Online Resource
Towards lignin-protein crosslinking: Amino acid adducts of a lignin model quinone methide
Brett G. Diehla*, Heath D. Wattsb, James D. Kubickic, Matthew R. Regnerd, John Ralphd, and
Nicole R. Browne
a
*Corresponding author
Department of Agricultural and Biological Engineering
226 Forest Resources Building
The Pennsylvania State University
University Park, PA 16802, United States
Tel: (814) 863-3446
Email: bgd115@psu.edu
b
Department of Geosciences
303 Deike Building
The Pennsylvania State University
University Park, PA 16802, United States
Tel: (801) 865-7394
Email: hdw115@psu.edu
c
Department of Geosciences, and The Earth & Environmental Systems Institute
335 Deike Building
The Pennsylvania State University
University Park, PA 16802, United States
Tel: (814) 865-3951
Email: jdk7@psu.edu
d
Department of Biochemistry, and DOE Great Lakes Bioenergy Research Center
Wisconsin Energy Institute
Madison, WI 153726, United States
Tel: (608) 890-2429
Email: jralph@wisc.edu, mregner@gmail.com
e
Department of Agricultural and Biological Engineering
209 Agricultural Engineering Building
The Pennsylvania State University
University Park, PA 16802, United States
Tel: (814) 865-7423
Email: nrb10@psu.edu
S1
Model compound properties
QM-Cys, 3 (2-tert-Butoxycarbonylamino-3-[3-hydroxy-1-(4-hydroxy-3-methoxy-phenyl)-2-(2methoxy-phenoxy)-propylsulfanyl]-propionic acid methyl ester). Pale white oil (yield: 73% after
purification). Theoretical mass: 537.20 g/mol (+ H+: 538.21 g/mol). Observed m/z + H+: 538.21.
Major isomer (86%): 1H NMR (400 MHz, acetone-d6): δ = 1.39 (9H, s, H7), 2.82 (2H, m,
H1), 3.53 (1H, m, Hγ), 3.67 (3H, s, H4), 3.74 (1H, m, Hγ), 3.80 (3H, s, OMeB), 3.87 (3H, s,
OMeA), 4.34 (1H, t, J = 5.78, Hα), 4.42 (1H, m, H2), 4.65 (1H, m, Hβ), 6.77 (1H, m, HA5), 6.85
(1H, m, HB6), 6.90 (1H, m, HB4), 6.94 (1H, m, HA6), 6.97 (1H, m, HB5), 7.05 (1H, m, HB3),
7.34 (1H, m, HA2). Minor isomer (14%): 1H NMR (400 MHz, acetone-d6): δ = 1.42 (9H, s,
H7), 2.74 (2H, m, H1), 3.58 (1H, m, Hγ), 3.65 (1H, s, H4), 3.72 (1H, m, Hγ), 3.79 (3H, s,
OMeB), 3.86 (3H, s, OMeA), 4.55 (1H, m, Hβ). Major isomer (86%): 13C NMR (75.5 MHz,
acetone-d6): δ = 28.50 (C7), 33.70 (C1), 51.41 (Cα), 52.40 (C4), 54.13 (C2), 56.11 (OMeA),
56.25 (OMeB), 62.01 (Cγ), 79.54 (C6), 83.87 (Cβ), 113.51 (CA2), 113.78 (CB5), 114.80 (CA5),
117.64 (CB3), 121.61 (CB6), 122.79 (CB4), 123.37 (CA6), 130.87 (CA1), 146.78 (CA4), 148.03
(CA3), 149.07 (CB1), 151.50 (CB2), 155.95 (C5), 172.20 (C3). Minor isomer (14%): 13C
NMR (75.5 MHz, acetone-d6): δ = 113.63 (CA2), 115.41 (CA5), 117.88 (CB3), 121.79 (CB6),
131.00 (CA1), 156.33 (C5). Major isomer (86%): 1H NMR (400 MHz, DMSO-d6/pyridined5): δ = 1.34 (9H, s, H7), 2.66-2.86 (2H, m, H1), 3.43 (1H, m, Hγ), 3.56 (3H, s, H4), 3.58 (1H,
m, Hγ), 3.70 (3H, s, OMeA), 3.78 (3H, s, OMeB), 4.18-4.27 (1H, m, H2), 4.33 (1H, m, Hα), 4.70
(1H, m, Hβ), 5.08 (1H, s, γ-OH), 6.76 (1H, m, HA5), 6.85 (1H, m, HB5), 6.88 (1H, m, HB4),
6.91 (1H, m, HA6), 6.92 (1H, m, HB6), 7.10 (1H, m, HB3), 7.34 (1H, m, HA1), 7.39 (1H, d, J =
8.07, NH), 9.19 (1H, s, A4-OH). 13C NMR (75.5 MHz, DMSO-d6/pyridine-d5): δ = 28.09
(C7), 32.23 (C1), 50.27 (Cα), 51.93 (C4), 53.47 (C2), 55.35 (OMeB), 55.74 (OMeA), 60.68 (Cγ),
81.78 (Cβ), 112.74 (CB6), 113.53 (CA2), 114.60 (CA5), 115.34 (CB3), 120.78 (CB5), 121.44
(CB4), 122.48 (CA6), 129.22 (CA1), 130.90 (C6), 146.05 (CA4), 147.43 (CA3), 148.01 (CB1),
149.87 (CB2), 155.37 (C5), 171.78 (C3). Minor isomer (14%): 1H NMR (400 MHz, DMSOd6/pyridine-d5): δ = 4.35 (1H, m, Hα), 4.61 (1H, m, Hβ). 13C NMR (75.5 MHz, DMSOd6/pyridine-d5): δ = 28.12 (C7), 32.42 (C1), 50.60 (Cα), 51.97 (C4), 53.69 (C2), 60.96 (Cγ),
81.65 (Cβ), 155.63 (C5), 171.74 (C3).
