几丁寡糖结构类似物β-1，3-乙酰氨基葡聚糖的化学合成及其诱导植物抗病性的研究
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摘要
本论文以氨基葡萄糖为起始原料，采用化学合成的方法，设计合成了一系列不同聚合度的几丁寡糖结构类似物β-1，3-乙酰氨基葡聚糖，并测定了几丁寡糖及其结构类似物对植物抗病性的诱导作用，对几丁寡糖及其结构类似物诱导处理后烟草植株体内抗性相关酶系活性动态进行了检测和分析，试图探讨上述寡糖类诱导物分子结构与其诱导抗病性的关系。
主要的研究结果如下：
1．几丁寡糖结构类似物β-1，3-乙酰氨基葡聚糖的化学合成
β-1，3-乙酰氨基葡聚二糖的合成：以氨基葡萄糖为化学合成的起始原料，用邻苯二甲酰基保护化合物7和8的2位氨基；以苄叉来保护其4，6位的羟基，避免了糖化反应发生在4位上，从而保证了糖苷键的连接具有特定的立体和区域选择性(β-1，3连接糖苷键)；化合物8还用硫烃基保护异头碳上羟基，使硫糖苷作为糖基供体，成为合成乙酰氨基葡聚糖的离去基团；化合物8的3位再用乙酰基加以保护，避免它同时作为糖基供体和糖基受体，只能充当糖基供体；由3位是自由羟基、异头碳上为甲氧基的化合物7作为糖基受体，以NIS-TfOH作催化剂，7和8发生糖基化反应，高区域、高立体选择性、高产率地合成了β-1，3连接的二聚糖9，其核磁共振氢谱也证实了新产生的糖苷键为β构型(J=8.40 Hz)。
β-1，3-乙酰氨基葡聚三糖和四糖的合成：将二聚糖9及随后得到的三聚糖15脱去3′或3″位的乙酰基，得到的化合物14和20，它们再作为糖基受体，与糖基供体8在NIS-TfOH催化下进行糖基化反应，即可得到β-1，3连接的乙酰氨基葡聚三糖15和乙酰氨基葡聚四糖21。核磁共振氢谱显示新产生的糖苷键为β构型(J=8.48Hz和J=8.40Hz)。
β-1，3-乙酰氨基葡聚五糖的合成：化合物8先转化为异头碳为游离羟基的化合物25，然后又与三氯乙腈加成形成端基活化中间体三氯乙酰亚胺酯26。糖基供体26与3位为自由羟基，硫烃基保护异头碳的糖基受体5发生缩合反应，形成双糖27；27又作为糖基供体与糖基受体20反应生成五聚糖28。其核磁共振氢谱显示新产生的糖苷键为β构型(J=8.54 Hz和J=8.37 Hz)。
β-1，3-乙酰氨基葡聚糖的脱保护：分别尝试了醋酸水解、催化氢化以及对甲苯磺酸(PTSA)水解的方法来脱去苄叉保护基，醋酸会使得乙酰氨基葡聚糖分解，催化氢化适用于二糖9、三糖15以及四糖21的脱保护，但五糖28在催化氢化时反应不完全，只能使用对甲苯磺酸的水解。邻苯二甲酰氨基的脱保护，均采用水合肼在水溶性的乙醇中回流，得到游离的氨基中间体；之后，或者直接全乙酰化，将全乙酰化的产物分离纯化后再脱去乙酰基即得到纯度较高的二糖和三糖的终产物13和19；或者游离的氨基中间体直接选择性乙酰化，得到终产物24和31，再分离纯化。
β-1，3-乙酰氨基葡聚糖的结构确定：化学合成的4个β-(1→3)-2-脱氧-2-乙酰氨基葡聚糖，聚合度分别是从2—5，其准确的化学结构均通过高分辨质谱以及核磁共振~1H NMR、~(13)C NMR的分析确证。上述4个化合物的总收率分别为10.56％、3.08％、1.28％和0.33％。与天然的几丁寡糖不同，合成的4个乙酰氨基葡聚糖均采取了β-1→3糖苷键的连接方式，是几丁寡糖的结构类似物，其结构清楚、组分单一，从而提供了研究几丁寡糖诱导植物抗病活性与寡糖区域异构体之间关系的理想材料。
化学合成的四个几丁寡糖结构类似物的化学结构Chemical structure of the four synthesized chitin oligosaccharide analogues
2．β-1，3-乙酰氨基葡聚糖诱导黄瓜对枯萎病的抗性研究
