Alkaline NBO. NBO was performed as previously described (46). Dimeric model compound D1 (5 mg) or extracted vanilla seed coat (40 mg) was mixed with nitrobenzene (0.4 ml) and 2 M NaOH (7 ml) in a 10-ml stainless steel reactor vessel (Taiatsu Techno Co.) and heated at 170°C for 2 hours in an oil bath. The reactor was then cooled in ice water, and 1 ml of freshly prepared ethyl vanillin (3-ethoxy-4-hydroxybenzaldehyde; 5 mg/ml) in 0.1 M NaOH solution was added to the reaction mixture as an internal standard. The mixture was transferred to a 100-ml separatory funnel and washed three times with 15 ml of DCM. The remaining aqueous layer was acidified with 2 M HCl until the pH was below 3.0 and extracted with 2 × 20 ml of DCM and 20 ml of diethyl ether. The combined organic layers were washed with DI water (20 ml) and dried over MgSO4. After filtration, the filtrate was collected in a 100-ml pear-shaped flask and dried under reduced pressure. For the TMS derivatization step, NBO products were transferred with pyridine (3 × 200 μl) into a GC vial, and N,O-bis(trimethylsilyl)trifluoroacetamide [BSTFA; 100 μl] was added. The mixture was heated to 50°C for 30 min. The silylated NBO products were analyzed by GC-MS and quantified by GC-FID using calibration curves (fig. S3, A and B).

Thioacidolysis followed by Raney nickel desulfurization. Thioacidolysis was performed as previously described (47). The thioacidolysis reagent was prepared freshly by adding 2.5 ml of EtSH and 0.7 ml of BF3 etherate to a 25-ml volumetric flask containing 20 ml of distilled 1,4-dioxane and then complemented with dioxane to exactly 25 ml. Freshly made thioacidolysis reagent (4.0 ml) was added to a 5-ml screw-cap reaction vial containing extractive-free CW (40 mg) or model compound (15 mg) and a magnetic stir bar. The vial cap was screwed on tightly, and the vial was kept in an oil bath containing a heating block at 100°C for 4 hours with stirring. After the reaction, the vial was cooled in an ice-water bath for 2 min. A solution of 4,4′-ethylidenebisphenol in dioxane was prepared and used as an internal standard. The product mixture was transferred to a separatory funnel and 10 ml of saturated NaHCO3 solution, along with internal standard solution, was added. Then, 5 ml of 1 M HCl solution was added to adjust the pH of the solution to below 3. The aqueous layer was extracted three times with 20 ml of DCM, and the combined organic phase was washed with saturated NH4Cl, dried over anhydrous MgSO4, and evaporated under reduced pressure at 40°C. The resulting products were desulfurized via Raney nickel. Briefly, the thioacidolysis products were dissolved in 3 ml of distilled dioxane with 1 ml of Raney nickel 3202 (Sigma-Aldrich) slurry. The mixture was heated at 80°C for 2 hours. After the reaction, nickel powder was removed using a magnet, and the reaction mixture was transferred quantitatively with DCM into a separatory funnel charged with 10 ml of NH4Cl and 10 ml of DCM. Then, 5 ml of 1 M HCl solution was added to adjust the pH of the solution to below 3. The aqueous layer was extracted twice with 10 ml of DCM, and the combined organic phase was washed with brine, dried over anhydrous MgSO4, and evaporated under reduced pressure at 40°C. For the TMS derivatization step, products were transferred with pyridine (3 × 200 μl) into a GC vial, and BSTFA (100 μl) was added. The mixture was heated to 50°C for 30 min. The silylated thioacidolysis products were analyzed by GC-MS and quantified by GC-FID using calibration curves (fig. S3, C and D).

Hydrogenolysis. Hydrogenolysis was performed as previously described (7). In cases in which isolated C-LBL was used as a feedstock, 200 mg of C-LBL was dissolved in 30 ml of methanol or dioxane/water (9:1, v/v) or THF/water (96:4, v/v) in a 100-ml high-pressure Parr reactor along with 100 mg of catalyst (5 wt % Pt/C, Pd/C, or Ru/C). The reactor was stirred with a mechanical propeller and heated via a high-temperature heating jacket. Once closed, the reactor was purged three times and then pressurized with H2 (40 bar, 4 MPa). The reactor was heated to the desired temperature and then held at that temperature for the specified residence time. After the reaction was completed, the reactor was cooled in a water bath to room temperature. The resulting liquid was filtered through a nylon membrane filter (0.8 μm, 47 mm; Whatman) and washed with EtOH. The solvent was removed under reduced pressure at 40°C with a rotary evaporator. The crude products were dissolved in EtOH and made up to 10 ml in a volumetric flask. A 1 ml of aliquot was transferred into three 5-ml vials and then dried under reduced pressure. The dried samples were used for GC, GPC, and NMR analyses. For GC sample preparation, the sample was dissolved in 0.9 ml of pyridine and 0.1 ml of BSTFA, incubated at 50°C for 30 min, and then subjected to GC-FID and GC-MS. For NMR sample preparation (fig. S4A), the sample was dissolved in 0.6 ml of DMSO-d6/pyridine-d5 (4:1, v/v) and then transferred to a 5-mm NMR tube for NMR. For GPC sample preparation (fig. S4B), the sample was dissolved in 1 ml of dimethylformamide (DMF) containing 0.1 M LiBr.

For the cases in which hydrogenolysis was performed directly on the CW material, 200 mg of preextracted vanilla seed coat was mixed with 30 ml of methanol and 100 mg of the catalyst (5 wt % Pt/C, Pd/C, or Ru/C). The remaining procedure was performed as described above.

For the cases in which the lignin model compound was used as the feedstock, a solution of 50 mg of dimer D1 in 30 ml of methanol or dioxane/water (9:1, v/v) was mixed with 50 mg of the catalyst (5 wt % Pt/C). The remaining procedure was performed as described above.

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