QM-Lys, 4 (2-Acetylamino-6-[3-hydroxy-1-(4-hydroxy-3-methoxy-phenyl)-2-(2-methoxyphenoxy)-propylamino]hexanoic acid methyl ester). Pale white oil (yield: quantitative, no
purification necessary). Theoretical mass: 504.25 g/mol (+ H+: 505.25 g/mol). Observed m/z +
H+: 505.25. 1H NMR (300 MHz, acetone-d6): δ = 1.4 (4H, m, H2, H3), 1.52 (2H, m, H4), 1.90
(3H, s, H7), 2.41 (2H, m, H1), 3.50 (1H, m, Hγ), 3.63 (3H, s, H9), 3.69 (1H, m, Hγ), 3.79 (3H, s,
OMeA), 3.86 (3H, s, OMeB), 3.97 (1H, d, J = 6.82 Hz, Hα), 4.18 (1H, m, Hβ), 4.38 (1H, m, H5),
6.77 (1H, m, HB6), 6.85 (1H, m, HA6), 6.93 (1H, m, HA5), 6.96 (1H, m, HB5), 6.96 (1H, m,
HB4), 7.09 (1H, m, HA2), 7.14 (1H, m, HB3), 7.39 (1H, d, J = 7.47, acetyl-NH). 13C NMR
(75.5 MHz, acetone-d6): δ = 22.55 (C7), 24.09 (C3), 30.24 (C2), 32.34 (C4), 47.34 (C1), 52.03
(C9), 52.97 (C5), 56.09 (OMeA), 56.09 (OMeb), 62.19 (Cγ), 64.62 (Cα), 87.12 (Cβ), 111.96
(CA2), 113.16 (CB5), 115.35 (CB6), 118.98 (CB3), 121.75 (CA6), 121.75 (CB4), 122.99 (CA5),
133.12 (CA1), 146.70 (CA4), 148.33 (CA3), 149.45 (CB1), 151.63 (CB2), 170.11 (C6), 173.60
(C8). 1H NMR (300 MHz, DMSO-d6/pyridine-d5): δ = 1.21 (4H, m, H2, H3), 1.58 (2H, m,
S2
H4), 1.88 (3H, s, H7), 2.31 (2H, m, H1), 3.45 (1H, m, Hγ), 3.59 (3H, s, H9), 3.67 (1H, m, Hγ),
3.71 (3H, s, OMeA), 3.79 (3H, s, OMeB), 3.90 (1H, d, J = 6.82 Hz, Hα), 4.21 (1H, m, Hβ), 4.27
(1H, m, H5), 5.00 (1H, s, γ-OH), 6.76 (2H, m, HA5, HA6), 6.84 (1H, m, HB5), 6.90 (1H, m,
HB4), 6.95 (1H, m, HB6), 6.98 (1H, m, HA2), 7.15 (1H, m, HB3), 8.30 (1H, d, J = 7.47, acetylNH), 9.12 (1H, s, A4-OH). 13C NMR (75.5 MHz, DMSO-d6/pyridine-d5): δ = 23.24 (C7),
23.26 (C3), 29.21 (C2), 30.94 (C4), 46.61 (C1), 51.62 (C9), 52.04 (C5), 55.41 (OMeA), 55.46
(OMeb), 60.82 (Cγ), 63.00 (Cα), 85.89 (Cβ), 111.66 (CA2), 112.29 (CB6), 115.15 (CA5), 116.99
(CB3), 120.71 (CA6), 120.78 (CB5), 121.57 (CB4), 131.4 (CA1), 145.83 (CA4), 147.58 (CA3),
148.60 (CB1), 149.93 (CB2), 169.56 (C6), 172.96 (C8).
