over Na₂SO₄, and evaporated. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc = 30/70 - 20/80 - 10/90) to afford 7 (2.83 g, 84%): [α]²¹_D + 0.315 (c = 0.42, CHCl₃); IR (thin film) ν_max = 2989, 2933, 2875, 1775, 1714, 1657, 1550, 1467, 1430, 1389, 1333, 1268, 1239, 1222, 1127, 1088, 1071, 1038, 991, 968, 879, 868, 850, 754, 722 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.83 (brs, 2H), 7.69 (dd, J = 5.5, 3.0 Hz, 2H), 6.00 (dd, J = 7.5, 4.3 Hz, 1H), 5.32 (d, J = 8.9 Hz, 1H), 5.25 (t, J = 9.8 Hz, 1H), 4.96 (d, J = 3.7 Hz, 1H), 4.69 (dd, J = 9.3, 4.9 Hz, 1H), 4.34 (t, J = 9.1 Hz, 1H), 4.20 (dd, J = 4.9, 2.2 Hz, 1H), 4.15 (ddd, J = 8.3, 4.2, 2.3 Hz, 1H), 4.02 (td, J = 10.2, 5.2 Hz, 1H), 3.88 (ddd, J = 14.1, 7.5, 4.1 Hz, 1H), 3.74 (dd, J = 10.5, 5.3 Hz, 1H), 3.66 (t, J = 10.5 Hz, 1H), 3.59 (t, J = 9.7 Hz, 1H), 3.44 (ddd, J = 14.1, 8.1, 4.3 Hz, 1H), 3.18 (dd, J = 10.2, 3.7 Hz, 1H), 2.00 (s, 3H), 2.00 (s, 3H), 1.69 (s, 3H), 1.41 (s, 3H), 1.35 (s, 3H), 1.33 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 170.51, 169.37, 133.99, 110.76, 99.91, 99.65, 98.83, 74.52, 73.90, 72.07, 71.86, 69.47, 64.36, 61.91, 61.71, 54.56, 40.55, 28.88, 27.87, 26.43, 23.18, 20.72, 18.89; HRMS (ESI+) m/z calcd for C₃₀H₃₇N₅NaO₁₂ [M + Na] 682.2336, found: 682.2362.

Sysnsthesis of (4aR,6R,7R,8R,8aS)-7-azido-6-(((3aS,4R,6S,7R,7aR)-7-(1,3-dioxoisoindolin-2-yl)-2,2-dimethyl-4-((N-nitrosoacetamido)methyl)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-8-yl acetate (8) (P8)

(4aR,6R,7R,8R,8aS)-6-(((3aS,4R,6S,7R,7aR)-4-(Acetamidomethyl)-7-(1,3-dioxoisoindolin-2-yl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-7-azido-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-8-yl acetate (7, 1.45 g, 2.20 mmol) was disolved in a 4:1 mixture of Ac₂O (17.6 mL) and AcOH (4.4 mL). The reaction mixture was cooled to 0 °C and NaNO₂ (1.52 g, 22.0 mmol) was added. After 2 h at 0 °C, the reaction was quenched with ice-water, and extracted with EtOAc. The combined organic extracts were washed with aq. NaHCO₃ and brine, dried over Na₂SO₄, and evaporated. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 80/20–60/40) to afford 8 (1.42 g, 94%): [α]²¹_D + 0.499 (c = 0.29, CHCl₃); IR (thin film) ν_max = 2991, 2924, 2853, 1776, 1742, 1715, 1387, 1339, 1268, 1221, 1200, 1173, 1123, 1096, 1074, 1040, 1028, 990, 968, 952, 895, 868, 755, 721 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.82 (brs, 2H), 7.68 (dd, J = 5.5, 3.0 Hz, 2H), 5.17 (t, J = 9.8 Hz, 1H), 5.11 (d, J = 8.9 Hz, 1H), 4.84 (d, J = 3.7 Hz, 1H), 4.66 (dd, J = 9.2, 4.9 Hz, 1H), 4.62 (dd, J = 14.1, 8.9 Hz, 1H), 4.33 (t, J = 9.1 Hz, 1H), 4.14 (dd, J = 5.0, 2.2 Hz, 1H), 4.03 (dt, J = 8.7, 2.7 Hz, 1H), 3.81 (d, J = 10.6 Hz, 1H), 3.80 (dd, J = 16.3, 7.1 Hz, 1H), 3.73 (dd, J = 10.4, 5.1 Hz, 1H), 3.57 (t, J = 10.3 Hz, 1H), 3.52 (t, J = 9.6 Hz, 1H), 3.18 (dd, J = 10.2, 3.7 Hz, 1H), 2.79 (s, 3H), 1.98 (s, 3H), 1.71 (s, 3H), 1.39 (s, 3H), 1.36 (s, 3H), 1.33 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 174.35, 169.26, 133.94 (2C), 131.84 (2C), 110.91, 99.72, 99.47, 99.07, 74.31, 73.41, 71.98, 70.49, 69.71, 64.34, 61.84, 61.76, 54.22, 38.64, 28.81, 27.88, 26.42, 22.51, 20.71, 18.99; HRMS (ESI+) m/z calcd for C₃₀H₃₆N₆NaO₁₃ [M + Na] 711.2238, found: 711.2259.

