【作者】 冉学光；

【导师】 曹德榕；

【作者基本信息】 华南理工大学 ， 应用化学， 2010， 博士

【摘要】 硒代蛋基酸是一种含硒的α-氨基酸,具有抗氧化、抗癌、增强繁殖等功能,同时也是生物体重要的补硒产品。本文研究了硒代蛋氨酸的合成与毒性及其对肥育猪生产性能的影响,同时研究了硒代蛋氨酸缩水杨醛席夫碱及其金属配合物、硒代蛋氨酸缩2-羟基-1-萘醛席夫碱及其金属配合物的合成与表征,并对上述席夫碱配合物的抗菌效果进行了初步探讨。以α-溴-γ-丁内酯为原料,经过氨解、氢氧化钡成环,再加入适量硫酸,得到α-氨基-γ-丁内酯氢溴酸盐,产率为78.2 %;α-氨基-γ-丁内酯氢溴酸盐在醋酸钾、无水乙醇条件下回流,通过分子间反应生成酰胺进而保护氨基和羧基的方法,得到3,6-二羟乙基-2,5-哌啶二酮,产率为70.0 %;3,6-二羟乙基-2,5-哌啶二酮与二氯亚砜发生氯代反应,得到3,6-二氯乙基-2,5-哌啶二酮,产率80.1 %;在氢氧化钠水溶液中用80 %水合肼还原硒粉,加入四丁基溴化铵,经硫酸二甲酯甲基化,得到二甲基二硒醚,产率为90.0 %;通过“一锅煮”方法制备硒代蛋氨酸:二甲基二硒醚在DMSO、碳酸钾作用下,水合肼还原得到甲硒基盐后,不经分离,直接与3,6-二氯乙基-2,5-哌啶二酮反应,得到硒代蛋氨酸分子间缩合物,进一步用酸水解、三乙胺调节pH= 6-7,得到硒代蛋氨酸,产率为60.1 %。产物经过元素分析、质谱、IR、1H NMR、13C NMR、COSPY谱等分析方法确认其结构。以亚硒酸钠为参照,饲喂小鼠不同浓度硒化合物,对比死亡率、体重、肝脏、肺等指标和器官变化来研究硒代蛋氨酸的毒性。结果表明当含硒量大于4.20 mg/kg体重时,相同水平的硒代蛋氨酸毒性小于亚硒酸钠,计算得到亚硒酸钠和硒代蛋氨酸的半致死量（LD50）值分别为7.85和16.57。研究硒代蛋氨酸在肥育猪生产中的应用,考察其对生产性能、胴体性状、肉质指标的影响。与亚硒酸钠比较,饲粮中添加不同水平硒代蛋氨酸的试验结果表明:①未能显著改善肥育猪日增重、日采食量、料重比、左侧胴体重、背膘厚,但增加了肥育猪的眼肌面积;②对肥育猪的肌纤维密度未产生影响,能提高肌纤维直径;③降低了宰后不同时间点肉色L*值,提高了不同时间点肉色a*值,降低了宰后12h肉色b*值;④提高肥育猪宰后12 h背最长肌pH值,降低滴水损失,但不影响影响大理石纹评分、肌内脂肪含量和剪切力。氨基酸席夫碱是一类重要的配体,这类席夫碱结构中含有富电子的氧原子和氮原子,具有较强的配位能力和多样的配位模式。硒代蛋氨酸是一种重要的氨基酸,除具有一般氨基酸的功能外,分子含有硒原子,是一种很有前景的补硒来源。金属铜、锌、钴是生物体必须的营养微量元素。理论上将硒代蛋氨酸、席夫碱、营养金属元素设计成一个多功能分子,期待其具有特殊的生物功能。基于此,通过硒代蛋氨酸与水杨醛反应生成相应的席夫碱钾盐[K（HL）]（硒代蛋氨酸席夫碱不稳定）。硒代蛋氨酸缩水杨醛金属配合物通过将硒代蛋氨酸、水杨醛、金属醋酸盐水合物“一锅煮”的方法制备。通过元素分析、摩尔电导率、热分析、磁化率、红外光谱、电子光谱和XRD对K（HL）和各金属配合物进行表征,确定席夫碱金属配合物的化学组成为:ML（H₂O）,M = Co（II）, Cu（II）和Zn（II）,L为硒代蛋氨酸缩水杨醛席夫碱。推断其可能的结构式:席夫碱铜配合物为平面四方形构型,席夫碱钴配合物为四面体构型。另外,设计合成硒代蛋氨酸与共轭性高且空间位阻较大的2-羟基-1-萘醛席夫碱反应生成相应的席夫碱,席夫碱不稳定,制备成对应的席夫碱锂盐[Li（HL）]。通过将硒代蛋氨酸、2-羟基-1-萘醛、金属醋酸盐水合物“一锅煮”的方法制备对应的席夫碱铜、锌、钴配合物。对各化合物进行元素分析、摩尔电导率、热分析、磁化率、红外光谱、核磁、电子光谱和XRD表征,确定席夫碱金属配合物为:ML（H2O）,M = Co（II）, Cu（II）和Zn（II）,L为硒代蛋氨酸与2-羟基-1-萘醛反应生成的席夫碱。推断配合物可能的结构式:席夫碱铜配合物为平面四方形构型,席夫碱钴配合物为四面体构型。采用抑菌圈测定法对硒代蛋氨酸缩水杨醛席夫碱钾盐及其席夫碱铜、锌、钴金属配合物、硒代蛋氨酸缩2-羟基-1-萘醛席夫碱锂盐及相应席夫碱铜、锌、钴金属配合物的抗菌活性进行了测定,研究结果表明:金属配合物的抗菌活性高于相应的配体,认为可能的原因是配合物中除了席夫碱、硒原子外,金属有可能参与协同作用,金属离子与席夫碱螯合减小了金属离子的极性,增加了金属螯合物的亲油特性,有利于穿过菌类的油脂层,从而提高配合物的抗菌活性。在所有的席夫碱金属配合物中,席夫碱铜配合物抗菌效果最好,可能是因为铜可破坏菌的机体组织。实验表明配合物的抑菌活性与其浓度呈正相关。更多还原

【Abstract】 Selenomethionine(Se-Met), anα-amino acid containing selenium, is a reagent protecting human systems against oxidation, radiations, cancer, aging and related diseases, as well as a hopeful source of selenium in the diets of both animals and human. The synthesis and toxicity of Se-Met as well as the effect of its addition on performance in finishing pigs were investigated. Moreover, the Schiff base ligands derived from Se-Met and salicylaldehyde as well as 2-hydroxy-1-naphthaldehyde and their Co(II), Cu(II) and Zn(II) complexes were synthesized and characterized. The biological activity of these metal complexes was screened.Preparation ofα-amino-γ-butyrolactone hydrobromide started with amination of commercially availableα-bromo-γ-butyrolactone by ammonia water, followed by treatment with barium hydroxide and sulphuric acid. The amination ofα-bromo-γ-butyrolactone described here was a more effective and simple route for the synthesis ofα-amino-γ-butyrolactone hydrobromide in one step with 78.2 % yield. 