Lignin characterization by 2D NMR spectroscopy. NMR spectra were acquired on a Bruker Biospin AVANCE III 700 MHz spectrometer fitted with a cryogenically cooled 5-mm QCI 1H/31P/13C/15N gradient probe with inverse geometry (proton coils closest to the sample), and spectral processing used Bruker’s TopSpin 3.5pl6 (Mac) software. For NMR experiments, ball-milled whole vanilla seed coat material was swelled in DMSO-d6/pyridine-d5, isolated lignins and C–DHP (dehydrogenation polymer) were dissolved in 4:1 v/v DMSO-d6/pyridine-d5, and model compounds were dissolved in acetone-d6. The central solvent peaks were used as the internal references (δCH: DMSO, 39.5/2.49; acetone, 29.84/2.05 ppm). Standard Bruker implementations of the traditional suite of 1D and 2D [gradient-selected and 1H-detected; for example, correlation spectroscopy (COSY), 1H–13C HSQC (Fig. 1), and heteronuclear multiple-bond correlation (HMBC)] NMR experiments were used for structural elucidation and assignment authentication for monomers and dimers. Adiabatic 2D HSQC (“hsqcetgpsisp2.2”) experiments for ball-milled seed coat material in a gel state were carried out using the parameters described previously (22). Processing used typical matched Gaussian apodization in F2 (LB = −0.5; GB = 0.001) and squared cosine-bell apodization in F1.

The characterization of the vanilla seed coat C-lignin was initially consistent with the previous report (19) but belied some issues. For both the CW and its EL (derived by removing polysaccharides via crude cellulases treatment) (21), characterization revealed each lignin to be an almost 100% benzodioxane polymer with only a trace level of the resinol (β–β) structure. Although not as high as we previously reported (~80%) (19), the seed coat sample had a very high KL value (~65%). However, the 2D HSQC NMR of the so-purified lignins contained many peaks in the aliphatic region that were not from the lignin itself (fig. S1). An alternative method (below) was therefore required for lignin quantification in these materials.

C-lignin quantification by 13C NMR. Samples for quantitative 13C NMR analysis were prepared by accurately weighing predried C-LBL samples (100 mg) dissolved in 1-ml internal standard [1,3,5-trioxane, DMSO-d6 (3.12 mg/ml)] solution. The C-LBL concentration was also 100.0 mg/ml. Relaxation reagent chromium(III) acetylacetonate [Cr(acac)3; ~2 mg] was added to the samples to facilitate the relaxation of the magnetization. Quantitative 13C NMR spectroscopy was performed as previously described (43). The NMR spectra were acquired on the 700-MHz spectrometer described above. Relaxation delays were set to be ~5 times the longest T1 values of carbon signals (for inverse-gated proton decoupled 13C NMR spectra); in our case, d1 = 12.5 s was used to fully relax of all of the carbons with the aid of the relaxation reagent. For the inverse-gated proton-decoupled 13C spectrum, at least 38 hours (10K scans) were required. Spectral processing used both Bruker’s TopSpin 3.5pl6 (Mac) and MestreNova 11.0 (Mac) software. The acquired FIDs were processed typically with a 5-Hz line broadening. The central solvent peaks were used as the internal references (δCH: DMSO, 39.5/2.49 ppm). Baseline was corrected manually over the 50- to 100-ppm region using TopSpin.

13C NMR is mostly used to quantify low–molecular weight technical lignins (such as kraft lignin and organosolv lignin) or milled wood lignins (43, 44). It is difficult to quantify native lignin with 13C NMR for two reasons. One is the poor solubility of lignin, and the other is the overlapping peaks from the lignin side chain with polysaccharide peaks. However, C-LBL is a perfect sample for 13C NMR analysis. First, the lignin structure is simple; there is only one type of structure in the lignin backbone—the benzodioxane derived from β–O–4-coupling. The chemical shifts of the benzodioxane carbons are unique (75 to 80 ppm), so that there is little chance of signal overlap with other components. Second, C-lignin is acid-resistant. Unlike the S-G–type lignins, harsh acid pretreatment can be applied to C-lignin without destroying the benzodioxane structure. Thus, we can easily remove the polysaccharides by acid pretreatment, further minimizing the signal overlap problem. According to the 2D HSQC spectrum of C-lignin (fig. S1), Cα and Cβ have the potential to allow 13C NMR quantification of the phenylpropanoid unit derived from caffeyl alcohol in the C-lignin (fig. S2). Cγ cannot be used for the quantification because of the signal overlap with the unknown peaks (δH, 4.00 to 4.35 ppm; δC, 60.0 to 62.5 ppm). The aromatic region of C-lignin cannot be used for the quantification because of the overlap with signals from protein residues (tyrosine and phenylalanine) (45). Cα and Cβ may seem equally good for the C-lignin quantification; however, when looking at the HSQC spectrum at a lower contour level, peaks from polysaccharide residues cannot be completely ignored even after the acidic LiBr pretreatment; the residual C3 and C5 of the cellulose overlap with the Cβ of the C-lignin. Because the relaxation reagent Cr(acac)3 was added to reduce the experiment time, the line broadening caused by the relaxation reagent made the overlap between Cβ and the cellulose residues even worse. As a result, Cα was chosen for the quantification as it had minimal peak overlap issues. Assuming that C-lignin is derived from pure caffeyl alcohol, the detailed calculation was as shown below (table S2)Embedded ImageIn the equations, cIS (mmol/ml) is the molar concentration of internal standard (IS; 1,3,5-trioxane), AIS is the peak integral of internal standard in the quantitative 13C NMR spectrum, c (mmol/ml) is the molar concentration of caffeyl alcohol unit in the C-lignin polymer, A is the peak integral of Cβ in the quantitative 13C NMR spectrum, ρLBL (mg/ml) is the mass concentration of C-LBL sample, YCA (mmol/mg) is the mole amount of caffeyl alcohol (CA) per milligram of C-LBL, MwCA (mg/mmol) is the molecular weight of caffeyl alcohol, Wlignin(LBL) is the weight percentage of C-lignin in C-LBL, LBL% is the weight percentage of C-LBL obtained from whole CW, and Wlignin(CW) is the weight percentage of C-lignin in whole CW.