QM-His, 5a (2-tert-Butoxycarbonylamino-3-{3-[3-hydroxy-1-(4-hydroxy-3-methoxy-phenyl)-2(2-methoxy-phenoxy)-propyl]-3H-imidazol-4-yl}-propionic acid methyl ester) and QM-His, 5b
(2-tert-Butoxycarbonylamino-3-{1-[3-hydroxy-1-(4-hydroxy-3-methoxy-phenyl)-2-(2-methoxyphenoxy)-propyl]-1H-imidazol-4-yl}-propionic acid methyl ester) (Note: Some of the NMR shift
assignments for this compound were based on interpretation of the 2D HMQC and HMBC NMR
spectra because, as noted above, the QM-His products could not be chromatographically
separated from a lignin QM dimer, leading to some shift degeneracy in the 1H and 13C 1D
spectra.) Pale white oil (yield: 45% by NMR). Theoretical mass: 571.25 g/mol (+ H+: 572.26
g/mol). Observed m/z + H+: 572.26. 1H NMR (400 MHz, acetone-d6): 1.35 (9H, s, H12), 2.97
(2H, m, H6), 3.44 (1H, m, Hγ), 3.54 (3H, s, H9), 3.56 (1H, m, Hγ), 3.63 (6H, s, OMe), 4.37 (1H,
m, H7), 4.96 (1H, m, Hβ), 5.66 (1H, m, Hα), 6.74 (1H, m, HA5), 6.77 (1H, m, HB3), 6.86 (1H,
m, HB4), 6.88 (1H, m, HA6), 6.89 (1H, m, HB5), 6.90 (1H, m, H5), 7.06 (1H, m, HB6), 7.11
(1H, m, HA2), 7.55 (1H, s, H5), 7.81 (1H, s, H2). 13C NMR (75.5 MHz, acetone-d6): 27.80
(C12), 29.40 (C6), 51.00 (C9), 54.06 (C7), 55.60 (OMe), 59.90 (Cγ), 60.97 (Cα), 78.40 (C11),
81.45 (Cβ), 111.40 (CB6), 111.60 (CA2), 112.60 (CB5), 115.00 (CA5), 116.56 (C5), 117.30
(C4), 120.20 (CA6), 120.80 (CB3), 122.40 (CB4), 129.80 (CA1), 135.20 (C5), 138.00 (C2),
147.50 (CA3), 147.60 (CB1), 147.80 (CB4), 150.80 (CB2), 155.40 (C10), 172.60 (C8). 1H NMR
(400 MHz, DMSO-d6/pyridine-d5): 1.33 (9H, m, H12), 2.89 (2H, m, H6), 3.46 (3H, s, OMeA),
3.47 (2H, m, Hγ), 3.47 (3H, s, H9), 3.67 (3H, s, OMeB), 4.37 (1H, m, H7), 5.02 (1H, m, Hβ),
5.70 (1H, m, Hα), 6.76 (1H, m, HA5), 6.76 (1H, m, H5), 6.82 (1H, m, HA6), 6.89 (1H, m, HB5),
7.05 (1H, m, HB3), 7.08 (1H, m, HB6), 7.13 (1H, m, HA2), 7.13 (1H, m, HB4), 7.62 (1H, s,
H2), 7.81 (1H, d, J = 8.42 Hz, acetyl-0NH), 9.23 (1H, s, A4-OH). 13C NMR (75.5 MHz,
DMSO-d6/pyridine-d5): 28.04 (C12), 29.77 (C6), 51.20 (C9), 53.98 (C7), 55.45 (OMeA), 55.63
(OMeβ), 59.31 (Cγ), 60.20 (Cα), 78.29 (C11), 80.20 (Cβ), 111.62 (CB6), 111.81 (CA2), 112.47
(CB5), 114.80 (C5), 114.83 (CA5), 116.60 (CB3), 120.69 (CA6), 130.00 (CA1), 134.98 (C2),
136.44 (C4), 147.20 (CA4), 147.88 (CB1), 148.05 (CA3), 150.66 (CB2), 155.41 (C10), 172.63
(C8).