Synthesis of (3aS,4S,6R,6aR)-6-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole-4-carbaldehyde (10) (P9)

3-((Bis(4-fluorophenyl)methoxy)methyl)-1-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (9, 3.58 g, 6.93 mmol) was disolved in CH₂Cl₂ (34.6 mL) and cooled to 0 °C. Into the solution, dichloroacetic acid (0.86 mL, 10.4 mmol), DMSO (3.46 mL), and N,N’-diisopropylcarbodiimide (1.63 mL, 10.4 mmol) were added. After 14 h, the reaction mixture was diluted with hexanes and filtered through a cotton, and evaportaed. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc = 80/20 - 50/50 - 20/80) to afford 10 (3.37 g, 95%): TLC (hexanes/EtOAc 50:50) R_f = 0.40; ¹H NMR (400 MHz, CDCl₃) δ 9.41 (s, 1H), 7.32–7.25 (m, 4H), 7.14 (d, J = 8.0 Hz, 1H), 7.00 (dt, J = 8.8, 6.9 Hz, 4H), 5.74 (d, J = 8.0 Hz, 1H), 5.65 (s, 1H), 5.42 (s, 2H), 5.36 (d, J = 9.9 Hz, 1H), 5.25 (dd, J = 6.3, 1.6 Hz, 1H), 4.99 (d, J = 6.3 Hz, 1H), 4.57 (d, J = 1.6 Hz, 1H), 1.54 (s, 3H), 1.37 (s, 3H); HRMS (ESI+) m/z calcd for C₂₆H₂₅F₂N₂O₇ [M + Na] 515.1630, found: 515.1655.

Synthesis of (4aS,6R,7S,8S,8aS)-7-azido-6-(((4R,6S,7aR)-4-(2-((3aS,4S,6R,6aR)-6-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-oxoethyl)-7-(1,3-dioxoisoindolin-2-yl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-8-yl acetate (11) (P10)