对5种植物病原真菌菌丝生长的抑制作用：在实验室条件下，分别测定了3个供试寡糖即β-1，3-乙酰氨基葡聚二糖、β-1，3-乙酰氨基葡聚三糖和Lewis~a三糖对黄瓜枯萎病菌(Fusarium oxysporum f. sp. cucumerium)、烟草黑胫病菌(Phytophthora parasitica var. nicotianae)、烟草赤星病菌(Alternaria alternata)、水稻纹枯病菌(Rhizoctonia solani)和稻瘟病菌(Magnaporthe grisea)等5种植物病原真菌菌丝生长的抑制作用。结果显示，3个供试寡糖对上述病原菌的菌丝生长没有显著的抑制作用。
黄瓜幼苗子叶期诱导对枯萎病的抗性：供试的3个寡糖，以5个不同浓度和2种诱导方法(叶面喷雾法和灌根法)对黄瓜幼苗分别进行诱导时，只有10μg／ml的β-1，3-乙酰氨基葡聚二糖和β-1，3-乙酰氨基葡聚三糖的叶面喷雾处理表现抗病性。而Lewis~a三糖的所有诱导处理均不表现抗性。
黄瓜胚根期诱导对枯萎病的抗性：在黄瓜胚根期，β-1，3-乙酰氨基葡聚二糖和β-1，3-乙酰氨基葡聚三糖浸泡处理种子胚根后，表现出抗病性，最低有效诱导浓度为5μg／ml。而Lewis~a三糖的5个诱导浓度处理均不表现诱导抗病性。3个供试寡糖在黄瓜胚根期与幼苗子叶期对枯萎病的诱导抗性结果相似。
3．β-1，4-乙酰氨基葡聚糖及β-1，3-乙酰氨基葡聚糖诱导烟草抗病性的研究
活体接种条件下寡糖诱导烟草对黑胫病的抗性：7个供试寡糖，以1μg／ml、10μg／ml和100μg／ml 3种不同浓度诱导烟草植株，再活体茎杆接种烟草黑胫病菌后，Lewis~a三糖、β-1，3-乙酰氨基葡聚二糖、β-1，3-乙酰氨基葡聚三糖、β-1，4-乙酰氨基葡聚二糖处理均不表现抗病性；β-1，3-乙酰氨基葡聚四糖、β-1，4-乙酰氨基葡聚三糖和β-1，4-乙酰氨基葡聚四糖的处理对黑胫病具有诱导抗性。在3种不同的诱导浓度中，10μg／ml是大多数供试寡糖适合的处理浓度。
离体接种条件下寡糖诱导烟草对黒胫病的抗性：7个供试寡糖以10μg／ml的浓度对烟草植株进行诱导，再离体接种黑胫病菌后，β-1，3-乙酰氨基葡聚二糖、β-1，3-乙酰氨基葡聚三糖、Lewis~a三糖和β-1，4-乙酰氨基葡聚二糖均无诱导抗性效果；只有β-1，3-乙酰氨基葡聚四糖、β-1，4-乙酰氨基葡聚三糖和β-1，4-乙酰氨基葡聚四糖表现出诱导抗性。
离体接种条件下寡糖诱导烟草对赤星病的抗性：7个供试寡糖以10μg／ml的浓度对烟草植株进行诱导，再离体接种赤星病菌后，β-1，3-乙酰氨基葡聚二糖、Lewis~a三糖和β-1，4-乙酰氨基葡聚二糖均无诱导抗性效果；β-1，3-乙酰氨基葡聚三糖、β-1，3-乙酰氨基葡聚四糖、β-1，4-乙酰氨基葡聚三糖和β-1，4-乙酰氨基葡聚四糖这4个供试寡糖处理则表现诱导抗性。
4．寡糖类诱导物的分子结构与其诱导抗病性之间的关系
甲苷化Lewis~a三糖的主链链接方式和糖残基类型对诱导效果的影响：甲苷化Lewis~a三糖对于黄瓜枯萎病、烟草黑胫病和赤星病，均不具有诱导抗病效果，可能是因为其主链的链接方式和糖残基的类型与几丁寡糖及其结构类似物不同，因而影响了其诱导活性。通过与相关资料的比较，可知Lewis寡糖类型的主链链接方式可能是决定诱导活性的主要因素，但不能排除糖残基的类型对诱导活性的影响。
C-1位的取代基团对诱导效果的影响：供试的7个寡糖均在还原端C-1含有取代的甲氧基，其诱导抗病效果却表现出极大差异，这说明了C-1位置上的甲氧基并非是决定这7个寡糖是否具有诱导活性的关键因素。
几丁寡糖及其结构类似物的聚合度对诱导效果的影响：几丁寡糖及其结构类似物诱导植物抗病性需要达到最低聚合度的阀值，因寡糖类型及植物病害系统不同而有差异。几丁寡糖结构类似物分别诱导黄瓜枯萎病，烟草赤星病和烟草黑胫病的抗性有效聚合度最低为2，3和4；几丁寡糖诱导对烟草黑胫病和烟草赤星病抗性的有效聚合度最低为3。