QM-Asp, 6 (2-tert-Butoxycaronylamino-succinic acid 1-benzyl ester 4-[3-hydroxy-1-(4hydroxy-3-methoxy-phenyl)-2-(2-methoxy-phenoxy)-propyl] ester). Pale white oil (yield: 58%
after purification). Theoretical mass: 625.25 g/mol (+ Na+: 648.24 g/mol). Observed m/z + Na+:
S3
648.24. Major isomer (74%): 1H NMR (400 MHz, acetone-d6): δ = 1.37 (9H, s, H6), 2.92
(2H, m, H2), 3.6 (1H, m, H3), 3.65 (1H, m, Hγ), 3.75 (1H, m, Hγ), 3.80 (3H, s, OMeB), 3.84
(3H, s, OMeA), 4.61 (1H, m, Hβ), 5.10 (2H, s, H8), 6.06 (1H, d, J = 4.42, Hα), 6.82 (1H, m,
HA5), 7.03 (1H, m, HB3), 6.84 (1H, m, HB5), 6.94 (1H, m, HA6), 6.94 (1H, m, HB4), 6.96 (1H,
m, HB6), 7.17 (1H, m, HA2), 7.34 (5H, m, H10, H11, H12, H13, H14), 7.65 (1H, s, A4-OH).
13C NMR (75.5 MHz, acetone-d6): δ = 28.42 (C6), 37.13 (C2), 51.23 (C3), 56.20 (OMe ),
A
56.16 (OMeB), 61.18 (Cγ), 67.37 (C8), 75.82 (Cα), 79.57 (C5), 83.60 (Cβ), 112.22 (CA2),
113.48 (CB6), 115.14 (CA5), 119.10 (CB3), 121.65 (CA6), 121.65 (CB5), 128.69 (CB4), 128.72
(CA1), 129.18 (C10-C14), 136.81 (C9), 147.32 (CA3), 147.97 (CA4), 151.75 (CB1), 151.80
(CB2), 156.11 (C4), 169.98 (C1), 171.71 (C7). Minor isomer (26%): 1H NMR (400 MHz,
acetone-d6): δ = 4.51 (1H, m, Hβ), 6.14 (1H, m, Hα). 13C NMR (75.5 MHz, acetone-d6): δ =
76.41 (Cα), 84.62 (Cβ), 119.27 (CB3), 169.81 (C1), 171.67 (C7). Major isomer (74%): 1H
NMR (400 MHz, DMSO-d6/pyridine-d5): δ = 1.33 (9H, s, H6), 2.82 (2H, m, H2), 3.57 (1H, m,
Hγ), 3.66 (1H, m, Hγ), 3.70 (3H, s, OMeB), 3.76 (3H, s, OMeA), 4.55 (1H, m, H3), 4.67 (1H, m,
Hβ), 5.09 (2H, s, H8), 6.04 (1H, m, Hα), 6.81 (1H, m, HA5), 6.83 (1H, m, HB3), 6.86 (1H, m,
HB5), 6.89 (1H, m, HA6), 6.91 (1H, m, HB6), 7.09 (1H, m, HA2), 7.11 (1H, m, HB4), 7.37 (5H,
m, H10, H11, H12, H13, H14), 7.48 (1H, d, J = 3.42, NH), 9.36 (1H, s, A4-OH). 13C NMR (75.5
MHz, DMSO-d6/pyridine-d5): δ = 27.98 (C6), 35.60 (C2), 50.24 (C3), 55.44 (OMeA), 55.54
(OMeB), 59.63 (Cγ), 65.98 (C8), 74.75 (Cα), 78.45 (C5), 81.39 (Cβ), 111.96 (CA2), 112.76
(CB6), 114.84 (CA5), 116.77 (CB4), 120.69 (CA6), 120.69 (CB3), 120.69 (CB5), 127.19 (CA1),
127.53 (C12), 127.66 (C13), 127.78 (C11), 127.95 (C14), 128.29 (C10), 135.98 (C9), 144.32
(C4), 146.65 (CA4), 147.30 (CA3), 147.67 (CB1), 150.12 (CB2), 169.12 (C1), 171.79 (C7).
Minor isomer (26%): 1H NMR (400 MHz, DMSO-d6/pyridine-d5): δ = 4.55 (1H, m, Hβ),
6.11 (1H, m, Hα). 13C NMR (75.5 MHz, DMSO-d6/pyridine-d5): δ = 75.08 (Cα), 82.70 (Cβ),
112.58 (CB6), 116.87 (CB4), 147.27 (CA3), 150.16 (CB2), 168.92 (C1).