(4aR,6R,7R,8R,8aS)-7-Azido-6-(((3aS,4R,6S,7R,7aR)-7-(1,3-dioxoisoindolin-2-yl)-2,2-dimethyl-4-((N-nitrosoacetamido)methyl)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-8-yl acetate (8, 1.42 g, 2.06 mmol) was dissolved in a 10:1 mixture of toluene/MeOH (20.6 mL) and cooled to 0°C. Into the reaction mixture, 40% KOH (2.89 mL, 20.6 mmol) was added. After 30 min, MgSO₄ (3.55 g) was added to the reaction mixture, during which the reaction temprature was maintained at ice-bath temparature. The reaction suspension was sttired for an additional 5 min, and the aldehyde 10 (1.27 g, 2.47 mmol) was added. After 12 h at r.t., the reaction mixture was filtered through a cotton and evaportaed. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 50/50–30/70) to afford 11 (2.08 g, 91%): [α]²¹_D + 0.334 (c = 1.02, CHCl₃); IR (thin film) ν_max = 2988, 2937, 1776, 1715, 1671, 1604, 1508, 1455, 1387, 1335, 1269, 1222, 1156, 1088, 1072, 990, 969, 868, 837, 754, 721 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.83 (brs, 2H), 7.72–7.66 (m, 2H), 7.34–7.27 (m, 4H), 7.21 (d, J = 8.0 Hz, 1H), 7.00 (q, J = 9.2 Hz, 4H), 5.71 (d, J = 8.1 Hz, 1H), 5.68 (s, 1H), 5.51 (s, 1H), 5.43 (d, J = 10.0 Hz, 1H), 5.31 (s, 1H), 5.29 (d, J = 4.8 Hz, 1H), 5.23–5.12 (m, 2H), 4.95 (d, J = 6.3 Hz, 1H), 4.80 (d, J = 3.8 Hz, 1H), 4.73–4.60 (m, 2H), 4.35 (t, J = 6.5 Hz, 1H), 4.29 (t, J = 9.0 Hz, 1H), 4.20 (d, J = 3.7 Hz, 1H), 3.87 (td, J = 10.3, 5.1 Hz, 1H), 3.67–3.59 (m, 1H), 3.52 (t, J = 9.9 Hz, 2H), 3.15 (dd, J = 10.2, 3.7 Hz, 1H), 3.01–2.92 (m, 2H), 1.97 (s, 3H), 1.67 (s, 3H), 1.58 (s, 3H), 1.40 (s, 3H), 1.37 (s, 3H), 1.31 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 203.47, 169.30, 163.39, 162.25, 160.94, 151.03, 142.00, 137.38, 133.92, 128.71, 128.64, 128.56, 115.40, 115.34, 115.19, 115.13, 114.08, 110.42, 102.32, 99.99, 99.69, 99.23, 98.43, 93.97, 84.34, 82.52, 81.93, 74.34, 74.19, 71.98, 69.64, 64.25, 61.79, 54.48, 39.83, 28.84, 27.92, 26.71, 26.46, 25.09, 20.71, 18.88; HRMS (ESI+) m/z calcd for C₅₄H₅₆F₂N₆NaO₁₈ [M + Na] 1137.3517, found: 1137.3549. The live procedure of the one-pot BCS reaction (∼300 mg scale) was posted at https://www.youtube.com/watch?v=WyEbXN8wWqc&ab_channel=Kurosulab.

Syntehsis of (4aS,6R,7S,8S,8aS)-7-azido-6-(((4R,6S,7aR)-4-((R)-2-((3aR,4R,6R,6aR)-6-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-hydroxyethyl)-7-(1,3-dioxoisoindolin-2-yl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-8-yl acetate (12) (P11)

