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Lepidimoide is a naturally occurring disaccharide reported to be an oligosaccharin, i.e. to exhibit ‘hormone-like’ biological activity. It was found in cress (Lepidium sativum) root exudates and exerts apparently allelopathic effects on neighbouring Amaranthus seedlings. In the present study the effect of cress root exudates on hypocotyl and root length of Amaranthus caudatus and Lactuca sativa was studied. The seedlings of both species grown with Lepidium sativum seedlings had longer hypocotyls and shorter roots as compared to the control. In this study I found an active principle with biological effects similar to those of lepidimoide to be more abundant in cress seed-coat mucilage than in root exudates. The active principle peaked 24 hours after seed soaking, and thereafter plateaued. I also for the first time confidently proved that the bioactive compound(s) were exuded by cress and were not microbial digestion products or seed treatment chemicals. Quantitative tests of cress root exudates and cress seed-coat mucilage showed the presence of hexoses, pentoses, uronic acids and unsaturated uronic acid. The presence of unsaturated uronic acid might be of interest because the known structure of lepidimoide includes an unsaturated uronic acid. Active principle from mucilage was partitioned into the aqueous phase when shaken with ethyl acetate at pH 2, 6.5 and 12, showing it to be hydrophilic, unlike auxins and gibberellins. The mucilage was also heated at 130°C for 48 h and severe heating did not affect its biological activity, suggesting that if the compound is lepidimoide then it is heat-resistant. In an attempt to test whether the compound is of high or low Mr, the mucilage was partitioned into 75% ethanol-precipitated and non-precipitated fractions. The biological activity in the non-precipitated fraction was very high, and was further separated by gel-permeation chromatography (GPC). GPC on Bio-Gel P-10 and P-2 suggested that the active principle had Mr ~500–750, compatible with oligosaccharide(s), suggesting that a particular oligosaccharide may be the active principle. TLC separation of bioactive fractions from P-2 showed that the bioactive compound migrated between GalA and Gal but co-migrated with sucrose; however, paper chromatography separation proved that the compound is not sucrose and might be a different disaccharide (lepidimoide). From the structure of lepidimoide, Fry et al. (1993) proposed that lepidimoide is formed by the lyase-catalysed cleavage of a pectic polysaccharide, rhamnogalacturonan-I (RG-I). So I tried to prepare lepidimoide or lepidimoide-like compounds by the action of RG-I lyase from Pichia pastoris on purified potato RG-I. The lyase showed its activity but the digest did not demonstrate biological activity, which might be due to presence of tris-HCl buffer in the solution. An attempt was also made to prepare lepidimoide by methyl esterification and -elimination of purified potato RG-I but again the product did not show any biological activity, which might be due to presence of borate buffer in the solution. This part of research might be useful for future work on preparation of lepidimoide and lepidimoide-like compounds.

Woody plants are the most abundant renewable resources on the earth. From the paper we consume to the house we live in, our daily lives rely heavily on woody plants. Over the past decades, enormous efforts have been expended to improve the utilization of fiber and wood. For example, much research has been conducted to develop environmentally benign, and economically feasible techniques for pulp and papermaking. The economical conversion of wood to useful sugars and alcohol has also been the subject of intensive research. Investigations aimed at the genetic manipulation of wood growth to better meet our needs are also underway. Nonetheless, harsh pulping and bleaching conditions are still required in the pulp and paper industry, and the bioconversion of polysaccharides in biomass to alcohol is still too expensive. An argument could be put forth that a major reason for this is the lack of basic knowledge concerning the structural and biochemical characteristics of the plant cell wall. The three major polymeric components of plant cell walls, cellulose, hemicellulose and lignin, are intimately associated with one another. Cellulose is associated with hemicellulose via non-covalent linkages, whereas lignin is theorized to be associated with cellulose and hemicellulose via both covalent and non-covalent linkages. The nature of associations between wood polymers is still poorly understood. However, it is these intimate