几丁寡糖及其结构类似物的糖苷键链接方式对诱导效果的影响：诱导抗性的产生，也许并不局限于乙酰氨基葡萄糖糖残基是否连接于C-4的位置，碳骨架主链是否采取β-1，4链接方式。β-1，4的糖苷键链接方式并非唯一的、可形成具有诱导活性的乙酰氨基葡聚糖的链接方式，碳骨架主链采取β-1，3链接方式而形成的几丁寡糖结构类似物——β-1，3-乙酰氨基葡聚糖同样具有诱导植物的抗病活性。
几丁寡糖及其结构类似物诱导抗病性的特点：几丁寡糖及其结构类似物的诱导抗病性属于系统的诱导抗病性，具有广谱性(非特异性)诱导抗性的特点。
5．β-1，3-乙酰氨基葡聚糖及β-1，4-乙酰氨基葡聚糖诱导烟草抗病性的生理机制
寡糖的诱导对3种防御酶活性变化的影响：5个供试寡糖诱导酶活性的不同效果具体表现在总体酶活水平、酶活峰值及到达最高峰的时间。β-1，3-乙酰氨基葡聚四糖和β-1，4-乙酰氨基葡聚四糖显著提高了POD、PPO和PAL的酶活性，诱导后48h达到酶活峰值，上述2寡糖对3种酶活性诱导效果分别为40％和99％；238％和186％；81％和73％，均高于相应的其他三种寡糖的诱导处理。
寡糖诱导的酶活性变化与烟草黑胫病诱导抗性效果之间的关联性：β-1，3-乙酰氨基葡聚四糖和β-1，4-乙酰氨基葡聚四糖可诱导烟草对黑胫病的抗病性；同时也可诱导过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)这3种酶活性的显著性增加。表明β-1，3-乙酰氨基葡聚四糖和β-1，4-乙酰氨基葡聚四糖诱导的POD、PPO和PAL这3种酶的活性提高与诱导烟草对黑胫病抗病性之间在是相对应的，具有关联性。
寡糖的分子结构对3种防御酶活性的影响：在5个供试寡糖中，聚合度均为4的β-1，3-乙酰氨基葡聚四糖和β-1，4-乙酰氨基葡聚四糖均可诱导POD、PPO和PAL这3种酶的酶活显著增加；而具有同样的化学结构，聚合度仅仅为2的β-1，3-乙酰氨基葡聚二糖和β-1，4-乙酰氨基葡聚二糖的总体酶活性与对照相接近。这表明了聚合度大小是影响几丁寡糖及其结构类似物对POD、PPO和PAL这3种酶活性诱导效果的因素之一。
β-1，3和β-1，4糖苷键分别链接形成的几丁寡糖结构类似物和几丁寡糖，均可诱导烟草的抗病性和POD、PPO和PAL 3种抗性酶的活性，说明了几丁寡糖及其结构类似物的化学结构具有影响POD、PPO和PAL这3种酶活性变化的化学分子结构特征。
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 oligosaccharides
Synthesis 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 BF_3·Et_2O/HgO to give 25 with a free hydroxyl at the reducing terminaln, followed by treatment with Cl_3CCN/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, ~(13)C 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 oligosaccharides
Induced 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 diseases
The 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 analogues
The 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 oligosaccharides
Dynamics 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.