QM-Glu, 7 (2-tert-Butoxycaronlyamino-pentanedioic acid 1-tert-butyl ester 5-[3-hydroxy-1-(4hyroxy-3-methoxy-phenyl)-2-(2-methoxy-phenoxy)-propyl] ester). Pale white oil (yield: 47%
after purification). Theoretical mass: 605.28 g/mol (+ Na+: 628.27 g/mol). Observed m/z + Na+:
628.25. Major isomer (74%): 1H NMR (400 MHz, acetone-d6): δ = 1.42 (9H, s, H10), 1.46
(9H, s, H7), 1.92, 2.08 (2H, m, H3), 2.47 (2H, t, J = 8.02, H2), 3.69, 3.79 (2H, m, Hγ), 3.84 (3H,
s, OMeB), 3.88 (3H, s, OMeA), 4.08 (1H, m, H4), 4.62 (1H, m, Hβ), 5.78 (1H, s, γ-OH), 6.06
(1H, d, J = 5.11, Hα), 6.79 (1H, m, HA5), 6.85 (1H, m, HB5), 6.94 (1H, m, HA6), 6.94 (1H, m,
HB4), 6.96 (1H, m, HB6), 7.02 (1H, m, HB3), 7.17 (1H, m, HA2). 13C NMR (75.5 MHz,
acetone-d6): δ = 27.69 (C3), 28.07 (C10), 28.49 (C7), 31.19 (C2), 54.57 (C4), 56.21 (OMeA),
56.21 (OMeB), 61.27 (Cγ), 75.23 (Cα), 79.17 (C9), 81.46 (C6), 83.89 (Cβ), 112.29 (CA2),
113.55 (CB6), 115.15 (CA5), 119.02 (CB3), 121.68 (CB5), 121.68 (CA6), 123.31 (CB4), 129.32
(CA1), 147.32 (CA4), 147.96 (CA3), 148.97 (CB1), 151.78 (CB2), 156.40 (C5), 171.93 (C1),
174.01 (C8). Minor isomer (26%): 1H NMR (400 MHz, acetone-d6): δ = 4.53 (1H, m, Hβ),
6.13 (1H, m, Hα). 13C NMR (75.5 MHz, acetone-d6): δ = 75.93 (Cα), 84.85 (Cβ), 115.53
(CA5), 119.26 (CB3), 129.72 (CA1), 147.53 (CA4), 148.20 (CA3), 172.10 (C1). Major isomer
S4
(74%): 1H NMR (400 MHz, DMSO-d6/pyridine-d5): δ = 1.34 (9H, s, H10), 1.36 (9H, s, H7),
1.83 (2H, m, H3), 2.39 (2H, m, H2), 3.58, 3.66 (2H, m, Hγ), 3.71 (3H, s, OMeB), 3.76 (3H, s,
OMeA), 3.93 (1H, m, H4), 4.67 (1H, m, Hβ), 5.10 (1H, s, γ-OH), 6.02 (1H, m, Hα), 6.81 (1H, m,
HA5), 6.82 (1H, m, HB3), 6.85 (1H, m, HB5), 6.89 (1H, m, HA6), 6.92 (1H, m, HB6), 7.08 (1H,
m, HA1), 7.10 (1H, m, HB4), 7.28 (1H, d, J = 7.86, NH), 9.34 (1H, s, A4-OH). 13C NMR (75.5
MHz, DMSO-d6/pyridine-d5): δ = 26.04 (C3), 27.48 (C10), 28.05 (C7), 30.36 (C2), 53.60
(C4), 55.45 (OMeA), 55.57 (OMeB), 59.71 (Cγ), 74.23 (Cα), 78.09 (C6), 80.37 (C9), 81.58 (Cβ),
111.91 (CA2), 112.77 (CB6), 114.90 (CA5), 116.73 (CB4), 120.02 (CB3), 120.56 (CB5), 120.70
(CA6), 127.53 (CA1), 146.61 (CA4), 147.30 (CA3), 147.72 (CB1), 150.13 (CB2), 155.65 (C5),
171.20 (C8), 171.38 (C1). Minor isomer (26%): 1H NMR (400 MHz, DMSO-d6/pyridine-d5):
δ = 1.97 (2H, m, H3), 4.56 (1H, m, Hβ), 6.08 (1H, m, Hα). 13C NMR (75.5 MHz, DMSOd6/pyridine-d5): δ = 26.31 (C3), 30.13 (C2), 53.86 (C4), 74.68 (Cα), 78.00 (C6), 80.24 (C9),
82.64 (Cβ), 111.35 (CA2), 115.21 (CA5), 128.32 (CA1), 146.72 (CA4), 147.47 (CA3), 148.42
(CB1), 149.98 (CB2), 154.74 (C5), 171.65 (C1).
