Supporting Information (S1) for
“Redox fluctuations increase the contribution of lignin to soil respiration”
Hall et al., Global Change Biology
Syntheses of 13C-labeled coniferyl alcohols used for lignin production
[methoxyl-13C]Coniferyl alcohol was synthesized in six steps: (1) regioselective monobenzylation of 3,4-dihydroxybenzaldehyde with benzyl bromide in the presence of K2CO3 and KI
in acetone at 57°C to give 4-benzyloxy-3-hydroxy-benzaldehyde, (2) methylation with 13CH3I in
the presence of K2CO3 in acetone at 43°C to give 4-benzyloxy-3-[13C]methoxybenzaldehyde, (3)
removal of the benzyl group with acetic acid/HCl (1:1) at 120°C to give 4-hydroxy-3[13C]methoxybenzaldehyde, (4) acetylation with acetic anhydride/pyridine (1:1) in CH2Cl2 at
room temperature to give 4-acetoxy-3-[13C]methoxybenzaldehyde, (5) Horner-WadsworthEmmons-Wittig olefination with triethyl phosphonoacetate in the presence of NaH in
tetrahydrofuran at 0°C to give a mixture of 3-(4-acetoxy-3-[13C]methoxyphenyl)-2-propenoic
acid ethyl ester and 3-(4-hydroxy-3-[13C]methoxyphenyl)-2-propenoic acid ethyl ester, (6)
reduction with diisobutylaluminum hydride in toluene at 0°C to afford a yellow solid.
Crystallization from CH2Cl2/hexane gave [methoxyl-13C]coniferyl alcohol as colorless needles in
a total yield of 60% based on 13CH3I). Diagnostic NMR spectra are as follows: H NMR (500
MHz, CDCl3) δ 6.91 – 6.90 (m, 1H, Ar-H), 6.89 – 6.88 (m, 1H, Ar-H), 6.85 – 6.84 (m, 1H, ArH), 6.54 – 6.50 (m, 1H, α-CH-), 6.23 – 6.18 (m, 1H, β-CH-), 5.60 (s, 1H, Ar-OH), 5.28 (s, 1H, γCH2-OH), 4.29 – 4.28 (m, 2H, γ-CH2-), 4.03 (s, 1.5H, -O13CH3), 3.74 (s, 1.5H, -O13CH3). 13C
NMR (126 MHz, CDCl3) δ 146.82, 146.81, 145.73, 145.70, 131.51, 129.38, 126.27, 120.43,
114.66, 108.52, 108.48, 63.98, 56.02.
[2-13C]Coniferyl alcohol was synthesized in two steps: (1) Horner-Wadsworth-EmmonsWittig olefination of 4-acetoxy-3-methoxybenzaldehyde with [2-13C]triethyl phosphonoacetate
in the presence of NaH in tetrahydrofuran at 0°C, followed by a gradual temperature increase to
60°C, to give 3-(4-acetoxy-3-methoxyphenyl)-2-[13C]propenoic acid ethyl ester, (2) reduction
with diisobutylaluminum hydride in toluene at 0°C to afford a yellow solid. Crystallization from
CH2Cl2/hexane gave [2-13C]coniferyl alcohol as colorless needles in a total yield of 83% based
on [2-13C]triethyl phosphonoacetate. Diagnostic NMR spectra are as follows: H NMR (500
MHz, acetone-d6) δ 7.61 (bs, 1H, Ar-OH), 7.05 – 7.04 (m, 1H, Ar-H), 6.86 – 6.84 (m, 1H, Ar-H),
6.76 – 6.75 (m, 1H, Ar-H), 6.50 – 6.47 (m, 1H, α-CH-), 6.39 – 6.34 (m, 0.5H, β-13CH-), 6.09 –
6.04 (m, 0.5H, β-13CH-), 4.20 – 4.17 (m, 2H, γ-CH2-), 3.85 (s, 3H, -OCH3), 3.76 (bs, 1H, γ-CH2OH). 13C NMR (126 MHz, acetone-d6) δ 148.40, 147.12, 130.63, 130.23, 130.05, 128.10,
121.49, 120.60, 120.56, 115.72, 109.92, 109.89, 63.56, 63.17, 56.11.
Lignin preparations
Synthetic guaiacyl lignins were produced by polymerizing the resultant coniferyl alcohols
in vitro with horseradish peroxidase (Sigma/Aldrich, type VI) (Kirk & Brunow, 1988). The
prepared synthetic lignins were fractionated by gel permeation chromatography on a 1.8 × 33 cm
column of Sephadex LH-20 in N,N-dimethylformamide to obtain a high molecular weight
fraction. The material eluting in the void volume of the column (i.e., excluded from the pores of
the Sephadex matrix, mol. wt. > 1000) (Kirk & Brunow, 1988), was used for the experiments
reported here. The collected lignin solutions were concentrated by rotary vacuum evaporation of
most of the N,N-dimethylformamide, and the lignins were then precipitated by adding the
concentrated solutions dropwise to 20 volumes of rapidly stirring distilled, deionized water. The
precipitates were collected by centrifugation (3000 × g), resuspended in distilled, deionized
water and centrifuged again likewise, resuspended once more in distilled, deionized water, and
finally lyophilized to yield in each case a beige powder. 13C NMR analysis of the lignins showed
chemical shifts and substructure frequencies consistent with those reported earlier for synthetic
guaiacyl lignin (Landucci et al., 1998).
References
Kirk TK, Brunow G (1988) Synthetic 14C-labeled lignins. Methods in Enzymology, 161, 65–73.
Landucci LL, Ralph SA, Hammel KE (1998) 13C NMR characterization of guaiacyl,
guaiacyl/syringyl and syringyl dehydrogenation polymers. Holzforschung, 52, 160–170.
Figure S1: Cumulative CO2 production for incubations of the methoxyl (a) and β-C labeled (b)
lignins, plotted by headspace treatment. Grey bars indicate anaerobic periods for the fluctuating
treatment. The extended anaerobic period for the β-C labeled lignins may have contributed to
differences in cumulative CO2 evolution in this treatment between the two incubations.
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