(4aS,6R,7S,8S,8aS)-7-azido-6-(((4R,6S,7aR)-4-(2-((3aS,4S,6R,6aR)-6-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-oxoethyl)-7-(1,3-dioxoisoindolin-2-yl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-8-yl acetate (11, 2.08 g, 1.87 mmol) was dissovled in a 1 : 3 : 6 mixture of HCO₂H : Et₃N : ⁱPrOH (18.7 mL). Into the reaction mixture, RuCl[(R,R)-TsDPEN](p-cymene) [6] (59.4 mg, 0.093 mmol) was added, and the raection mixture was stiired for17 h at r.t. The reaction mixture was quenched with H₂O, and extracted with CHCl₃. The combined organic extracts were dried over Na₂SO₄ and evaporated. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc = 50/50–20/80) to afford 12 (2.08 g, 100%): [α]²²_D + 0.142 (c = 0.60, CHCl₃); IR (thin film) ν_max = 3428 (br), 2932, 2875, 2858, 1776, 1715, 1669, 1604, 1508, 1456, 1386, 1269, 1222, 1156, 1074, 1040, 969, 941, 879, 867, 837, 779, 721 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.84 (brs, 2H), 7.69 (dd, J = 5.5, 3.0 Hz, 2H), 7.32–7.27 (m, 4H), 7.21 (d, J = 8.1 Hz, 1H), 6.99 (t, J = 8.7 Hz, 4H), 5.71 (d, J = 9.4 Hz, 1H), 5.67 (d, J = 8.1 Hz, 1H), 5.51 (s, 2H), 5.48 (d, J = 3.1 Hz, 1H), 5.36 (d, J = 8.9 Hz, 1H), 5.20 (t, J = 9.8 Hz, 1H), 5.00 (dd, J = 6.7, 3.7 Hz, 1H), 4.96 (d, J = 3.7 Hz, 1H), 4.90 (dd, J = 6.7, 3.1 Hz, 1H), 4.73 (dd, J = 9.2, 4.9 Hz, 1H), 4.37 (t, J = 9.0 Hz, 1H), 4.30 (dd, J = 9.7, 2.7 Hz, 1H), 4.19 (d, J = 10.3 Hz, 1H), 4.14 (dd, J = 4.8, 2.0 Hz, 1H), 4.12–4.08 (m, 1H), 3.98 (td, J = 10.0, 5.0 Hz, 1H), 3.77 (dd, J = 10.5, 5.2 Hz, 1H), 3.57 (t, J = 10.4 Hz, 1H), 3.53 (t, J = 9.7 Hz, 1H), 3.20 (dd, J = 10.2, 3.8 Hz, 1H), 2.22–2.14 (m, 1H), 1.98 (s, 3H), 1.86–1.78 (m, 1H), 1.70 (s, 3H), 1.59 (s, 3H), 1.38 (s, 3H), 1.35 (s, 6H), 1.31 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 169.29, 163.39, 162.22, 160.95, 141.41, 128.57, 128.52, 128.49, 115.36, 115.15, 114.70, 110.66, 102.20, 100.14, 99.75, 99.67, 96.10, 89.08, 83.32, 82.09, 78.55, 75.62, 74.56, 72.06, 70.13, 69.92, 69.56, 67.58, 64.51, 62.00, 61.84, 54.64, 33.75, 30.96, 29.69, 28.85, 27.99, 27.28, 26.52, 25.27, 20.72, 18.90; HRMS (ESI+) m/z calcd for C₅₄H₅₈F₂N₆NaO₁₈ [M + Na] 1139.3673, found: 1139.3711.

Syntehsis of (4aS,6R,7S,8S,8aS)-7-acetamido-6-(((4R,6S,7R,7aR)-4-((R)-2-((3aR,4R,6R,6aR)-6-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-hydroxyethyl)-7-(1,3-dioxoisoindolin-2-yl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-8-yl acetate (P12)