主要的研究结果如下：
1．几丁寡糖结构类似物β-1，3-乙酰氨基葡聚糖的化学合成
β-1，3-乙酰氨基葡聚二糖的合成：以氨基葡萄糖为化学合成的起始原料，用邻苯二甲酰基保护化合物7和8的2位氨基；以苄叉来保护其4，6位的羟基，避免了糖化反应发生在4位上，从而保证了糖苷键的连接具有特定的立体和区域选择性(β-1，3连接糖苷键)；化合物8还用硫烃基保护异头碳上羟基，使硫糖苷作为糖基供体，成为合成乙酰氨基葡聚糖的离去基团；化合物8的3位再用乙酰基加以保护，避免它同时作为糖基供体和糖基受体，只能充当糖基供体；由3位是自由羟基、异头碳上为甲氧基的化合物7作为糖基受体，以NIS-TfOH作催化剂，7和8发生糖基化反应，高区域、高立体选择性、高产率地合成了β-1，3连接的二聚糖9，其核磁共振氢谱也证实了新产生的糖苷键为β构型(J=8.40 Hz)。
β-1，3-乙酰氨基葡聚三糖和四糖的合成：将二聚糖9及随后得到的三聚糖15脱去3′或3″位的乙酰基，得到的化合物14和20，它们再作为糖基受体，与糖基供体8在NIS-TfOH催化下进行糖基化反应，即可得到β-1，3连接的乙酰氨基葡聚三糖15和乙酰氨基葡聚四糖21。核磁共振氢谱显示新产生的糖苷键为β构型(J=8.48Hz和J=8.40Hz)。
β-1，3-乙酰氨基葡聚五糖的合成：化合物8先转化为异头碳为游离羟基的化合物25，然后又与三氯乙腈加成形成端基活化中间体三氯乙酰亚胺酯26。糖基供体26与3位为自由羟基，硫烃基保护异头碳的糖基受体5发生缩合反应，形成双糖27；27又作为糖基供体与糖基受体20反应生成五聚糖28。其核磁共振氢谱显示新产生的糖苷键为β构型(J=8.54 Hz和J=8.37 Hz)。
β-1，3-乙酰氨基葡聚糖的脱保护：分别尝试了醋酸水解、催化氢化以及对甲苯磺酸(PTSA)水解的方法来脱去苄叉保护基，醋酸会使得乙酰氨基葡聚糖分解，催化氢化适用于二糖9、三糖15以及四糖21的脱保护，但五糖28在催化氢化时反应不完全，只能使用对甲苯磺酸的水解。邻苯二甲酰氨基的脱保护，均采用水合肼在水溶性的乙醇中回流，得到游离的氨基中间体；之后，或者直接全乙酰化，将全乙酰化的产物分离纯化后再脱去乙酰基即得到纯度较高的二糖和三糖的终产物13和19；或者游离的氨基中间体直接选择性乙酰化，得到终产物24和31，再分离纯化。
β-1，3-乙酰氨基葡聚糖的结构确定：化学合成的4个β-(1→3)-2-脱氧-2-乙酰氨基葡聚糖，聚合度分别是从2—5，其准确的化学结构均通过高分辨质谱以及核磁共振~1H NMR、~(13)C NMR的分析确证。上述4个化合物的总收率分别为10.56％、3.08％、1.28％和0.33％。与天然的几丁寡糖不同，合成的4个乙酰氨基葡聚糖均采取了β-1→3糖苷键的连接方式，是几丁寡糖的结构类似物，其结构清楚、组分单一，从而提供了研究几丁寡糖诱导植物抗病活性与寡糖区域异构体之间关系的理想材料。
化学合成的四个几丁寡糖结构类似物的化学结构Chemical structure of the four synthesized chitin oligosaccharide analogues
2．β-1，3-乙酰氨基葡聚糖诱导黄瓜对枯萎病的抗性研究
对5种植物病原真菌菌丝生长的抑制作用：在实验室条件下，分别测定了3个供试寡糖即β-1，3-乙酰氨基葡聚二糖、β-1，3-乙酰氨基葡聚三糖和Lewis~a三糖对黄瓜枯萎病菌(Fusarium oxysporum f. sp. cucumerium)、烟草黑胫病菌(Phytophthora parasitica var. nicotianae)、烟草赤星病菌(Alternaria alternata)、水稻纹枯病菌(Rhizoctonia solani)和稻瘟病菌(Magnaporthe grisea)等5种植物病原真菌菌丝生长的抑制作用。结果显示，3个供试寡糖对上述病原菌的菌丝生长没有显著的抑制作用。
