It must be emphasized that one of Freudenberg’s major contributions to the study of lignins was the demonstration that coniferyl alcohol (3) coupling in vitro, using a “redoxase” from mushroom, afforded the racemic products (Figure 7.17A), (±)-dehydrodiconiferyl alco­hols (68), (±)-pinoresinols (69), and (±)-threo/erythro guaiacylglycerol 8-O-4′ coniferyl alcohol ethers (71) in yields of circa 26, 13, and 9%, respectively (287). [By contrast, dehy­drogenation of sinapyl alcohol (5) mainly afforded the (±)-syringaresinols (70) (288,289).] Freudenberg then considered a gymnosperm lignin structure which was initially based on the dimeric ratios obtained for coniferyl alcohol (3) coupling, but later changed the propor­tions (ratios) to a predominance of 8- O—4 interunit linkages without further experimental verification from his own laboratory (Figure 7.3A) (12, 290).

At around the same time, Freudenberg and other researchers reported that lignins were present in other living systems, such as mosses (Polytrichum commune, Sphagnum) (11, 12, 291, 292), algae (Cystoseira barbata, Fucus vesicolosus) (293-295) and possibly in fungi (e. g., Polyporus) (296,297). The “conclusions” were largely a consequence of the inadequate technologies then available and the consequently misleading indications so obtained; there is no evidence that lignins are present in such organisms (298-301). It was also reported that native lignins and synthetic dehydropolymerisates were identical (53, 54), but once again this was not the case (31, 302). Freudenberg further reported that mistletoe (Viscum album), an angiosperm, formed a gymnosperm (G-enriched) lignin when parasitizing gymnosperms (Pinus silvestris/Abies alba), whereas an angiosperm (G-S) lignin was formed when it parasitized an angiosperm [white hawthorn (Crataegus oxyacantha)] (11, 12). This prompted him to conclude that “these are the examples of roles that lignins play in taxonomy” (11). Such claims did not, however, survive further experimental scrutiny either: Mistletoe has since been demonstrated to only biosynthesize an angiosperm (G-S) lignin through

its own biosynthetic processes (303) regardless of plant source for parasitism. The findings in these studies thus serve to illustrate the quality of the data largely being obtained in the 1950s/1960s, and which accordingly contributed to the unproven notion of random assembly.

The Freudenberg laboratory also reported that monolignol glycosides (and dimeric lig­nans) were first formed in the cambial regions of various tree species (53, 54), and that these diffused into adjacent cells. These were then considered to be hydrolyzed back to glu­cose and monolignols (from the monolignol glucosides) by action of a (3-glucosidase, with both the monolignols and the lignans subsequently employed for lignification (53, 54, 290). Goldschmid and Hergert (304) were unable to confirm such observations using western hemlock (Tsuga heterophylla), however, and later Grisebach’s group (305) established that, based on turnover experiments and pool size determinations, only a part of lignin synthesis could be attributed to coniferin (72) metabolism. Thus, in contrast to Freudenberg’s earlier assumptions, it had not in fact been determined as to how either lignin precursor transport

occurred prior to lignification, or in what form, or in what specific cells. These data, when taken together, contributed to the now long-held view of lignins being randomly assembled.

On a very different tact, experimental work undertaken on delignification of the gym — nosperm, spruce (Picea abies) using sulphite-based chemical reagents was also carried out in the Forss laboratory. These researchers noted, based on distinct molecular size chro­matographic elution profiles of lignin derivatives proper, that there were two broad classes of metabolic products solubilized (306). Forss and Fremer considered that they were de­rived from “hemi-lignins” and polydisperse higher molecular weight lignins, respectively, i. e., in a somewhat analogous (polyphenolic) structural relationship as for the polymeric hemicelluloses and cellulose in (woody) plant tissue. Subsequent chemical analyses of the “hemilignin” fraction by ourselves (147-150), however, established that they were instead hydrolytically cleavable mono-, di-, and tri-sulphonic acids, such as 38-41 (Figure 7.10A), etc., and were thus presumably cleaved from the native lignin macromolecule(s). That is, no evidence was obtained that they were distinct “hemilignin” biopolymers, as suggested by Forss and Fremer.

The higher molecular weight polydisperse sulphonated lignins examined by Forss and Fremer displayed some interesting differences in their chromatographicbehavior, as well as in their elemental analyses, suggesting the possibility of discrete molecular species. A structural basis for these differences was, however, not actually established. Nevertheless, a speculative structure emerged from their studies in the proposal of a lignin repeating unit derived from 18 monomeric [p-coumaryl (1] and coniferyl (3) alcohol] entities (structure not shown), albeit without any supporting spectroscopic evidence (307, 308). Taken together, the studies by Freudenberg and Forss had thus led to two widely divergent and highly speculative depictions or views of lignins, each of which is discussed further below.

Given the limitations of the technologies and considerations about lignin formation at that time, it is perhaps not surprising that the Freudenberg depiction of lignin structure had several fatal flaws: (i) Of the 24 aromatic monomeric subunit linkages in his model (Figure 7.3A) (11), six — then revised to five in 1968 (12) — had potentially thioacidolysis cleavable monomeric units, of which one was sinapyl alcohol (5)-derived; gymnosperms are generally not considered to biosynthesize sinapyl alcohol (5) (substructure 16 in Figure 7.3A); (ii) many of the interunit linkages now known to be present in native lignins were absent in the proposed structural depiction. Absent, for example, are the potentially cleavable thioacidolysis 3-3′, 3-5′, or 5-5′, linked dimeric entities (substructure VII, Figure 7.2D), as well as dibenzodioxocin (5-5′,8-O-4′,7-O-4′) moieties (substructure V, Figure 7.2D); (iii) Eight of the 24 aromatic moieties, linking 2/3, 13a/14a, 13c/14c, and 15/17 (Figure 7.3A) also contain substructures that have never been reported as occurring in lignins proper; yet, these accounted for one-third of the linkages shown; and (iv) Between 1965 and 1968, an 8-O-4′ interunit linkage was also replaced by an 8-1′ substructure (11, 12).

In an analogous manner, the Forss and Fremer (307) gymnosperm lignin structure also contained similar serious structural limitations; the proposed repeating unit had eight po­tentially cleavable linkages [by thioacidolysis] which would also lead to monomer release, as well as a non-cleavable ten-monomeric entity containing 3-3′, 8-3′, 8-5′, and 5-5′ carbon — carbon linkages. This linkage distribution does not agree with subsequent experimental evidence obtained to the present date.

In hindsight, the depictions of lignin structures being derived from either random or non-random assembly thus lacked a sound experimental footing, and both were premature

and incorrect representations. Nevertheless, they served as a useful historical starting point, but can be eliminated from further consideration as a reasonable facsimile for either lignin structure or as an explanation as to how lignins were being formed.

At the same time, it should be emphasized that the proposed regularity in gymnosperm lignin structure was arbitrarily dismissed, with no definitive experiments, for example, being conducted and/or designed to determine the molecular basis for the intriguing molecular size chromatographic profile differences observed. More to the point, in both the Freudenberg and Forss/Fremer lignin structural depictions, experiments had not been designed to either probe lignin structure proper, or to distinguish between “random” versus “non-random” coupling, and/or to establish how lignins were indeed formed in vivo.