associations that make delignification difficult, and make the bioconversion of polysaccharides to alcohol inefficient. It is also believed that the linkages between lignin and polysaccharides are responsible for the reduced digestibility of grasses by ruminants. Besides cellulose, hemicellulose and lignin, there are many secondary metabolites such as lignans, neolignans, tannins and terpenoids. The structures of lignans and neolignans are analogous to the interunits of lignin. Lignin is considered an optically inactive polymer, whereas lignans and neolignans are optically active small molecules. Although it has been proposed that the biosynthesis of lignin, lignans and neolignans are via the same oxidative coupling mechanism, it is still unclear that how the plant cell wall differentiates the formation of lignans, neolignans and lignin. How and why plant cell wall generates so many lignans and neolignans having broad structural variation is also unknown. As a matter of fact, it is still uncertain which enzymes are actually involved in the biosynthesis of lignin. A better understanding of biosynthetic pathways of lignin, lignans and neolignans is a prerequisite for the genetic manipulation of plant growth. Investigations described in this dissertation were an effort to better understand the fundamental aspects of covalent linkages between lignin and hemicellulose in wood. Enantiomeric synthesis of neolignans provides a tool for investigating the optically active nature of neolignans, and may be helpful to study the biosynthetic pathways of neolignans. Chapter | describes chemical structures of wood components and the biosynthesis of lignin, lignans and neolignans. The mechanisms of lignin-carbohydrate bond formation are also discussed, and a concise review of lignin-carbohydrate linkages proposed in the literature concludes Chapter 1. Chapter 2 presents the methods used in investigating covalent linkages in wood, which include methods of isolating lignin-carbohydrate complexes, chemical cleavage methods, DDQ oxidation and model compound/NMR methods. The synthesis of plant cell wall model compounds and neolignans are reviewed in Chapter 3. The experimental work performed for the completion of this thesis is described in Chapters 4-8. A method which provides β-𝘖-4 lignin model dimers with complete threo stereospecificity is described in Chapter 4. This method is complementary to the current method for the preparation of erythro lignin model dimers. Chapter 5 presents a practical synthesis of methyl 4-𝘖-methy] α-D-glucopyranosiduronic acid. Methyl 4-𝘖-methyl-α-D-glucopyranosiduronic acid was prepared from methyl α-D-glucopyranoside in 4 steps (74% overall yield). Previous preparations of this compound were much lengthier, and had very low overall yields. Chapter 6 deals with the synthesis and rearrangement reactions of ester-linked lignin-carbohydrate model compounds. A series of ester-linked lignin-carbohydrate model compounds were synthesized, and migration of the uronosy] group between the primary (γ) and benzyl (α) position of lignin side chain is discussed. Several approaches to synthetic neolignans are described in Chapter 7. Chapter 8 presents a novel approach for the preparation of chiral aryl alkylethers. The successful application of this novel approach to synthesis of several optically active 8-𝘖-4 neolignans and a 1,4- benzodioxane neolignan is described, as is the introduction of an alkyl] aryl ether bond in carbohydrate molecules. Some of the material of this dissertation has been reported in the following papers: 1. Li, K. and Helm, R. F. Approaches to Synthetic Neolignans. J. Chem. Soc. Perkin Trans 1. Accepted. 6. Li, K. and Helm, R. F. Use of Carbohydrates as Building Blocks to Synthesize Neolignans. 211th ACS National ACS Meeting, New Orleans, March 24-28, 1996. CELL-079. 2. Li, K. and Helm, R. F. A Practical Synthesis of Methyl 4-𝘖-Methylα-D-Glucopyranosiduronic Acid. Carbohydr. Res. 273(1995), 249-253. 3. Li, K. and Helm, R. F. Synthesis and Rearrangement Reactions of Ester-Linked Lignin-Carbohydrate Model Compounds. J. Agric. Food Chem. 48(1995), 2098-2103. 4. Helm, R. F. and Li, K. Complete threo Stereospecificity for the Preparation of β-𝘖-4 Lignin Model Dimers. Holzforschung. 49(1995), 533-536. 5. Helm, R. F. and Li, K. Synthesis and Rearrangement Reactions of Lignin-uronic Acid Model Compounds Related to Hardwood Cell Wall Structure. The 8th International Symposium on Wood and Pulping Chemistry. Helsinki, Finland, June 1995, vol. 1, pp107-114. 7. Li, K. and Helm, R. F. Approaches to Synthetic Neolignans, 34th National Organic Symposium, Williamsburg, VA. June 11-15, 1995. Poster 281. / Ph. D.