3,6-Bis(2-hydroxyethy1)-2,5-diketopiperazine was readily produced fromα-amino-γ-butyrolactone hydrobromide by treatment with CH3COOK in absolute alcohol solution with 70.0 % yield. This route not only effectively protected carboxylic hydroxy and amino groups, but also prevented deprotection process through formation of two amide bonds in diketopiperazine. 3,6-Bis(2-hydroxyethy1)-2,5-diketopiperazine treated with thionyl chloride gave 3,6-bis(-2chloroethyl)-2,5-diketopiperazine in 80.1 % yield.A vital procedure for preparation of Se-Met is introduction of methylselenide. Hydrazine hydrate was used as reduction on selenium to give diselenide anion in water. Subsequently, addition of tetrabutyl ammonium bromide and dimethyl sulfate to above solution smoothly provided dimethyl diselenide in 90.0 % yield. The reduction of dimethyl diselenide was performed using hydrazine hydrate in DMSO in the presence of potassium carbonate. The nucleophilic methylselenide anions were not isolated and reacted directly with 3,6-bis(-2chloroethyl)-2,5-diketopiperazine to give 3,6-bis(β-methylselenoethyl)-2,5- diketopiperazine intermediate in a one-pot procedure. Followed by treatment with aqueous HCl and neutralization with triethylamine at pH= 6-7, Se-Met was obtained as white solid in 60.1 % yield. Se-Met was characterized by elemental analysis, mass spectrum, IR, NMR and COSPY spectra.The toxicity of Se-Met was evaluated through death rate, body weight, liver, lung and body organs change of rats when rats were fed with basal diets containing different concentration of Se-Met and sodium selenite (SS). Results showed that the toxicity of Se-Met was lower than that of SS at same level of dietary Se when the rats were fed with more than 4.20 mg/kg diet of Se. The data of LD50 were 7.85 for SS and 16.57 for Se-Met, respectively. This indicated that the toxicity of Se-Met was lower than that of SS.Finishing pigs were slaughtered to investigate the effects of Se-Met addition on performance, carcass traits, pork quality, and muscle antioxidant capacity in finishing pigs. A SS group was used as a standard of comparison in a control experiment. Results showed that①there was no significant differences in average daily gain, average daily feed intake and feed, back-fat thickness among all groups (P>0.05). However, addition of 0.30 mg/kg Se significantly improved eye muscle area compared to that of 0.15 mg/kg Se and SS added groups (P<0.05);②there was no significant differences in muscle fiber density among all groups (P>0.05). Addition of 0.30mg/kg Se significantly increased muscle fiber diameter of longissimus muscle (P<0.05);③meat color L* value was significantly decreased and a* value was significantly improved at 12 h, 24 h, 48 h post-slaughter in all Se-added groups in contrast with the control (P<0.05), also 0.15 mg/kg Se supplementation significantly decreased meat b* value at 12 h post-slaughter (P<0.05);④marbling score, shear force, drip loss and intramuscular fat content of porcine Longissimus muscle were not affected by Se addition. All Se-added groups presented significantly higher pH value at 12 h post-slaughter than the control (P<0.05).Amino acid Schiff bases are an important class of ligands containing electron-rich oxygen and nitrogen atoms, showing