DFRC Method for Lignin Analysis. 1. New Method for -Aryl Ether Cleavage: Lignin Model Studies Fachuang Lu and John Ralph* U.S. Dairy Forage Research Center, USDA-Agricultural Research Service, 1925 Linden Drive West, and Department of Forestry, University of WisconsinsMadison, Madison, Wisconsin 53706 A new method for selective and efficient cleavage of arylglycerol--aryl (-O-4) ether linkages in lignins is described and applied to several lignin -ether models. The term “DFRC” was coined for derivatization followed by reductive cleavage. Derivatization, accompanied by cell wall solubilization, is accomplished with acetyl bromide (AcBr); reductive cleavage of resulting -bromo ethers utilizes zinc in acetic acid. Degradation monomers, 4-acetoxycinnamyl acetates, from -ether cleavage by the DFRC method were identified by NMR, GC-MS, and comparison of GC retention times with authentic compounds. Under the conditions used in this study, the -ether linkage of all models was cleaved in very high (>92%) yield. The DFRC method produces simpler mixtures of monomers with higher yields than alternative hydrolytic methods. Because of its relative simplicity, mild conditions, and exceptional selectivity, this method should become a powerful analytical method for lignin characterization. Keywords: Acetyl bromide; lignin; lignin model compound; -aryl ether; thioacidolysis; -bromo- ether; cleavage; quantitative analysis; gas chromatography; reductive elimination; acetylation; bromination INTRODUCTION Although a great deal of progress has been made in lignin chemistry, many ambiguities remain regarding lignin structure and its cross-linking with other cell wall components (Dence and Lin, 1992; Ralph and Helm, 1993). As a highly abundant, renewable raw material that is currently underutilized, lignin has attracted increasing interest in wood chemistry, plant biochem- istry, and related fields (Fengel and Wegener, 1989; Chen, 1991). Unlike other natural polymers such as proteins, polysaccharides, and nucleic acids, which have interunit linkages susceptible to enzymic and chemical hydroly- ses, lignin contains resistant carbon-carbon and diphe- nyl ether bonds (Morohoshi, 1991; Sakakibara, 1991). It is a common practice to degrade the polymer to low molecular weight compounds in order to obtain struc- tural information. When this strategy is applied to lignins, however, significant limitations arise such as the low yield of degradation products, interference from other contaminants, and side reactions (Lapierre, 1993). Permanganate oxidation, alkaline-nitrobenzene oxi- dation, and acidolysis are traditional methods for lignin characterization, although all have shortcomings. Thio- acidolysis (Lapierre, 1993; Rolando et al., 1992) was a major improvement in achieving relatively selective cleavage reactions to form diagnostic products in good yields. Recently several iodine-containing reagents have been used in attempts to degrade lignins under milder conditions; these include trimethylsilyl iodide (Makino et al., 1990; Meshitsuka et al., 1987; Shevchen- ko and Akim, 1995), acetyl iodide (Shevchenko and Akim, 1995), pivaloyl iodide (Fukagawa et al., 1992), and dry hydrogen iodide (Shevchenko et al., 1991; Shevchenko and Akim, 1995). Complicated mixtures of unstable degradation products formed under those conditions made it difficult to isolate, identify, and quantitate degradation products. The main degradation monomer from dry HI treatment of spruce lignin, for example, was 1,3-diiodo-1-(4-hydroxy-3-methoxyphen- yl)propane which was stable for only 1-2 h (Shevchenko and Akim, 1995). Currently, thioacidolysis remains probably the most effective diagnostic method for lignin characterization (Rolando et al., 1992). However thio- acidolysis is not a simple technique to perform; it requires optimization in a user’s lab, and utilizes the malodorous ethane thiol. There remains a need to develop a more selective, simpler, and more powerful method for degradation of lignin to provide detailed information about lignin structure. During recent research on selective methods for cleavage of R-ether linkages in lignins, Figure 1 (R ) aryl), it was found that AcBr is an ideal reagent for this purpose. AcBr treatment of R,-diaryl ether lignin models in dioxane or acetic acid at room temperature resulted in complete formation of acetylated R-bromo derivatives along with selective R-ether cleavage/bro- mination (Lu and Ralph, 1996b). It is well established that -bromo ethers or esters can be reductively cleaved by zinc dust in polar solvents (Rowlands et al., 1952; Soday and Boord, 1933; Schmitt and Boord, 1932; Kato et al., 1987), or Cr(II)en complex in dimethylformamide (DMF) (Kochi et al., 1968; Greene et al., 1987) to form alkenes. Experiments have shown that the lignin derivatives from AcBr treatment possess the -bromo ether skeleton required for the reductive elimination (Lu * Author to whom correspondence should be ad- dressed [telephone (608) 264-5407; fax (608) 264-5147; e-mail jralph@facstaff.wisc.edu]. Figure 1. -Ether units, major substructures in lignins. 4655 J. Agric. Food Chem. 1997, 45, 4655-4660 S0021-8561(97)00539-6 CCC: $14.00 © 1997 American Chemical Society