Chemical components in wood contain multiple anionic groups, including carboxyl groups and hydroxyl groups. During kraft cooking and bleaching, such structures are also formed, degraded or modified by the action of reactions both deliberate and unwanted. It has previously been found by Laine that anionic groups with pKa values of 3.3 and 5.5 can explain the observed anionic charge on chemical fibres, corresponding to carboxylic acids on hemicelluloses as well as oxidized lignin structures, respectively. It has further been found that most of the fibre charge can be accounted for through the contribution from methylglucuronic acids as well as hexenuronic acids. This study aimed to provide additional information regarding the charge component unaffiliated with either of the aforementioned uronic acids by studying kraft pulps procured from an industrial kraft mill corresponding to unit operations through a fibreline using the ODHot(EOP)D1D2 bleaching sequence. Each sample had its total charge determined by means of conductometric titration, its content of methylglucuronic acid determined by means of methanolysis followed by GC, and its content of hexenuronic acids determined by means of the HUT-method for the determination of hexenuronic acids, followed by either UV absorbance measurements or calculations based on the reduction in kappa number during the selective acid hydrolysis. The lignin content was determined by means of kappa number analysis as well as by calculations based on the kappa number after selective acid hydrolysis, which was assumed to be characteristic of the kappa number addition from the lignin content of the samples. The hexenuronic acid content was successfully determined according to the HUT-method. A difference in content was observed when results for the unbleached and oxygen delignified samples that was obtained from UV measurements were compared with the observed difference in kappa number prior and after selective acid hydrolysis for said samples. In both cases, the results based on the difference in kappa number was higher. No correlation between remaining charge and lignin content could be made. It was instead observed that individual bleaching steps had binary effects on each of the analysed components of the fibre charge. A significant increase in charge unaffiliated with methylglucuronic acid or hexenuronic acid was observed during the EOP step, indicating an oxidation of chemical structures in the pulp: this effect could not be correlated to lignin content. / Kemisk massa kan ses som ett kompositmaterial bestående av cellulosa, hemicellulosa och lignin. Dessa beståndsdelar, liksom separata lågmolekylära föreningar, innehåller anjoniska kemiska grupper som bidrar till att ge träfibrer en anjonisk laddning. Ytterligare sådana grupper kan bildas under reaktioner vid kokning och blekning, tillika kan sådana grupper modifieras eller brytas ner. Laine har tidigare visat att fiberladdningen kan förklaras genom två skilda kategorier av kemiska grupper med pKa värden på 3,3 samt 5,5, vilka kan kopplas till karboxylsyragrupper på hemicellulosor respektive till lignin. Man har därtill funnit att stora delar av fiberladdningen kan förklaras genom tillskott från de två uronsyrorna metylglukuronsyra och hexenuronsyra. Denna studie syftar till att undersöka det laddningstillskott som inte kan länkas till någon av dessa två uronsyror. Detta har gjorts genom att studera kemiska massor från industrin, tagna från de olika stegen i en bleksekvens av typen ODHot(EOP)D1D2. Massornas totalladdning mättes medelst konduktometrisk titrering, emedan metylglukuronsyrahalten mättes via methanolysis följt av GC och hexenuronsyrahalten mättes via selektiv syrahydrolys per HUT-metoden följt av UV-karakterisering respektive beräkningar baserade på differensen i kappa tal före och efter sagda hydrolys. Ligninhalten i samtliga prov mättes via kappatalsmätningar samt genom beräkningar baserade på kappatalet efter selektiv syrahydrolys, vilket anses vara kappatalsbidraget från ligninföreningarna. Hexenuronsyrahalterna uppmätta medelst UV-karakterisering respektive kappatalsdifferens jämfördes mot varandra och fanns ge olika resultat för de oblekta och syrgasdelignifierade massorna. I bägge fallen gav mätningen baserade på kappatalsdifferens högre utslag. Ingen korrelation mellan kvarvarande laddning - totalladdning minus tillskottet från uronsyrorna - och ligninhalt kunde observeras. Istället observerades binära effekter för varje uppmätt komponent under vardera blekningssteg. Under EOP-steget observerades ett tillskott till den kvarvarande laddningen, vilket skulle kunna förklaras genom oxidation av trästrukturer. Dock kan ej heller detta relateras till ligninhalten i proverna.