QM-Ser, 8 (2-Benzyloxycarbonylamino-3-[3-hydroxy-1-(4-hydroxy-3-methoxy-phenyl)-2-(2methoxy-phenoxy)-propoxy]-propionic acid methyl ester). Pale white oil (39% after
purification). Theoretical mass: 555.21 g/mol (+ Na+: 578.20 g/mol). Observed m/z + Na+:
578.20. Major isomer (68%): 1H NMR (400 MHz, acetone-d6): δ = 3.65 (2H, m, H1), 3.70
(3H, s, H4), 3.76 (3H, s, OMeB), 3.81 (3H, s, OMeA), 3.81 (2H, m, Hγ), 4.32 (1H, m, Hβ), 4.43
(1H, m, H2), 4.60 (1H, m, Hα), 5.09 (2H, m, H6), 6.80 (1H, m, HB6), 6.82 (1H, m, HB3), 6.88
(1H, m, HA5), 6.89 (1H, m, HA6), 6.89 (1H, m, HB4), 6.92 (1H, m, HB5), 7.00 (1H, m, HA2),
7.15, (1H, m, H10), 7.38 (5H, m, H8, H9, H11, H12). 13C NMR (75.5 MHz, acetone-d6): δ =
52.35 (C4), 55.59 (C2), 56.20 (OMe), 61.74 (Cγ), 66.87 (C6), 69.92 (C1), 82.25 (Cα), 85.28
(Cβ), 111.70 (C10), 112.00 (CA2), 113.49 (CB5), 115.23 (CB6), 119.27 (CA5), 121.73 (CA6),
122.03 (CB3), 123.09 (CB4), 128.67 (C8, C12), 129.22 (C9, C11), 130.62 (CA1), 138.05 (C7),
147.23 (CA4), 148.17 (CA3), 149.20 (CB1), 151.76 (CB2), 157.06 (C5), 171.87 (C3). Minor
isomer (32%): 1H NMR (400 MHz, acetone-d6): δ = 3.72 (2H, m, Hγ), 3.79 (3H, s, OMeB),
3.83 (3H, s, OMeA), 4.55 (1H, m, Hα). 13C NMR (75.5 MHz, acetone-d6): δ = 55.42 (C2),
61.33 (Cγ) 69.47 (C1), 82.40 (Cα), 85.68 (Cβ), 111.94 (CA2), 119.15 (CA5), 130.19 (CA1),
147.29 (CA4), 148.95 (CB1), 156.95 (C5). Major isomer (68%): 1H NMR (400 MHz, DMSOd6/pyridine-d5): δ = 3.39 (1H, m, Hγ), 3.56-3.65 (2H, m, H1), 3.65 (3H, s, H4), 3.67 (1H, m,
Hγ), 3.65 (3H, s, OMeB), 3.72 (3H, s, OMeA), 4.41 (1H, m, H2), 4.46 (1H, m, Hβ), 4.59 (1H, m,
Hα), 5.08 (2H, s, H6), 6.73-6.83 (1H, m, HA5), 6.74-6.80 (1H, m, HA6), 6.80-6.87 (1H, m
HB4), 6.81 (1H, m, HB6), 6.82-6.93 (1H, m, HB5), 6.94-7.07 (1H, m, HA2), 6.97-7.06 (1H, m,
HB3), 7.25-7.38 (5H, m, H7-H12), 7.81 (1H, d, J = 8.42, NH), 9.23 (1H, s, A4-OH). 13C NMR
(75.5 MHz, DMSO-d6/pyridine-d5): δ = 54.20 (C1), 55.29 (OMeA), 55.38 (OMeB), 51.81 (C4)
60.08 (Cγ), 65.80 (C6), 67.90 (C1), 80.91 (Cα), 82.50 (Cβ), 111.48 (CA2), 112.75 (CB5), 114.82
(CA5), 115.06 (CB6), 116.38 (CB4), 120.57 (CA6), 120.59 (CB3), 127.80-128.38 (C7-C12),
128.72 (CA1), 137.05 (C7), 146.36 (CA4), 147.84 (CB1), 149.55 (CB2), 149.93 (CA3), 156.30
(C5), 170.81 (C3). Minor isomer (32%): 1H NMR (400 MHz, DMSO-d6/pyridine-d5): δ =
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4.42 (1H, m, Hβ), 4.55 (1H, m, Hα). 13C NMR (75.5 MHz, DMSO-d6/pyridine-d5): δ = 80.69
(Cα), 82.90 (Cβ), 111.84 (CA2), 115.90 (CB4), 128.54 (CA1).
