Synthesis of highly deuterated coniferyl alcohol for silencing of NMR signals in the resulting dehydrogenative polymer – Journal of Wood Science

Unless otherwise stated, all commercially available chemicals were used without further purification. Tetrahydrofuran (THF) was distilled from benzophenone ketyl radical under argon. Thin layer and column chromatography were performed with Silica Gel 70F₂₅₄ Wako (Fujifilm Wako Pure Chemicals Co. Ltd., Osaka, Japan) and Silica Gel 60N –spherical, neutral– (Kanto Chemicals Co., Tokyo, Japan), respectively. NMR spectra were measured on a JEOL JMN EX-270 (JEOL, Tokyo, Japan) or Bruker AVANCE Neo (Bruker, Billerica, MA, USA) spectrometer. Chemical shifts are reported in ppm (δ-scale) using tetramethylsilane (CDCl₃) or solvent peaks (CD₃OD or dimethyl sulfoxide (DMSO)-d₆) as the internal standards. Coupling constants (J) are given in Hz. Mass spectra were acquired with FI mode on a JEOL JMS-T100GCV equipment. All MS and NMR spectra on AVANCE Neo were measured at the GC–MS and NMR Laboratory, Faculty of Agriculture, Hokkaido University.

4-(Benzyloxy)-3-hydroxybenzaldehyde (1)

To a solution of 3,4-dihydroxybenzaldehyde (7.37 g, 53.4 mmol) in 100 mL of MeCN, NaHCO₃ (5.83 g, 1.3 eq.) was added at room temperature. The suspension was allowed to stir at 60 °C for 1 h, and 6.77 g of benzyl chloride (BnCl, 1.0 eq.) was added. The reaction was continued at 80 °C overnight. The mixture was acidified with 1 M aqueous HCl and washed with EtOAc (×3). The combined organic layer was neutralized with brine and dried over anhydrous Na₂SO₄. After removal of the solvent, the crude product was purified by silica gel column chromatography (hexane then EtOAc:hexane = 1:2). After evaporation of the combined target fractions, the product was recrystallized from EtOAc (×3) to give a total of 8.50 g of the 4-O-benzylated product as a pale-yellow solid (70%).

4-(Benzyloxy)-3-(²H₃)methoxybenzaldehyde (2)

Sodium hydride (5.6 g, ca.127 mmol) was added to a THF (100 mL) solution of CD₃OD (3.24 g, 90 mmol) portionwise at 0 °C. After gas generation stopped, 20.6 g of TsCl (1.2 eq.) was added at the same temperature. After the reaction was completed, 100 mL of Et₂O and cold water were added to the mixture. The water layer was recovered and again washed with Et₂O (×2). The combined organic layer was washed with aqueous sat.NaHCO₃ and brine. The solution was dried over anhydrous Na₂SO_4, and the solvent was removed in vacuo to afford 19.1 g of (²H₃)methyl p-toluenesulfonate as a mixture with mineral oil.

To the solution of 1 (8.35 g, 36.6 mmol) in N,N-dimethylformamide (DMF, 80 mL), NaH (1.76 g, ca. 44 mmol) was added portionwise at room temperature. The crude (²H₃)methyl p-toluenesulfonate (8.31 g) and KI (600 mg, 0.1 eq.) were added to the mixture, which was stirred at room temperature. After the overnight reaction, the mixture was partitioned between water and EtOAc (×3). The combined organic layer was washed with water and brine and dried over anhydrous Na₂SO₄. After removal of the solvent by evaporation, the product was purified by silica gel column chromatography (EtOAc:hexane = 1:1), giving 7.83 g of the product (87%) as a pale yellow solid.¹H NMR (CDCl₃, 500 MHz): δ 5.24 (2H, s, CH₂), 6.98 (1H, d, J = 8.0 Hz, H-5), 7.30–7.46 (7H, m, H-2, H-6 and benzyl aromatic), 9. 83 (1H, s, formyl); ¹³C NMR (CDCl₃, 125 MHz): δ 55.2 (OCD₃, m, J = 22 Hz), 70.8 (CH₂), 109.3 (C-2), 112.4 (C-5), 126.5 (C-6), 127.1 (benzyl), 128.1 (benzyl), 128.7 (benzyl), 130.2 (C-1), 136.0 (benzyl), 150.0 (C-3), 153.5 (C-4), 190.8 (formyl). HR-FI-MS: m/z = 245.11312 [M]⁺ calcd. for C₁₅¹H₁₁²H₃O₃, found 245.11358.

