Study On Chemical Synthesis Of Chitin Oligosaccharide Analogues β-1,3-N-Acetyl-Glucosamine Oligosaccharides And Their Induced Resistance Of Plants To Diseases

Through chemical synthesis, a series of novel Chitin oligosaccharide analogues,β-1, 3-N-acetyl-glucosamine oligosaccharides, with different degree of polymerization, were designed and synthesized by glucosamine as starting material. The induced resistance of cucumbers and tobacco to the diseases by the synthesizedβ-1, 3-N-acetyl-glucosamine oligosaccharides together with methyl glycoside Lewis^a trisaccharide and three Chitin oligosaccharides were investigated, and three enzyme activities in the tobacco plants induced by the five synthetic oligosaccharides was tested and analyzed.The main results were as follows:1. Chemical Synthesis ofβ-1, 3-N-acetyl-glucosamine oligosaccharidesSynthesis ofβ-1, 3-N-acetyl-glucosamine disaccharide: The phthalic group was used for both compounds 7 and 8 as amino protecting group, and the benzylidene was used to protect the hydroxy groups at 4, 6 position. The glycosylation reaction was proceeded, catalyzed with NIS-TfOH, using compound 8 as donor,and compound 7 as acceptor, So a novel chitin oligosaccharide analogue,β-1, 3-N-acetyl-glucosamine disaccharide 9 was obtained. The stereochemistry of the newly introduced glycosidic linkage was determined to beβon the basis of the ¹H NMR of H-1', H-2' coupling constant (J= 8.40 Hz).Synthesis ofβ-1, 3-N-acetyl-glucosamine trisaccharide and tetrasaccharide:Compound 14 and 20 could be readily prepared from disaccharide 9 and trisaccharide 15 by deacetylation at the 3' or 3" position with NaOMe solution. The compound 14 and 20, with a free hydroxy group in C-3' or C-3", respectively, and a methyl group at C-1, could be used as acceptors ; and compound 8, with a thiophenyl as leaving group at the reducing terminal, could be used as donor. The glycosylation reaction was performed and gave the expected trisaccharide 15 and tetrasaccharide 21, respectively. The stereochemistry of the newly introduced glycosidic linkage was determined to beβon the basis of the ¹H NMR of H-1", H-2" or H-1''', H-2''' coupling constant (J= 8.48 Hz, J = 8.40 Hz, respectively). Synthesis ofβ-1, 3-N-acetyl-glucosamine pentasaccharide: The compound 8, protected at C-3 with acetyl, and at the reducing terminal with thiophenyl, was treated first with BF₃·Et₂O/HgO to give 25 with a free hydroxyl at the reducing terminaln, followed by treatment with Cl₃CCN/DBU to give trichloroacetimidate 26 with an active leaving group at reducing terminal. Condensation of 26 with the previously described 5, in the presence of TMSOTf and dichloromethane, gaveβ(1→3) linked disaccharide 27. The glycosylation of 20 with donor 27 was achieved under the conditions described above, providing the desired pentasaccharide 28. The stereochemistry of the newly introduced linkage of 27 and 28 was determined to beβon the basis of the coupling constant (J= 8.54 Hz, J= 8.37 Hz, respectively).Deprotection ofβ-1, 3-N-acetyl-glucosamine oligosaccharides: Acetic acid hydrolysis, PTSA hydrolysis, and catalyzed hydrogenation were used to remove the 4, 6-O-benzylidene protective groups. Acetic acid hydrolysis could cause decompose of saccharide, catalyzed hydrogenation was fit for the disaccharide 9, trisaccharide 15 and tetrasaccharide 21, but was not applicable for the pentasaccharide 28 to which the PTSA hydrolysis was proved to be feasible. The compounds were then treated with hydrazine hydrate and water in boiling ethanol, to remove the phthalic groups. Finally the peracetylation was performed by acetic anhydride and pyridine, followed by de-O-acetylation to giveβ-1, 3-N-acetyl-glucosamine disaccharide 13 and trisaccharide 19; theβ-1, 3-N-acetyl-glucosamine tetrasaccharide 24 and pentasaccharide 31 were obtained using a selective N-acetylation by acetyl anhydride in dichloromethane and methanol.Structure confirmation ofβ-1, 3-N-acetyl-glucosamine oligosaccharides: Allsynthesized novel compounds were characterized and confirmed by ¹H NMR, ¹³C NMR and HRMS, and the total yields for the di-, tri-, tetra- and pentasaccharide were 10.56%,3.08%,1.28% and 0.33%, respectively. Being different with the natural chitin oligosaccharide, these four synthesized chitin oligosaccharide analogues have a backbone of 1→3 linked structure, with a degree of polymerization from 2 to 5. These compounds could be applied to study the relationship between induced resistance to plant diseases and the molecular structures of tested Chitin oligosaccharides and their analogues.2. Induced Resistance of Cucumber to Fusarium Wilt Disease by synthesizedβ-1, 3-N-acetyl-glucosamine oligosaccharides Inhibition on mycelial growth by three chemosynthesised oligodaccharides:The inhibition in vitro on mycelial growth to five plant pathogens were showed that there was no inhibition on the mycelial growth by these three oligosaccharides.Induced Resistance of cucumber in seedling phase to Fusarium Wilt : Incucumber seedling phase, after treating with five different concentrations and two methods (Foliar spaying and irrigating roots), only the plants induced byβ-1, 3-N-acetyl-glucosamine disaccharide andβ-1, 3-N-acetyl-glucosamine trisaccharide at 10μg/ml by foliar spraying showed resistance to the wilt disease.Induced Resistance of cucumber in radicle phase to Fusarium Wilt Disease: Inradicle