QM-Tyr, 9 (2-tert-Butoxycarbonlyamino-3-{4-[3-hydroxy-1-(4-hydroxy-3-methoxy-phenyl)-2(2-methoxy-phenoxy)-propoxy]-phenyl}-propionic acid methyl ester). Pale white oil (yield: 45%
after purification). Theoretical mass: 597.26 g/mol (+ H+: 598.27 g/mol). Observed m/z + H+:
598.29. 1H NMR (300 MHz, acetone-d6): δ = 1.33 (9H, d, J = 3.28 Hz, H13), 2.88 (1H, m, H7),
3.00 (1H, m, H7), 3.62 (3H, d, J = 3.74, H10), 3.78 (3H, s, OMeB), 3.81 (3H, s, OMeA), 3.81
(1H, m, Hγ), 3.91 (1H, m, Hγ), 4.31 (1H, m, H8), 4.55 (1H, m, Hβ), 5.13 (1H, d, J = 5.13, Hα),
6.07 (1H, d, J = 7.66, γ-OH), 6.78 (1H, m, HA5), 6.81 (1H, m, HB5), 6.87 (2H, m, H3, H5), 6.93
(1H, m, HB4), 6.95 (2H, m, HB3, HB6), 6.97 (1H, m, HA6), 7.06 (2H, m, H2, H6), 7.17 (1H, m,
HA2), 7.55 (1H, s, Ph-OH). 13C NMR (100 MHz, acetone-d6): δ = 28.42 (C13), 37.29 (C7),
52.07 (C10), 56.02 (C8), 56.17 (OMe), 61.19 (Cγ), 79.22 (C12), 79.42 (Cα), 85.40 (Cβ), 112.09
(CA2), 113.50 (CB6), 115.29 (CA5), 116.81 (C3, C5), 119.21 (CB5), 121.43 (CA6), 121.70
(CB3), 123.22 (CB4), 130.32 (CA1), 130.82 (C2, C4, C6), 147.12 (CA4), 148.13 (CA3), 149.10
(CB1), 151.82 (CB2), 156.10 (C11), 157.77 (C1), 173.22 (C9). 1H NMR (400 MHz, DMSOd6/pyridine-d5): δ = 1.27 (9H, s, H13), 2.71-2.95 (2H, m, H7), 3.62 (3H, s, H4), 3.67 (3H, s,
OMeB), 3.71 (3H, s, OMeA), 3.73 (1H, m, Hγ), 3.55 (3H, s, H10), 3.79 (1H, m, Hγ), 4.18 (1H, m,
H8), 4.66 (1H, m, Hβ), 5.08 (1H, s, γ-OH), 5.49 (1H, d, J = 4.20, Hα), 6.76 (1H, m, HA5), 6.81
(3H, m, HB3, H3, H5), 6.84 (1H, m, HB5), 6.89 (2H, m, HA6, HB6), 7.05 (2H, m, H2, H6), 7.06
(1H, m, HB4), 7.11 (1H, m, HA2), 9.21 (1H, s, A4-OH). 13C NMR (75.5 MHz, DMSOd6/pyridine-d5): δ = 28.10 (C13), 35.50 (C7), 51.60 (C10), 55.47 (OMeB), 55.50 (C8), 55.60
(OMeA), 59.78 (Cγ), 78.26 (C12), 78.30 (Cα), 82.69 (Cβ), 112.02 (CA2), 112.81 (CB6) 115.01
(CA5), 115.79 (C3, C5), 116.43 (CB4), 120.73 (CA6), 120.75 (CB3), 121.57 (CB5), 128.42
(CA1), 129.65 (C2, C6), 129.96 (C4), 146.35 (CA4), 147.37 (CA3), 148.03 (CB1), 150.06
(CB2), 156.30 (C1), 172.56 (C9).
QM-Thr, 10 (2-tert-Butoxycarbonylamino-3-[3-hydroxy-1-(4-hydroxy-3-methoxy-phenyl)-2-(2methoxy-phenoxy)-propoxy]-butyric acid methyl ester). Not experimentally observed. DFTcalculated 1H NMR (syn-isomer, DMSO force field): 0.8 (H1), 3.2 (Hβ), 3.3 (Hγ), 3.3 (H3),
3.5 (OMeB), 3.6 (Hγ), 3.6 (H2), 3.6 (OMeA), 4.4 (Hα), 6.1 (HB6), 6.7 (HA5), 6.7 (HB3), 6.7
(HB5), 6.9 (HA2), 6.9 (HA6), 7.0 (HB4). DFT-calculated 13C NMR (syn-isomer, DMSO force
field): 15.2 (C1), 52.1 (OMeA), 52.1 (OMeB), 55.9 (Cγ), 60.7 (C3), 69.2 (C2), 70.7 (Cα), 88.5
(Cβ), 109.0 (CA2), 112.4 (CB3), 113.4 (CA5), 121.5 (CB5), 123.6 (CA6), 124.5 (CB6), 125.7
(CB4), 131.4 (CA1), 144.5 (CA4), 145.8 (CA3), 145.9 (CB1), 151.9 (CB2). DFT-calculated 1H
NMR (anti-isomer, DMSO force field): 0.9 (H1), 3.1 (Hγ), 3.2 (H3), 3.3 (H2), 3.3 (OMeA), 3.4
(Hβ), 3.5 (OMeB), 3.9 (Hγ), 4.3 (Hα), 4.8 (HB6), 6.4 (HB5), 6.6 (HB3), 6.8 (HA5), 6.8 (HA6),
6.8 (HB4), 7.2 (HA2). DFT-calculated 13C NMR (anti-isomer, DMSO force field): 51.9
(OMeB), 52.6 (OMeA), 73.7 (Cα), 60.9 (Cγ), 90.4 (Cβ), 112.1 (CB3), 113.9 (CA5), 114.3 (CA2),
121.2 (CA6), 121.5 (CB5), 122.4 (CB6), 123.8 (CB4), 130.1 (CA1), 144.8 (CA4), 146.1 (CA3),
149.3 (CB2), 149.3 (CB1).
