Chemoenzymatic Synthesis of Tenofovir

Abstract

We report on novel chemoenzymatic routes toward tenofovir using low-cost starting materials and commercial or homemade enzyme preparations as biocatalysts. The biocatalytic key step was accomplished either via stereoselective reduction using an alcohol dehydrogenase or via kinetic resolution using a lipase. By employing a suspension of immobilized lipase from Burkholderia cepacia (Amano PS-IM) in a mixture of vinyl acetate and toluene, the desired (R)-ester (99% ee) was obtained on a 500 mg scale (60 mM) in 47% yield. Alternatively, stereoselective reduction of 1-(6-chloro-9H-purin-9-yl) propan-2-one (84 mg, 100 mM) catalyzed by lyophilized E. coli cells harboring recombinant alcohol dehydrogenase (ADH) from Lactobacillus kefir (E. coli/Lk-ADH Prince) allowed one to reach quantitative conversion, 86% yield and excellent optical purity (>99% ee) of the corresponding (R)-alcohol. The key (R)-intermediate was transformed into tenofovir through "one-pot" aminolysis-hydrolysis of (R)-acetate in NH₃-saturated methanol, alkylation of the resulting (R)-alcohol with tosylated diethyl(hydroxymethyl) phosphonate, and bromotrimethylsilane (TMSBr)-mediated cleavage of the formed phosphonate ester into the free phosphonic acid. The elaborated enzymatic strategy could be applicable in the asymmetric synthesis of tenofovir prodrug derivatives, including 5'-disoproxil fumarate (TDF, Viread) and 5'-alafenamide (TAF, Vemlidy). The molecular basis of the stereoselectivity of the employed ADHs was revealed by molecular docking studies.

Figure 1

Chemical structures of tenofovir (TFV), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF), as well as chiral building blocks, derived from (S)-lactic acid, mostly employed in the manufacturing processes of the TFV, TDF, and TAF.

Scheme 1. Chemoenzymatic Synthesis of Tenofovir [(R)-(−)-11]

Reagents and conditions: (i) 1 (18.44 mmol), ice cold 57% HI_aq. (10 equiv), 2 h at 0–5 °C with standing; (ii) 1 (12.9 mmol) or 2 (8.1 mmol), anhydrous K₂CO₃ (1 equiv), chloroacetone (1.1 equiv), dry DMF, 12 h at 25 °C; (iii) 3a (7.5 mmol) or 3b (3.3 mmol), NaBH₄ (1.2 equiv), MeOH/CH₃CN (2:1 v/v), 40 min at 0–5 °C; (iva) rac-4a (2.4 mmol), acetyl chloride (1.5 equiv), Et₃N (1.5 equiv), DMAP (cat.), dry CH₂Cl₂, 15 min at 0–5 °C, then 48 h at 25 °C; (ivb) rac-4b (0.20 mmol), Ac₂O (1.5 equiv), Et₃N (1.5 equiv), DMAP (cat.), dry CH₂Cl₂, 15 min at 0–5 °C, then 24 h at 25 °C; (v) rac-4a (0.19 mmol) or rac-4b (0.13 mmol), N,O-bis(trimethylsilyl) acetamide (BSA, 4 equiv), CH₂Cl₂, 20 min at 25 °C; (vi) rac-4a (2.4 mmol, 60 mM final concentration), Amano PS-IM (42 mg/mmol), PhCH₃/vinyl acetate (60 mL; 2:1 v/v), 26 h at 40 °C, 800 rpm (magnetic stirrer); (vii) 3a (0.4 mmol, 100 mM final concentration), E. coli/(Lk-ADH-A or Lk-ADH Prince) (60 mg), NAD(P)H (1.0 mM final concentration), 50 mM Tris-HCl buffer (pH 7.5), 2-PrOH (10% v/v), DMSO (2.5% v/v), 4 mL (final volume), 24 h at 30 °C, 250 rpm (orbital shaker); (viiia–b) (R)-(−)-4a (0.47 mmol) or (R)-(−)-5a (0.40 mmol), NH₃-saturated MeOH, 48 h at 150 °C (pressure tube); (ix) 8 (3.15 mmol), TsCl (1.2 equiv), Et₃N (1.2 equiv), dry CH₂Cl₂, 15 min at 0–5 °C, then 24 h at 25 °C; (x) (R)-(−)-7 (0.26 mmol), 9 (1.5 equiv), Mg(O^tBu)₂ (1.5 equiv), dry DMF, 1 h at 65 °C, then 24 h at 75 °C; (xi) (R)-(−)-10 (0.3 mmol, 99% ee), TMSBr (23 equiv), dry CH₂Cl₂, 48 h at 0–5 °C (pressure tube).

Scheme 2. Stereocomplementary Bioreductions of 3a Catalyzed by E. coli/ADH-A or E. coli/Lk-ADH Prince

Figure 2

Representative three-dimensional (3D) binding modes of 1-(6-chloro-9H-purin-9-yl) propan-2-one (3a) with stereocomplementary alcohol dehydrogenases, namely, ADH-A (PDB code: 2XAA; A and B) and Lk-ADH (PDB code: 4RF2; C and D), with close contacts to amino acid residues and cofactors located in the active sites. The docked ligand 3a and the cofactors are shown as sticks representation, where 3a is white, NADH is yellow, and NADPH is violet. The overall receptor structures are shown as a semitransparent cartoon diagram (left; A and C), where ADH-A is wheat and Lk-ADH is gray. The most significant amino acid residues contributing to the stabilization of the ligand 3a molecule in the complex with ADH-A or Lk-ADH by polar interactions, alkyl–alkyl (CH–CH), van der Waals (vdW,) and/or π–alkyl (CH−π) interactions are shown in lines representations. Nitrogen atoms are shown in blue, oxygen atoms in red, chlorine atoms in green, and phosphorus atoms in orange. All hydrogens were omitted for clarity. The zinc ion is presented as a semitransparent slate sphere. The formation of intermolecular hydrogen bonds is represented by magenta dashed lines, whereas the plausible trajectory of the hydride transfer from cofactors to a carbon atom of the carbonyl group is shown as red dashed lines. Mutual distances between the amino acid residues and the respective ligand’s atoms are given in Ångströms (right column; B and D). The figure was prepared using the program PyMOL (http://www.pymol.org/).

Scheme 3. Transformation of (R)-1-(6-Chloro-9H-purin-9-yl) Propan-2-ol [(R)-(−)-4a] and/or (R)-1-(6-Chloro-9H-purin-9-yl) Propan-2-yl Acetate [(R)-(−)-5a] into Tenofovir [(R)-(−)-11]