phase, the cucumber plants, roots of which were soaked withβ-1, 3-N-acetyl-glucosamine disaccharide andβ-1, 3-N-acetyl-glucosamine trisaccharide solution, showed resistance to the wilt disease. The effective concentration of the oligosaccharides was at least 5μg/ml.3. Induced Resistance of tobacco to the diseases by synthesizedβ-1, 3 -N-acetyl-glucosamine oligosaccharides andβ-1, 4-N-acetyl-glucosamine oligosaccharidesInduced resistance of tobacco in vivo to black shank (Phytophthora parasUica van nicotianae) : Induced resistance of tobacco to black shank disease treated by seven synthesized oligosaccharides at three different concentrations (1μg/ml 10μg/ml and 100μg/ml) was investigated. The tobacco treated byβ-1, 3-N-acetyl-glucosamine tetrasaccharide,β-1, 4-N-acetyl-glucosamine trisaccharide andβ-1, 4-N-acetyl -glucosamine tetrasaccharide showed resistance to the disease. The effective concentration of oligosaccharides for induction was 10μg/ml mostly.Induced resistances of tobacco detached leaves to black shank disease and brown spot disease: The tobacco plants were treated by seven tested oligosaccharides at concentration of 10μg/ml, and then inoculated with Phytophthora parasitica or Alternatia alternataon the detached leaves. The tobacco treated byβ-1, 3-N-acetyl-glucosamine tetrasaccharide,β-1, 4-N-acetyl-glucosamine trisaccharide, andβ-1, 4-N-acetyl-glucosamine tetrasaccharide showed resistance to Phytophthora parasUica var. nicotianae; The tobacco treated byβ-1, 3-N-acetyl-glucosamine trisaccharide,β-1, 3-N-acetyl-glucosamine tetrasaccharide,β-1, 4-N-acetyl-glucosamine trisaccharide andβ-1, 4-N-acetyl-glucosamine tetrasaccharide showed resistance to Alternatia alternate.4. The relationship between the molecular structures of the synthesized oligosaccharides and induced resistance of plants to diseasesThe backbone linked structure and sugar residues of methylated Lewis^a trisaccharide: All the treatments with methylated Lewis^a trisaccharide showed no resistance to both cucumber and tobacco. That would be due to the difference of backbone linkage structure and sugar residues between methylated Lewis^a trisaccharide and Chitin oligosaccharides / Chitin oligosaccharide analogues. Compared with documents, it showed that the backbone linkage structures were the main factors influencing the induced resistance. However, the effect of sugar residues can not be excluded.The substituents on the reducing terminal C-1: Although there was a substituent methyl on the reducing terminal C-1 of all the seven tested oligosaccharides, the induced resistance by these oligosaccharides was diverse. It was saggested that the substituent methyl on the C-1 was not the key factor for the induced resistance by the seven tested oligosaccharides.The degree of polymerization of Chitin oligosaccharides / Chitin oligosaccharide analoguesThe effective degree of polymerization (DP) of Chitin oligosaccharides / Chitin oligosaccharide analogues to induce plant resistance was varied with the type of oligosaccharides and with the plant-disease systems. That of Chitin oligosaccharide analogues for the induced resistance to cucumber Fusarium wilt disease, to tobacco brown spot disease and to tobacco black shank disease was at least 2, 3 and 4. The effective degree of polymerization of Chitin oligosaccharides for the induce resistance to tobacco brown spot disease and black shank disease was at least 3.The backbone linkage structure of Chitin oligosaccharides / Chitin oligosaccharide analogues: Bpth Chitin oligosaccharide and Chitin oligosaccharide analogues showed induced resistance to cucumber and/or tobacco. It can be concluded that induced resistance was not limited in the structure of Chitin oligosaccharide; Chitin oligosaccharide analogues were another kind of N-acetyl-glucosamine oligosaccharides, which show induced resistance to plants.The induced resistance characteristics of Chitin oligosaccharides and Chitin oligosaccharide analogues: the induced resistance by Chitin oligosaccharide analogues and Chitin oligosaccharides appeared in both induced and non-induced leaves as a systemic induced resistance, and it was also no-specific.5. The physiological mechanism of tobacco induced resistance ofβ-1, 3-N-acetyl-glucosamine oligosaccharides andβ-1, 4-N-acetyl-glucosamine oligosaccharidesDynamics of three defense enzymes, POD, PPO and PAL, in the tobacco plants induced by five tested oligosaccharides: The enhanced activity of POD, PPO and PAL would be one of mechanisms of the induced resistance. The tobacco plants treated withβ-1, 3-N-acetyl-glucosamine tetrasaccharide andβ-1, 4-N-acetyl-glucosamine tetrasaccharide showed significantly higher activity of POD, PPO and PAL in comparison to the control, and reached the peak after 48h with induced efficiencies in enzyme activity of 40% and 99%; 238% and 186%; 81% and 73%, respectively. Most of the total enzymes activities induced by other three saccharides were similar to the enzyme activity of control tobacco.The differences of induced efficiencies in the enzyme activity of the five tested oligosaccharides displayed in these aspects: the total enzyme activity level, the time to reach enzyme activity peak, the enzyme activity peak value. There was relativity between the enzymes activity change of POD, PPO, PAL and Induced resistances of tobacco to Phytophthora parasitica var. nicotianae disease.