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QM-Hyp, 11 (4-[3-Hydroxy-1-(4-hydroxy-3-methoxy-phenyl)-2-(2-methoxy-phenoxy)propoxy]-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester). Not experimentally
observed. DFT-calculated 1H NMR (syn-isomer, DMSO force field): 3.2 (Hβ), 3.5 (OMeB),
3.6 (OMeA), 4.7 (Hα), 3.4 (Hγ), 3.5 (Hγ), 6.0 (HB6), 6.1 (H2), 6.6 (HA5), 6.7 (HB3), 6.7 (HB5),
6.8 (HA6), 7.0 (HB4), 7.2 (HA2), 7.2 (H4). DFT-calculated 13C NMR (syn-isomer, DMSO
force field): 52.0 (OMeB), 52.2 (OMeA), 56.3 (Cγ), 80.5 (Cα), 87.7 (Cβ), 105.1 (C3), 109.8
(CA2), 112.4 (CB3), 112.5 (C1), 113.0 (CA5), 119.1 (C4), 121.6 (CB5), 122.3 (CA6), 124.5
(CB6), 125.5 (CB4), 132.7 (CA1), 144.1 (CA4), 145.3 (CA3), 147.1 (CB1), 147.6 (C2), 151.2
(CB2). DFT-calculated 1H NMR (anti-isomer, DMSO force field): 3.4 (OMeA), 3.4 (Hγ), 3.5
(OMeB), 3.6 (Hβ), 3.7 (Hγ), 4.0 (H2), 4.6 (Hα), 4.8 (HB6), 6.1 (H4), 6.5 (HB5), 6.6 (HB3), 6.8
(HA5), 6.8 (HA6), 6.9 (HB4), 7.2 (HA2). DFT-calculated 13C NMR (anti-isomer, DMSO
force field): 51.9 (OMeB), 52.4 (OMeA), 60.9 (Cγ), 78.7 (Cα), 90.9 (Cβ), 95.7 (C3), 99.8 (C1),
112.2 (CB3), 112.3 (C4), 113.0 (CA2), 113.9 (CA5), 119.5 (CA6), 121.8 (CB5), 122.4 (CB6),
124.1 (CB4), 131.8 (CA1), 144.5 (CA4), 145.9 (CA3), 139.0 (C2), 149.1 (CB1), 149.2 (CB2).
Supplemental Fig. 1 Overlaid HMQC side chain regions of compounds 3 and 5. The α- and βshifts are labeled; methoxyl and γ-shifts are not labeled due to substantial shift degeneracy. Grey
shifts are non-diagnostic. DFT calculated α-shifts (red squares) and β-shifts (blue squares) are
shown for compounds 3, 5a and 5b (both threo and erythro stereoisomers are shown). Calculated
13
C shifts correlate relatively well with experimentally observed 13C shifts, though not well
enough to allow for assignment of stereochemistry in the experimentally observed product shifts.
Calculated 1H shifts are underestimated by about 0.5-1.0 ppm. Further research is necessary to
refine the predicative abilities of DFT for 1H shifts of lignin compounds.
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Supplemental Table 1 Regression statistics for δ13C DFT NMR and experimental 13C NMR of
compound 3.
slope
intercept
R2
MUE
RMSE
max error
Compound 3 (threo)
1.01
-0.46
0.9972
2.0
2.7
7.0
Compound 3 (erythro)
1.01
-0.19
0.9969
1.9
2.8
8.0
1H, 13C,
HMQC, and HMBC Spectra
Single and multi-dimensional NMR spectra of compounds 3-9 (below) were collected in
acetone-d6 as described in the Experimental section of the manuscript. NMR spectra were also
collected in DMSO-d6/pyridine-d5 (4:1 v/v) because it is a preferred lignin and plant cell wall
NMR solvent system; full shift assignments in this solvent are listed above and discussed in the
main text. In general, negligible shift migration was observed between the acetone-d6 and
DMSO-d6/pyridine-d5 solvent systems. HMQC spectra are shown in blue and HMBC spectra in
red. Only the aliphatic shifts of the HMQC spectra are labeled (aromatic shifts are reported
above); only the α-1 cross-peaks of the HMBC spectra, indicative of QM-amino acid
crosslinking, are labeled.
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