黄瓜幼苗子叶期诱导对枯萎病的抗性：供试的3个寡糖，以5个不同浓度和2种诱导方法(叶面喷雾法和灌根法)对黄瓜幼苗分别进行诱导时，只有10μg／ml的β-1，3-乙酰氨基葡聚二糖和β-1，3-乙酰氨基葡聚三糖的叶面喷雾处理表现抗病性。而Lewis~a三糖的所有诱导处理均不表现抗性。
黄瓜胚根期诱导对枯萎病的抗性：在黄瓜胚根期，β-1，3-乙酰氨基葡聚二糖和β-1，3-乙酰氨基葡聚三糖浸泡处理种子胚根后，表现出抗病性，最低有效诱导浓度为5μg／ml。而Lewis~a三糖的5个诱导浓度处理均不表现诱导抗病性。3个供试寡糖在黄瓜胚根期与幼苗子叶期对枯萎病的诱导抗性结果相似。
3．β-1，4-乙酰氨基葡聚糖及β-1，3-乙酰氨基葡聚糖诱导烟草抗病性的研究
活体接种条件下寡糖诱导烟草对黑胫病的抗性：7个供试寡糖，以1μg／ml、10μg／ml和100μg／ml 3种不同浓度诱导烟草植株，再活体茎杆接种烟草黑胫病菌后，Lewis~a三糖、β-1，3-乙酰氨基葡聚二糖、β-1，3-乙酰氨基葡聚三糖、β-1，4-乙酰氨基葡聚二糖处理均不表现抗病性；β-1，3-乙酰氨基葡聚四糖、β-1，4-乙酰氨基葡聚三糖和β-1，4-乙酰氨基葡聚四糖的处理对黑胫病具有诱导抗性。在3种不同的诱导浓度中，10μg／ml是大多数供试寡糖适合的处理浓度。
离体接种条件下寡糖诱导烟草对黒胫病的抗性：7个供试寡糖以10μg／ml的浓度对烟草植株进行诱导，再离体接种黑胫病菌后，β-1，3-乙酰氨基葡聚二糖、β-1，3-乙酰氨基葡聚三糖、Lewis~a三糖和β-1，4-乙酰氨基葡聚二糖均无诱导抗性效果；只有β-1，3-乙酰氨基葡聚四糖、β-1，4-乙酰氨基葡聚三糖和β-1，4-乙酰氨基葡聚四糖表现出诱导抗性。
离体接种条件下寡糖诱导烟草对赤星病的抗性：7个供试寡糖以10μg／ml的浓度对烟草植株进行诱导，再离体接种赤星病菌后，β-1，3-乙酰氨基葡聚二糖、Lewis~a三糖和β-1，4-乙酰氨基葡聚二糖均无诱导抗性效果；β-1，3-乙酰氨基葡聚三糖、β-1，3-乙酰氨基葡聚四糖、β-1，4-乙酰氨基葡聚三糖和β-1，4-乙酰氨基葡聚四糖这4个供试寡糖处理则表现诱导抗性。
4．寡糖类诱导物的分子结构与其诱导抗病性之间的关系
甲苷化Lewis~a三糖的主链链接方式和糖残基类型对诱导效果的影响：甲苷化Lewis~a三糖对于黄瓜枯萎病、烟草黑胫病和赤星病，均不具有诱导抗病效果，可能是因为其主链的链接方式和糖残基的类型与几丁寡糖及其结构类似物不同，因而影响了其诱导活性。通过与相关资料的比较，可知Lewis寡糖类型的主链链接方式可能是决定诱导活性的主要因素，但不能排除糖残基的类型对诱导活性的影响。
C-1位的取代基团对诱导效果的影响：供试的7个寡糖均在还原端C-1含有取代的甲氧基，其诱导抗病效果却表现出极大差异，这说明了C-1位置上的甲氧基并非是决定这7个寡糖是否具有诱导活性的关键因素。
几丁寡糖及其结构类似物的聚合度对诱导效果的影响：几丁寡糖及其结构类似物诱导植物抗病性需要达到最低聚合度的阀值，因寡糖类型及植物病害系统不同而有差异。几丁寡糖结构类似物分别诱导黄瓜枯萎病，烟草赤星病和烟草黑胫病的抗性有效聚合度最低为2，3和4；几丁寡糖诱导对烟草黑胫病和烟草赤星病抗性的有效聚合度最低为3。
几丁寡糖及其结构类似物的糖苷键链接方式对诱导效果的影响：诱导抗性的产生，也许并不局限于乙酰氨基葡萄糖糖残基是否连接于C-4的位置，碳骨架主链是否采取β-1，4链接方式。β-1，4的糖苷键链接方式并非唯一的、可形成具有诱导活性的乙酰氨基葡聚糖的链接方式，碳骨架主链采取β-1，3链接方式而形成的几丁寡糖结构类似物——β-1，3-乙酰氨基葡聚糖同样具有诱导植物的抗病活性。
