Thioacidolysis

Thioacidolyis is an acidolytic degradation method used to analyze lignin subunit composition. The method relies on the use of boron trifluoride etherate ((C2H5)2-O-BF3) and ethanethiol (C2H5-SH) to cleave |3-O-4 bonds (1.82) in the lignin (4.19). The reaction mechanism is shown in Figure 4.4.

First the a-carbon is substituted by BF3, resulting in compound 4.20. The activated a-carbon makes this compound reactive with ethanethiol, resulting in the formation of 4.21. The thioethyl group now attacks the activated P-carbon of 4.21, resulting in the intermediate 4.22. Attack of ethanethiol results in the formation of 4.23, and through a similar mechanism the y-carbon is substituted, resulting in the formation of 1,2,3-trithioethane phenylpropanoid monomer 4.25 (Lapierre et al., 1986; Chen, 1991; Rolando et al., 1992; Lapierre, 1993).

The thioacidolysis reagent is prepared fresh for each analysis by mixing 2.5 mL BF3-etherate and 10 mL of ethanethiol in a flask. The volume is adjusted to 100 mL with dioxane. The cell wall extract (20 mg) is added to 10 mL of the thioacidolyis reagent, along with an internal control in order to be able to quantify the products later on. Docosane dissolved in methylene chloride is a suitable internal standard. The reaction is performed in glass tubes that can be closed with Teflon-lined screw caps. This is important, because the reagent is very odiferous. The actual thioacidolysis is performed for 4 h. at 100°C in an oil bath. Deionized water is added to the cooled reaction to get a volume of 30 mL. Sodium bicarbonate (0.4 M NaHCO3) is added to increase the pH to a value between 3 and 4. The reaction mixture is then extracted in 3 x 30 mL methylene chloride. Water is removed from the combined organic extracts by addition of Na2SO4 and the methylene chloride is removed via evaporation at 40°C. The resulting residue is redisolved in 0.5 mL methylene chloride, silylated, and injected in a GC-MS for quantitation. The internal standard is used to correct for loss of compounds during the extraction.

Thioacidolysis allows the distinction between products derived from lignin and products derived from p-coumaric and ferulic acids, and the distinction between products derived from cinnamaldehydes and cinnamyl alcohols. Recent improvements have made it possible to estimate the fraction of free phenolic groups in uncondensed lignin (see Section 1.3.1), and to depolymerize the dimers, so that they can be included in the analysis of the lignin composition.

Thioacidolysis allows the distinction between products derived from lignin and products derived from p-coumaric and ferulic acids, and the distinction between products derived from cinnamaldehydes and cinnamyl alcohols. Recent improvements have made it possible to estimate the fraction of free phenolic groups in uncondensed lignin (see Section 1.3.1), and to depolymerize the dimers, so that they can be included in the analysis of the lignin composition.

Isolation and identification of phenolic compounds HO . HO.

R5 R3

C2H5S

C2H5S

R5 R3

C2H5S, oH

C2H5S,

sc2h

R5 R3

sc2h

R5 R3

(C2H5)2O-BF3 C2H5-SH

H2O R5

sc2h:

Figure 4-4. Reaction mechanism for the formation of 1,2,3- trithioethane phenylpropanoid monomers (4.25) from lignin (4.19). R1 is either an aryl group or a hydrogen atom. In H-residues both R3 and R5 are hydrogen atoms, in G-residues R3 is a methoxyl group and R5 is a hydrogen atom, and in S-residues both R3 and R5 are methoxyl groups. R4 is either a hydrogen atom or an alkyl group. The wavy bonds indicate that both the S- and R-stereo-isomers are present.

C2H5S

Examples of the use of thioacidolysis can be found in Hoffmann et al. (2004), who describe the effects on lignin subunit composition resulting from silencing the gene encoding hydroxycinnamoyl-CoA shikimate/quinate hydroxyltransferase (HCT) in tobacco, and in O'Connell et al. (2002) who investigated the effects of down-regulating cinnamoyl-CoA reductase in tobacco on lignin subunit composition.

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