(4aS,6R,7S,8S,8aS)-7-Azido-6-(((4R,6S,7aR)-4-((R)-2-((3aR,4R,6R,6aR)-6-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-hydroxyethyl)-7-(1,3-dioxoisoindolin-2-yl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-8-yl acetate (12, 2.08 g, 1.86 mmol) was dissolved in pyridine (24.8 mL) and the reaction flask was covered with a tin foil. Into the reaction mixture, AcSH (12.4 mL) was added. After 5 days at r.t. under dark, the reaction mixture was concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 20/80 - 0/100) to afford (4aS,6R,7S,8S,8aS)-7-acetamido-6-(((4R,6S,7R,7aR)-4-((R)-2-((3aR,4R,6R,6aR)-6-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-hydroxyethyl)-7-(1,3-dioxoisoindolin-2-yl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-8-yl acetate (2.03 g, 97%): [α]²⁰_D - 0.079 (c = 0.30, CHCl₃); IR (thin film); ν_max = 3710, 2981, 2973, 2922, 2865, 2844, 2826, 1717, 1672, 1509, 1455, 1392, 1345, 1331, 1221, 1054, 1033, 1015, 832, 820, 804, 789, 773, 721, 700, 675 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.90–7.82 (m, 2H), 7.75 (dd, J = 5.6, 3.0 Hz, 2H), 7.30 (dd, J = 8.7, 5.4 Hz, 4H), 7.23 (d, J = 8.2 Hz, 1H), 7.01–6.97 (m, 5H), 5.83 (d, J = 2.0 Hz, 1H), 5.74–5.71 (m, 2H), 5.53 (s, 2H), 5.26 (d, J = 8.8 Hz, 1H), 4.91 (dd, J = 9.0, 5.1 Hz, 1H), 4.81 (t, J = 9.7 Hz, 1H), 4.69 (dd, J = 6.5, 2.0 Hz, 1H), 4.65 (dd, J = 6.5, 3.7 Hz, 1H), 4.57 (d, J = 7.8 Hz, 1H), 4.42 (t, J = 3.4 Hz, 1H), 4.34–4.26 (m, 2H), 3.89 (dd, J = 4.6, 2.2 Hz, 1H), 3.47 (t, J = 2.6 Hz, 1H), 3.42–3.37 (m, 2H), 3.33 (t, J = 9.5 Hz, 1H), 3.25 (dd, J = 9.8, 7.9 Hz, 1H), 3.07 (td, J = 9.7, 5.0 Hz, 1H), 3.00–2.93 (m, 1H), 2.06 (s, 3H), 2.05 (s, 3H), 1.64 (s, 3H), 1.59 (s, 3H), 1.37 (s, 3H), 1.37 (s, 3H), 1.27 (s, 3H), 1.23 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 169.70, 162.33, 150.62, 138.98, 137.65, 134.47, 134.12, 131.89, 128.57, 128.49, 115.34, 115.13, 114.70, 110.97, 102.26, 100.31, 99.64, 97.21, 93.07, 85.09, 84.92, 81.99, 78.92, 73.27, 71.99, 71.15, 70.86, 69.91, 67.09, 63.75, 60.41, 55.40, 54.72, 54.24, 30.96, 29.69, 28.73, 27.99, 27.11, 26.52, 25.33, 21.07, 20.82, 18.66, 14.19; HRMS (ESI+) m/z calcd for C₅₆H₆₃F₂N₄O₁₉ [M + H] 1133.4055, found: 1133.4074.

Synthesis of N-((4aS,6R,7S,8S,8aS)-6-(((4R,6S,7R,7aR)-7-amino-4-((R)-2-((3aR,4R,6R,6aR)-6-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-hydroxyethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-8-hydroxy-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-7-yl)acetamide (13) (P13)

(4aS,6R,7S,8S,8aS)-7-acetamido-6-(((4R,6S,7R,7aR)-4-((R)-2-((3aR,4R,6R,6aR)-6-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-hydroxyethyl)-7-(1,3-dioxoisoindolin-2-yl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-8-yl acetate (see P12, 2.03 g, 1.80 mmol) was dissoved in EtOH (35.9 mL) and ethylenediamine (3.6 mL) was added. The reaction mixture was heated at 60°C for 5 h, and was concentrated in vacuo. The residue was purified by a short path silica gel column (CHCl₃/MeOH/ Et₃N = 90/10/0.1) to afford 13 (1.56 g, 91%): ¹H NMR (400 MHz, MeOD) δ 7.60 (d, J = 8.1 Hz, 1H), 7.33 (dd, J = 8.6, 5.5 Hz, 4H), 7.05–6.99 (m, 4H), 5.84 (d, J = 2.9 Hz, 1H), 5.73 (s, 1H), 5.63 (d, J = 8.0 Hz, 1H), 5.57 (d, J = 6.3 Hz, 1H), 5.04 (d, J = 3.7 Hz, 1H), 4.76 (dd, J = 6.6, 2.9 Hz, 1H), 4.27 (d, J = 8.7 Hz, 1H), 4.08 (dt, J = 10.7, 2.3 Hz, 1H), 4.05–4.00 (m, 2H), 3.98 (dd, J = 6.2, 3.0 Hz, 2H), 3.91 (dd, J = 8.2, 5.2 Hz, 2H), 3.89–3.81 (m, 3H), 3.77–3.71 (m, 2H), 3.60 (d, J = 8.9 Hz, 1H), 2.81 (t, J = 8.5 Hz, 1H), 2.15–2.06 (m, 1H), 1.99 (s, 3H), 1.67–1.60 (m, 1H), 1.58 (s, 3H), 1.48 (s, 3H), 1.45 (s, 3H), 1.38 (s, 3H), 1.33 (s, 6H): ¹³C NMR (100 MHz, MeOD) δ 128.24, 114.65, 114.44, 114.07, 109.49, 104.06, 100.86, 99.55, 92.53, 89.01, 84.14, 81.74, 79.74, 77.99, 75.12, 74.69, 69.14, 68.16, 64.34, 61.89, 55.88, 54.44, 29.29, 28.03, 27.16, 26.03, 25.11, 24.01, 21.12, 17.87; HRMS (ESI+) m/z calcd for C₄₆H₅₉F₂N₄O₁₆ [M + H] 961.3894, found: 961.3928.