strong coordination ability and various coordination behavior. Se-Met, anα-amino acid containing selenium, is a potential source of selenium in the diets. On the other hand, cobalt, copper and zinc are necessary nutritious trace elements. Thus, we designed novel molecules which contained Se-Met, Schiff bases and nutritional metal elements, expecting their special biological activity. Certain problem of instability of the Se-Met Schiff base was encountered, which was resolved by making an equimolar potassium salt of the Schiff base ligand [K(HL)]. The synthesis of metal complexes proved to be straightforward in simple by one-pot reactions of Se-Met, salicylaldehyde and metal(II) acetate monohydrate. New cobalt(II), copper(II) and zinc(II) complexes of Schiff base derived from Se-Met and salicylaldehyde were characterized by elemental analysis, IR, NMR, electronic spectra, conductance magnetic measurements and powder XRD. The analytical data showed that the Schiff base ligand acted as tridentate towards divalent metal ions (cobalt, copper, zinc) via the azomethine-N, carboxylate oxygen and phenolato oxygen by a stoichiometric reaction of M:L (1:1) to form metal complexes [ML(H2O)], where L was the Schiff base ligand derived from Se-Met and salicylaldehyde, M = Co(II), Cu(II) and Zn(II). The proposed structures of metal complex were square planar geometry for Cu(II) complex and tetrahedral geometry for the Co(II) complex.2-Hydroxy-1-naphthaldehyde shows special property due to its high conjugation and steric hindrance. In the same way , due to instability of Schiff base of Se-Met and 2-hydroxy-1-naphthaldehyde, it was made an equimolar lithium salt of the Schiff base ligand [Li(HL)]. The novel zinc(II), copper(II), and cobalt(II) complexes of the Schiff base derived from 2-hydroxy-1-naphthaldehyde and Se-Met were synthesized by one-pot reactions and characterized by elemental analysis, IR, electronic spectra, conductance measurements, powder XRD, NMR and magnetic measurements. The analytical data displayed the composition of the metal complex to be ML(H2O), where L was the Schiff base ligand and M = Co(II), Cu(II) and Zn(II). The proposed structures of metal complex were square planar geometry for Cu(II) complex and tetrahedral geometry for the Co(II) complex.The ligands salts derived from Se-Met and salicylaldehyde as well as 2-hydroxy-1-naphthaldehyde and their metal complexes had been screened for their antibacterial activities. All the metal complexes were more potent bactericides than the ligand. This enhancement of metal complexes in the activity can be explained on the basis of chelation theory. Chelation reduces the polarity of the metal atom mainly because of partial sharing of its positive charge with the donor groups and possibleπelectron delocalization within the whole chalete ring. Such a chelation also enhances the lipophililic character of the central metal atom, which subsequently favors its permeation through the lipid layers of cell membrane and blocking the metal binding sites on enzymes of microorganism. Co(II) and Zn(II) complexes are much less microbial active than the Cu(II) complex.更多还原