Selective modification of the-linkage in DDQ-treated Kraft lignin analysed by 2D NMR spectroscopy

The depolymerisation of the biopolymer lignin has the potential to provide access to a range of high value and commodity chemicals. However, research in this increasingly important area of green chemistry is hindered by the lack of analytical methods. The key challenge in using NMR is the throughput that can be achieved without the need for high field spectrometers fitted with cryoprobes. Here, we report the use of a relatively fast 2D HSQC NMR experiment performed on a 500 MHz spectrometer fitted with a BBFO+ probe to obtain high quality spectra. The use of the developed protocol to study the selective modification of the β–β linkage in Kraft lignin is also reported.


References
Electronic Supplementary Material (ESI) for Green Chemistry.This journal is © The Royal Society of Chemistry 2014

NMR methods
NMR spectra were acquired on a Bruker Avance III 600 MHz spectrometer fitted with a 5 mm CPTCI cryoprobe.The central DMSO solvent peak was used as internal reference (δ C 39.5, δ H 2.49 ppm).The 1 H, 13 C-HSQC experiment was acquired using standard Bruker pulse sequence 'hsqcetgpsp.3'(phase-sensitive gradient-edited-2D HSQC using adiabatic pulses for inversion and refocusing).Composite pulse sequence 'adiabatic' was used for broadband decoupling during acquisition.2048 data points was acquired over 12 ppm spectral width (acquisition time 142 ms) in F2 dimension using 4 scans with 1 s interscan delay and the d4 delay was set to 1.8 ms (1/4J, J = 140 Hz).The spectrum was processed using squared cosinebell in both dimesnions and LPfc linear prediction (32 coefficients) in F1.Volume integration of cross peaks in the HSQC spectra was carried out using MestReNova software.
Standard HSQC experiments For spectral width of 150 ppm 256 increments were acquired in F1 dimension (acquisition time 5.6 ms) that resulted in the total experimental time of 20 min.
Short HSQC experiments For spectral width of 40 ppm 76 increments were acquired in F1 dimension (acquisition time 6.3 ms) that resulted in the total experimental time of 6 min.HSQC experiments including the aromatic region For spectral width of 86 ppm 156 increments were acquired in F1 dimension (acquisition time 5.9 ms) that resulted in the total experimental time of 12 min.As expected, overlay of the respective 2D HSQC spectrum of eudesmin (3) and epieudesmin (S1) with the spectrum of Kraft lignin suggested that the epimer naturally occurring in lignin had the same relative configuration as eudesmin (3) (Figure S2). 2

Fig. S1
Fig. S1Summary of the ratios obtained using a 600 MHz spectrometer fitted with a cryoprobe at a concentration of 100 mg of substrate in 0.6 mL of DMSO-d6.A. Full width 2D HSQC NMR spectrum ( c / H 50-95/2.5-6.0) of isolated Kraft lignin.Contours are colour coded according to the linkage they are assigned to (see Figure2 legend).Black cross peaks currently correspond to unassigned signals; B. Table comparing the ratios of integral intensities obtained for specific linkages relative to the aromatic region using the 600 MHz spectrometer fitted with a cryoprobe (coloured in red); C. Representative comparison of the different ratios of linkages obtained for Kraft lignin using a 600 MHz (with cryoprobe) and a 500 MHz spectrometer (with BBFO+ probe).

Fig. S2
Fig. S2 Partial 2D HSQC NMR spectra ( c / H 50-95/2.5-6.0) of A. isolated Kraft lignin after stirring in DMF overnight; B. Kraft lignin after treatment with 0.25 weight equivalents of DDQ; C. Kraft lignin after treatments with 0.5 weight equivalents of DDQ; D. Kraft lignin after treatment with 0.75 weight equivalents of DDQ; E. Kraft lignin after treatment with 1 weight equivalent of DDQ.Contours are colour coded according to the linkage they are assigned to (see Figure 2 legend).Black cross peaks currently correspond to unassigned signals.NMR samples were run on a 500 MHz spectrometer at a concentration of 100 mg of substrate in 0.6 mL of DMSO-d6.

Fig. S3
Fig. S3 Overlay of partial 2D HSQC ( c / H 68-90/3.2-4.8)spectra of Kraft lignin (shown in blue and pink) and a mixture of epimers 3/S1 (shown in purple/black).Contours are colour coded according to the linkage/structure they are assigned to.For clarity the signals assigned to the C, 3 and S1 protons are shown.

Fig. S5
Fig. S5 Partial 2D HSQC NMR spectra of a mixture of Kraft lignin after treatment with 0.75 weight equivalents of DDQ (100 mg) and an authentic sample of pyran-4-one 5 (10 mg): A. aromatic region ( c / H 120-150/7.4-8.4) and B. aliphatic region ( c / H 50-90/4.0-5.6).Overlap of the cross peaks both at  c / H 147.3/8.21ppm and  c / H 52.9/4.65 ppm corresponding to the aromatic pyran-4-one 5 CH group and methylene group in 5 are shown in orange and red boxes respectively.Contours are colour coded according to the linkage they are assigned to (see Figures 2 and 5 legends).

Table S1
1ean ratios of the -O-4, - and -5 linkages, relative to the aromatic region, in Kraft lignin following treatment with different amounts of DDQ To confirm the relative configuration of the  linkage in Kraft lignin, eudesmin (3) was treated with BF 3 .Et 2 O to give a 1:1 mixture of eudesmin (3) and epieudesmin (S1) (Scheme S1).1