Synthesis of (E)-N-((4R,6S,7R,7aR)-6-(((4aS,6R,7S,8S,8aS)-7-acetamido-8-hydroxy-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-4-((R)-2-((3aR,4R,6R,6aR)-6-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-hydroxyethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)-13-methyltetradec-2-enamide (P14) and tunicamycin V (1) (P15)

N-((4aS,6R,7S,8S,8aS)-6-(((4R,6S,7R,7aR)-7-Amino-4-((R)-2-((3aR,4R,6R,6aR)-6-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-hydroxyethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-8-hydroxy-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-7-yl)acetamide (13, 94.8 mg, 0.099 mmol) was diisoved in DMF (0.5 mL). Into the reaction mixture, (E)-13-methyltetradec-2-enoic acid (14, 35.6 mg, 0.15 mmol) [1], NHS (22.7 mg, 0.20 mmol), NMM (59.9 μL, 0.59 mmol), and EDCI (47.3 mg, 0.25 mmol) were added. After 13 h at r.t., the reaction mixture was quenched with aq. NaHCO₃, and extracted with CHCl₃. The combined organic extracts were wsahed with brine, dried over Na₂SO₄, and evaporated. The crude product was purified by a short path silica gel column (hexanes/EtOAc = 50/50 - CHCl₃/MeOH = 96/4) to afford the title compound (108.1 mg, 93%, P14). (E)-N-((4R,6S,7R,7aR)-6-(((4aS,6R,7S,8S,8aS)-7-acetamido-8-hydroxy-2,2-dimethylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-4-((R)-2-((3aR,4R,6R,6aR)-6-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-hydroxyethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)-13-methyltetradec-2-enamide (see P14, 108.1 mg, 0.091 mmol) was dissoved in a 4:1 mixture of AcOH and H₂O (0.91 mL) and the reaction mixture was stirred for 8 h at 60°C. The reaction mixture was filtered through a membrane filter and concentrated in vacuo. The crude mixture was purified by a short path silica gel column (CHCl₃/MeOH = 90/10 - CHCl₃/MeOH/H₂O/28%NH₄OH = 56/42/7/3). The obtained product was further purified by reverse-phase HPLC [column: HYPERSIL GOLD^TM (C18, 12 μm, 175 Å, 250 × 10 mm), solvents: 90:10 MeOH:H₂O, flow rate: 2.0 mL/min, UV: 254 nm, retention time: 10.3 min] to afford 1 (67.5 mg, 89%): [α]²¹_D - 0.035 (c = 0.17, MeOH); IR (thin film) ν_max = 3445, 3384, 3277, 2922, 2851, 1663, 1461, 1377, 1274, 1091, 1025, 917, 844, 816, 758, 736 cm⁻¹; ¹H NMR (400 MHz, DMSO) δ 7.77 (t, J = 7.8 Hz, 2H), 6.64 (d, J = 8.7 Hz, 1H), 6.62–6.54 (m, 1H), 5.87 (d, J = 15.2 Hz, 1H), 5.82 (d, J = 6.6 Hz, 1H), 5.69 (d, J = 8.0 Hz, 1H), 5.33 (s, 1H), 5.09–5.01 (m, 3H), 4.77–4.71 (m, 2H), 4.70–4.65 (m, 2H), 4.61–4.57 (m, 1H), 4.41 (d, J = 8.4 Hz, 1H), 4.05–3.95 (m, 2H), 3.75–3.67 (m, 2H), 3.64–3.55 (m, 4H), 3.46–3.40 (m, 2H), 3.25–3.15 (m, 2H), 2.11 (q, J = 7.1 Hz, 2H), 1.99 (s, 1H), 1.90–1.82 (m, 1H), 1.76 (s, 3H), 1.49 (Hep, J = 6.5 Hz, 1H), 1.41–1.33 (m, 2H), 1.24 (s, 14H), 1.20–1.11 (m, 3H), 0.84 (d, J = 6.5 Hz, 6H); ¹³C NMR (100 MHz, DMSO) δ 173.14, 170.08, 167.02, 164.03, 151.89, 146.27, 143.46, 142.24, 141.34, 125.78, 103.14, 101.95, 99.81, 89.49, 87.22, 85.44, 83.22, 74.40, 74.07, 71.89, 71.60, 71.24, 70.22, 67.81, 67.32, 65.14, 61.51, 53.86, 48.87, 48.66, 48.45, 36.98, 34.71, 32.22, 30.29, 30.07, 30.04, 29.96, 29.90, 29.87, 29.68, 28.86, 28.37, 27.77, 27.46, 23.94, 23.51, 20.08, 14.96, 12.22; HRMS (ESI+) m/z calcd for C₃₈H₆₃N₄O₁₆ [M + H] 831.4239, found: 831.4255.