4-Hydroxy-3-(²H₃)methoxybenzene(²H)carbaldehyde (4, Vanillin-d
₄)

Compound 2 (5.20 g, 21.2 mmol) was dissolved in 16.6 mL of toluene. To the solution, 40 mL of D₂O, 2.94 g of K₂CO₃ (1.0 eq.) and 897 mg of 1,3-bis(2,5-diisopropylphenyl)imidazolium chloride ([bis(dipp)Im]Cl, 0.1 eq.) were added. The reaction was allowed to stir at 40 °C overnight and then partitioned with EtOAc (×2). The water layer was acidified with 1 M aqueous HCl and washed with EtOAc. The combined organic layer was washed with 1 M aqueous HCl, aqueous sat.NaHCO₃ and brine. The solution was dried over anhydrous Na₂SO_4, and the solvent was removed in vacuo. The purification by flash silica gel column chromatography eluting with EtOAc:hexane = 1:2 afforded 5.29 g of crude product, of which deuteration efficiency was 98.5%D at the formyl group. This could be estimated by the comparison with an integration value of H-5 (7.10 ppm in ¹H NMR). The same treatment was repeated for the recovered product with half amounts of the reagents, which gave 5.18 g of product with > 99%D of deuteration efficiency in the formyl group.

The crude product was dissolved in 50 mL of CH₂Cl_2, and 2.0 mL of TfOH (22.7 mmol) was added dropwise to the solution at − 20 °C. After the completion of the reaction, 2.3 mL of 10 M aqueous NaOH was added to the mixture. The mixture was partitioned between 1 M aqueous HCl and EtOAc (×3). The combined organic layer was neutralized with brine and dried over anhydrous Na₂SO₄. After removal of the solvent, the crude product was purified with flash silica gel column chromatography eluting with EtOAc:hexane = 1:2 to give 1.65 g of vanillin-d₄ (50% from 2). ¹H NMR (CDCl₃, 500 MHz): δ 7.04 (1H, d, J = 8.4 Hz, H-5), 7.42–7.45 (2H, m, H-2 and H-6); ¹³C NMR (CDCl₃, 125 MHz): δ 55.2 (OCD₃, m, J = 22 Hz), 108.8 (C-2), 114.3 (C-5), 127.4 (C-6), 129.7 (t, J = 3 Hz, C-1), 147.1 (C-3), 151.7 (C-4), 190.6 (t, J = 27 Hz, formyl). HR–FI–MS: m/z = 156.07167 [M]⁺ calcd. for C₈¹H₄²H₄O₃, found 156.07245.

(2,2-²H₂)Propane(²H₂)dioic acid (Malonic acid-d
₄, 5)

Malonic acid (2.08 g, 20.0 mmol) was dissolved in 6.0 mL of D₂O, and the solution was allowed to stir overnight at 80 °C. The solution was frozen, and the solvent was removed by freeze-drying. Deuteration efficiency was evaluated by ¹H NMR: Forty-two point zero milligrams of the product were dissolved in 0.45 mL of DMSO-d₆ containing 2.2 mg of benzaldehyde as an internal standard. The integration values of CH₂ at 3.22 ppm and COOH at 12.57 ppm were compared with those of intact malonic acid, respectively. The treatment and analysis were repeated four times, and 1.73 g of white solid was obtained (80%).

GC–MS analyses for the optimization of reaction conditions

A portion of the reaction mixture equivalent to 1.0 mg of the starting material was sampled and diluted with 800 µL of EtOAc. The resultant solution was mixed with 200 µL of N,O-bis(trimethylsilyl)trifluoroacetamide and filtered with a 0.45 µm PTFE membrane filter. The sample was allowed to stand for 1 h at room temperature. One microliter of each solution was analyzed on GC-2010/QP-2010 system (Shimadzu, Kyoto, Japan) under the following conditions: Column: BPX5 30 m, ϕ0.25 mm, df 1.0 µm (SGE Analytical Science, Melbourne, VIC, Australia); Injection unit: 250 °C; Column oven: 80 °C for 3.0 min, 40 °C/min for 6.0 min then 320 °C for 4.0 min; Carrier gas control: constant linear velocity (40.0 cm/s); Carrier gas: helium; Interface: 250 °C; Ion source: 250 °C. Total ion chromatograms (TIC) were obtained, and the yields were calculated simply based on the area of each compound per total of areas. Retention times of trimethylsilyl-derivatized compounds were as follows: Vanillin: 8.17 min; α, β-dihydroconiferyl alcohol: 8.81 min; Coniferyl alcohol: 9.22 min; Ferulic acid: 9.73 min.

(E)-3-[4-Hydroxy-3-(²H₃)methoxyphenyl](2,3-²H₂)prop-2-enoic acid (6, Ferulic acid-d
₅)

Prior to the reaction, the phenolic H of 4 was exchanged for D by repeating the addition and evaporation of CD₃OD three times to obtain vanillin-d₅.

To 2.4 mL of pyridine, 168 mg of (ND₄)₂SO₄ (0.5 eq.) and 520 mg of malonic acid-d₄ were added, and which was stirred for 10 min at room temperature. To the mixture was added 377.2 mg of vanilline-d₅ (2.40 mmol) and the reaction was allowed to stir at 70 °C overnight. Pyridine was azeotropically removed with toluene, and the residue was dissolved in a small quantity of acetic acid (AcOH)/methanol (MeOH). The mixture was subjected to silica gel column chromatography (CHCl₃, then MeOH:CHCl₃ = 10:90). The evaporation of the target fractions gave 391.4 mg of white solid (82%). ¹H NMR (CD₃OD, 500 MHz): δ 6.29 (0.08H, s, H-β), 6.80 (1H, d, J = 8.2 Hz, H-5), 7.04 (1H, dd, J = 8.2 and 2.0 Hz, H-6), 7.15 (1H, d, J = 2.0 Hz, H-2); ¹³C NMR (CD₃OD, 125 MHz): δ 55.6 (OCD₃, m, J = 22 Hz), 111.6 (C-2), 115.5 (t, J = 24 Hz, Cβ-D), 115.8 (Cβ-H), 116.4 (C-5), 123.9 (C-6), 127.7 (C-1), 146.5 (t, J = 24 Hz, Cα), 149.3 (C-4), 150.5 (C-3), 171.0 (carbonyl). HR–FI–MS: m/z = 199.08929 [M]⁺ calcd. for C₁₀¹H₅²H₅O₄, found 199.08870.

