Apr 06, 2023
Synthetic Procedure of Pinoresinol
- Lisa.Stanley1,
- Rui Katahira1,
- Gregg T. Beckham1
- 1National Renewable Energy Laboratory, Renewable Resources and Enabling Sciences Center
Protocol Citation: Lisa.Stanley, Rui Katahira, Gregg T. Beckham 2023. Synthetic Procedure of Pinoresinol. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpbnojlzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 19, 2022
Last Modified: August 21, 2023
Protocol Integer ID: 67085
Keywords: lignin model compounds, dimers, nuclear magnetic resonance, pinoresinol, lignin, synthesis, beta-beta linkage
Funders Acknowledgement:
U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office
Grant ID: DE-AC36-08GO28308
Disclaimer
This work was authored by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. Funding provided by U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. The views expressed herein do not necessarily represent the views of the DOE or the U.S. Government.
Abstract
A direct understanding of the degradation reaction pathways of lignin polymers in biomass is difficult due to the complexity of lignin’s structure. To overcome the difficulty, simple lignin dimeric and trimeric model compounds which include typical lignin interunit linkages are useful to clarify reaction mechanisms. The following procedure describes the synthetic procedure of a β-β dimeric lignin model compound: pinoresinol. Lignin model compounds are useful for screening the effectiveness of catalysts and microoganisms. As well as determining the effect of a treatment on the lignin fraction, in particular the effect on the degree of depolymerization in the lignin polymer.
Materials
Coniferyl alcoholSigma AldrichCatalog #223735 Step 1.1 (Can also be synthesized in a two-step reaction from ferulic acid)
AcetoneFisher ScientificCatalog #A18P-4 Step 1.1
Iron (III) chloride hexahydrateSigma AldrichCatalog #236489 Step 1.1
Ethyl acetateFisher ScientificCatalog #E145 Step 1.1 and 2.1
Sodium chlorideSigma AldrichCatalog #S9888 Step 1.1 [see Note 1]
Sodium sulfateSigma AldrichCatalog #239313 Step 1.1
Hexane mixture of isomersSigma AldrichCatalog #178918 Step 2.1
Silca gelSigma AldrichCatalog #60737 Step 2.1
Acetone-D6Cambridge Isotope Laboratories, Inc.Catalog #DLM-9-25ML Step 3.1
Chloroform-DCambridge Isotope Laboratories, Inc.Catalog #DLM-7-100 Step 3.1
Safety warnings
Almost all chemicals used for this procedure are hazardous. Read the Safety Data Sheet (SDS) for all chemicals and follow all applicable chemical handling and waste disposal procedures.
Before start
All glassware is dried in an oven set to 105ºC then cooled in a desiccator prior to use.
Synthetic Procedure
Synthetic Procedure
Figure 1. Reaction scheme for the synthesis of pinoresinol (β-β dimer).
Coniferyl alcohol (2.0782 g , 11.5 mmol) was dissolved in acetone (15 mL ) then diluted with deionized (D.I.) water (70 mL ). A solution of iron (III) chloride hexahydrate (3.4289 g , 12.7 mmol, 1.1 eq) in D.I. water (30 mL ) was then added to the reaction mixture. The mixture was stirred at room temperature for 1 hour. After an hour, the reaction was extracted five times with ethyl acetate (70 mL x5). It was then washed with a saturated solution of brine (350 mL ) [see Note 1], dried over sodium sulfate, and concentrated in vacuo.[1,2] The crude mixture was purified via flash chromatography to yield pinoresinol (0.4971 g, 24%) as well as β-O-4 (0.5507 g, 25%) and β-5 (0.261 g, 13%) dimer side products.
Figure 2. Chemical structures of β-5 and β-O-4 dimer side products.
Note
Note 1. Preparation of saturated brine solution: Fill a container partially with D.I. water. Add a spatula full of sodium chloride (NaCl) and stir until dissolved. Repeat until excess NaCl begins to settle onto the bottom of the container.
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Purification
Purification
Flash chromatography was performed using a Teledyne Isco Combiflash® NextGen 300+. Collected fractions were determined using a UV detector with wavelengths set at 254 and 280 nm. Samples were prepared by dissolving the crude material in the smallest amount of compatible solvent. Silica gel (mesh size 70-230) was then added to adsorb the material. Excess solvent was vacuum evaporated and the sample was loaded into a RediSep® Rf 25 g sample cartridge (catalog # 69-3873-240).