Additional information

Previously, four groups reported the total synthesis of tunicamycin V (Suami et al. 1984; Myers et al. 1994; Li et al. 2015; and Yamamoto et al. 2018) [2], [3], [4], [5]. Their syntheses required 21–35 chemical steps and the overall yields to accomplish their syntheses were 0.037–3.5% in the longest linear sequences. We achieved the total synthesis of tunicamycin V in 15 steps from D-galactal with 21% overall yield [1]. The overall efficiency of the original synthetic scheme could further be improved by refining each step; particularly, azidonitration of the acetamide derivative 3 provided only the desired diastereomer 4 in 90% yield. A plausible mechanism of highly selective azidonitration of 3 is illustrated in Scheme 1. The acetamide group is likely participated in addition reaction of the azido radical spp. to the vinyl ether moiety of 4. The re-face attack of the azido spp. is prevented by the acetamide group, producing the 3S-azido radical specie 4l exclusively. The azido radical intermediate is further oxidized by CAN to form the cation specie 4ll, which is trapped by the nitrate anion to form an α/β-mixture of 4. Büchner–Curtius–Schlotterbeck (BCS) -type reaction applied to couple 8I and 10 provided the desired ketone 11 in excellent yield. We have optimized the procedure of BCS reaction to be readily able to set-up a gram-scale reaction; BCS reaction using a mixture of solvents (toluene and MeOH) for the generation of the diazo intermediate 8l followed by addition of MgSO₄ to attenuate basicity of KOH enables a one-pot reaction. These protochols simplify the BCS reaction procedures and enable us to synthesize gram-quantity of the advanced intermediate 12 towards tunicamycin analogues. The optimized synthetic route illustrated in Scheme 1 was demonstrated to synthesize 2 grams of 12 and 100 mg of tunicamycin V (1). The synthese of the intermediates 13 and 14 were previousely reported [1]. The intermediates 3-12 are new and characterized for the first time in this article. The synthesis of tunicamycin V demostarted here is over 10-times larger scale than our previously reported synthesis.

Method validation

Each step was repeated at least three times in different scales (100-200 mg, 300-1,000 mg, and 1-4 g for 2 → 13 in Scheme 1) and (5 mg, 20 mg, and 100 mg for 13 → 1 in Scheme 1) by the first and second authors in this manuscript. Yields provided in Scheme 1 are averages of three-five times of each reaction.

CRediT authorship contribution statement

Katsuhiko Mitachi: Visualization, Investigation, Methodology, Writing – original draft, Writing – review & editing. David Mingle: Visualization, Investigation, Writing – review & editing. Michio Kurosu: Conceptualization, Methodology, Supervision, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

• Data will be made available on request.