4-[(E)-3-Hydroxy(1,2,3,3-²H₄)prop-1-enyl]-2-(²H₃)methoxyphenol (7, Coniferyl alcohol-d
₇)

Ferulic acid-d₅ (347.3 mg, 1.74 mmol) was dissolved in 5.0 mL of THF. To the solution, one drop of anhydrous DMF and 150 µL of (COCl)₂ (1.0 eq.) were added in this order at 0 °C. Anhydrous CeCl₃ (429 mg, 1.0 eq.) was suspended in 10 mL of NMP, which was sonicated for 15 min, and 146 mg of NaBD₄ (2.0 eq.) was added to the suspension. At − 20 °C, the acid chloride solution was added dropwise to the reductant suspension. The reaction was then allowed to stir overnight at room temperature. The solvents were removed under reduced pressure, and the resultant residue was resuspended in MeOH containing AcOH (minimum quantity for neutralization). The crude product was directly subjected to silica gel column chromatography (CHCl₃, then MeOH:CHCl₃ = 10:90). Careful evaporation of the target fractions gave 224.5 mg of coniferyl alcohol-d₇ (65%) as a mixture with 6% NMP. ¹H NMR (CD₃OD, 500 MHz): δ 6.16 (0.08H, s, H-β), 6.72 (1H, d, J = 8.0 Hz, H-5), 6.83 (1H, dd, J = 8.0 and 2.0 Hz, H-6), 6.98 (1H, d, J = 2.0 Hz, H-2); ¹³C NMR (CD₃OD, 125 MHz): δ 55.5 (m, J = 22 Hz, OCD₃), 63.3 (quintet, J = 21 Hz, Cγ), 110.5 (C-2), 116.3 (C-5), 120.9 (C-6), 126.4 (t, J = 23 Hz, Cβ-D), 126.7 (C β-H), 130.4 (C-1), 131.9 (t, J = 23 Hz, Cα), 147.8 (C-4), 149.2 (C-3). HR–FI–MS: m/z = 187.12333 [M]⁺ calcd. for C₁₀¹H₅²H₇O₃, found 187.12258.

DHP preparation

Monolignols (coniferyl alcohol: 100.2 mg; coniferyl alcohol-d₇: 107.1 mg as a mixture with NMP) were separately dissolved in 1.0 mL of acetone, and to the solutions were added 49 mL of phosphate-buffered saline (PBS, pH 6.1, 50 mM phosphate). The solutions containing 0.5 mg of horseradish peroxidase (Fujifilm Wako Pure Chemicals Co. Ltd., Osaka, Japan) in 25 mL PBS and 4.0 mmol H₂O₂ in 25 mL PBS were added dropwise to the monolignol solutions over 3 h, and the reaction was continued for another 13 h. The resulting suspensions were centrifuged, and the precipitates were washed with Milli-Q water three times. The residues were freeze-dried to give DHPs (56.8 mg from coniferyl alcohol; 63.1 mg from coniferyl alcohol-d₇).

NMR analyses

The obtained DHP products were treated by the addition/evaporation of CD₃OD three times. Fifty milligrams of samples were dissolved in 0.45 mL of DMSO-d₆, and the complete dissolution of the samples was visually confirmed. The edited HSQC spectra were obtained using 256 scans using the standard Bruker pulse program (hsqcedetgpsp.3) with the following parameters for acquisition: TD = 2048 (F2), 192 (F1); SW = 11.1082 ppm (F2), 209.9969 ppm (F1); SWH = 5555.556 Hz (F2); 26,411.512 Hz (F1); IN_F = 37.8623 ms; AQ 0.1843200 s (F2), 0.0036348 s (F1); FIDRES = 5.425347 Hz (F2), 275.119934 Hz (F1); FW = 240,000,000.000 Hz. The resulting data were processed with the TopSpin 4.1.3. The thresholds in Fig. 3a–d were set as follows: Base level 1.4E10 (Positive) and − 1.4E10 (Negative); Level increment 1.200 (Positive and Negative); Number of levels 16. Threefold magnification in Fig. 3b was obtained at the Base level 1.4/3E10 (Positive) and − 1.4/3E10 (Negative). The thresholds in Fig. 3e, f were set as follows: Base level 1.4E9 (Positive) and − 1.4E9 (Negative); Level increment 1.200 (Positive and Negative); Number of levels 16.