Pinoresinol was purified via flash chromatography. Column used was a RediSep® Silver 80 g silica gel flash column (catalog # 69-2203-380). Solvent system was hexane (Solvent A) and ethyl acetate (Solvent B). Pinoresinol was collected starting five minutes into the run using a ratio of 60% ethyl acetate: 40% hexane. The β-5 dimer side product was collected starting twenty minutes into the run using a ratio of 85% ethyl acetate:15% hexane. The β-O-4 dimer side product was collected starting thirty-two minutes into the run using a ratio of 95% ethyl acetate:5% hexane.
Figure 3. Run program from Combiflash® NextGen 300+ of pinoresinol separation as well as the β-5 and β-O-4 dimer side products.
NMR Spectroscopy
NMR Spectroscopy
Nuclear magnetic resonance (NMR) spectra are acquired in suitable deuterated NMR solvent at 25°C on a Bruker AVANCE 400MHz spectrometer equipped with a 5 mm BBO probe. Chemical shifts (δ) are reported in ppm. 1H-NMR spectra are recorded with a relaxation delay of 1.0 s and an acquisition time of 4.09 s. The acquisition parameters for 13C-NMR include a 90˚ pulse width, a relaxation delay of 1.0 s, and an acquisition time of 1.36 s. Tetramethylsilane is used as a reference.
Figure 4. 1HNMR spectrum of pinoresinol.
1H NMR (400 MHz, d6-acetone) δ 7.52 (s, 1H, ArOH), 6.99 (d, J=1.8 Hz, 2H, H2), 6.85-6.78 (m, J = 8.2, 1.7 Hz, 4H, H5/6), 4.67 (d, J = 4.1 Hz, 2H, H), 4.22 – 4.18 (m, J= 6.9, 2.0 Hz, 2H, H), 3.91 (s, 6H, OMe), 3.84 – 3.78 (m, 2H, H), 3.10 – 3.07 (m, J= 4.6, 1.8 Hz, 2H, H).
Figure 5. 13C NMR spectrum of pinoresinol.
13C NMR (100 MHz, CDCl3): 146.77 (3), 145.28 (4), 132.87 (1), 118.96 (6), 114.33 (5), 108.66 (2), 85.88 (α), 71.66 (γ), 55.94 (OMe), 54.14 (β).
β-5 1H NMR (400 MHz, d6-acetone): δ 7.67 (s, 1H, ArOH), 6.94-6.85 (m, 5H, aromatic region), 6.40 (d, J=1.6 Hz, 1H, Hα), 6.18 (dt, J=9.2, 6.6 Hz, 1H, Hβ), 5.10 (d, J=9.3 Hz, 1H, Hα'), 4.31 (d, J=5.9 Hz, 2H, Hγ), 3.86 (s, 3H, OMe), 3.85 (s, 3H, OMe), 3.84 (m, 2H, Hγ'), 3.46 (m, 1H, Hβ').
β-O-4 1H NMR (400 MHz, d6-acetone): δ 7.42 (s, 1H, ArOH), 6.91-6.87 (m, 6H, aromatic region), 6.56-6.50 (m, 1H, Hα'), 6.34-6.31 (m, 1H, Hβ'), 4.91-4.87 (m, 1H, Hα), 4.53 (s, 2H, Hγ'), 4.22 (ddd, J=5.6, 3.6, 1.4 Hz, 0.5H, OHγ), 4.08 (dt, J=7.1 Hz, 0.5H, OHγ), 3.89-3.85 (m, 2H, Hγ), 3.82 (s, 3H OMe), 3.81 (s, 3H, OMe), 3.57-3.44 (m, 0.5H, OHα), 2.92 (dd, J=7.1, 5.6 Hz, 0.5H, OHγ'), 2.77 (dd, J=8.4, 5.6 Hz, 0.5H, OHγ').
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Citations
Step 1.1
C. S. Lancefield, N. J. Westwood. The synthesis and analysis of advanced lignin model polymers
10.1039/c5gc01334hStep 1.1
Ciaran W. Lahive, Paul C. J. Kamer, C. S. Lancefield, Peter J. Deuss. An introduction to model compounds of lignin linking motifs; synthesis and selection considerations for reactivity studies
10.1002/cssc.202000989Step 3.1
S. A. Ralph, L. L. Landucci, J. Ralph. NMR Database of Lignin and Cell Wall Model Compounds
https://www.glbrc.org/databases_and_software/nmrdatabase/NMR_DataBase_2009_Complete.pdf