几丁寡糖及其结构类似物诱导抗病性的特点：几丁寡糖及其结构类似物的诱导抗病性属于系统的诱导抗病性，具有广谱性(非特异性)诱导抗性的特点。
5．β-1，3-乙酰氨基葡聚糖及β-1，4-乙酰氨基葡聚糖诱导烟草抗病性的生理机制
寡糖的诱导对3种防御酶活性变化的影响：5个供试寡糖诱导酶活性的不同效果具体表现在总体酶活水平、酶活峰值及到达最高峰的时间。β-1，3-乙酰氨基葡聚四糖和β-1，4-乙酰氨基葡聚四糖显著提高了POD、PPO和PAL的酶活性，诱导后48h达到酶活峰值，上述2寡糖对3种酶活性诱导效果分别为40％和99％；238％和186％；81％和73％，均高于相应的其他三种寡糖的诱导处理。
寡糖诱导的酶活性变化与烟草黑胫病诱导抗性效果之间的关联性：β-1，3-乙酰氨基葡聚四糖和β-1，4-乙酰氨基葡聚四糖可诱导烟草对黑胫病的抗病性；同时也可诱导过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)这3种酶活性的显著性增加。表明β-1，3-乙酰氨基葡聚四糖和β-1，4-乙酰氨基葡聚四糖诱导的POD、PPO和PAL这3种酶的活性提高与诱导烟草对黑胫病抗病性之间在是相对应的，具有关联性。
寡糖的分子结构对3种防御酶活性的影响：在5个供试寡糖中，聚合度均为4的β-1，3-乙酰氨基葡聚四糖和β-1，4-乙酰氨基葡聚四糖均可诱导POD、PPO和PAL这3种酶的酶活显著增加；而具有同样的化学结构，聚合度仅仅为2的β-1，3-乙酰氨基葡聚二糖和β-1，4-乙酰氨基葡聚二糖的总体酶活性与对照相接近。这表明了聚合度大小是影响几丁寡糖及其结构类似物对POD、PPO和PAL这3种酶活性诱导效果的因素之一。
β-1，3和β-1，4糖苷键分别链接形成的几丁寡糖结构类似物和几丁寡糖，均可诱导烟草的抗病性和POD、PPO和PAL 3种抗性酶的活性，说明了几丁寡糖及其结构类似物的化学结构具有影响POD、PPO和PAL这3种酶活性变化的化学分子结构特征。
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 oligosaccharides
Synthesis 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 BF_3·Et_2O/HgO to give 25 with a free hydroxyl at the reducing terminaln, followed by treatment with Cl_3CCN/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, ~(13)C 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 oligosaccharides
Induced 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 diseases
The 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 analogues
The 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 oligosaccharides
Dynamics 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.
引文
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