紫外分光光度法快速测定发酵液中的L-色氨酸

建立了一种紫外分光度法测定发酵液中L-色氨酸的方法。L-色氨酸和甲酸在HCl介质中生成蓝紫色化合物,该化合物最大吸光度为364 nm,L-色氨酸测定范围为0~2.5mg/mL,工作曲线为Y=0.382X+0.0278,R2=0.9999,其它氨基酸均不干扰L-色氨酸的测定。该方法与氨基酸分析仪测定结果一致,测定改造色氨酸合成酶酶促合成的L-色氨酸浓度达到46mg/mL。     

铜L-色氨酸配合物的合成与表征

利用液相反应合成法制得了铜(Ⅱ)和L-色氨酸的配合物,结合元素分析、差示扫描量热与热重分析联用、粉末X射线衍射法以及红外光谱实验手段对该配合物进行表征。结果表明,1个铜离子能够与2个L-色氨酸分子通过侧链氨基上的氮原子和羧基上的氧原子配合,形成稳定的配合物。利用太赫兹时域光谱法获得了室温条件下铜-L-色氨酸配合物在低频波段的光谱特征,并结合密度泛函理论计算对太赫兹光谱进行分析。该配合物在太赫兹波段的吸收对应于分子整体的振动,涉及吲哚环和侧链的扭曲振动。研究结果有助于深入了解铜离子与氨基酸的相互作用,以及铜离子在复杂生物体中所起的作用。     

萘乙二胺法测定色氨酸及应用于DL-色氨酸拆分过程

在酸性条件下,萘乙二胺与色氨酸形成偶氮化合物,最大吸收波长在545nm处。100倍的丝氨酸、丙氨酸、天冬氨酸、精氨酸、苯丙氨酸、酪氨酸及10倍底物N-乙酰-色氨酸均不干扰显色,甲硫氨酸对显色影响较大,相对误差达到23.9%;拆分过程所需的钴离子和10倍量的酶蛋白对显色反应也没有影响,相对误差可控制在10%以内。采用萘乙二胺法检测色氨酸更适合于色氨酸拆分过程的检测,便于控制拆分过程。     

分子印迹电化学传感器测定L-色氨酸的研究

报道了一种由溶胶-凝胶法制备的分子印迹电化学传感器,并用于L-色氨酸的测定.印迹聚合物是由四乙氧基硅烷、甲基三甲氧基硅烷、苯基三甲氧基硅烷等聚合而成,L-色氨酸为模板分子.通过循环伏安法验证了印迹膜与模板分子的结合和去除.该传感器对L-色氨酸具有良好的选择性以及高的灵敏度,线性范围为1.0×10-9～1.0×10-5m...     

催化NH吲哚的C2异戊烯基化反应:肽后期多样化和两步全合成tryprostatin B

异戊烯基化吲哚生物碱是一类同时具有吲哚环和类异戊二烯基团的天然产物,主要来源于各种真菌中.异戊烯基的存在可以增强化合物的亲脂性,使其能够更容易地穿过脂溶性的细胞膜与靶蛋白相结合,因此,这类天然产物往往表现出优异的生物活性.例如,从烟曲霉中分离的吲哚生物碱tryprostatins A和B是由L-色氨酸和L-脯氨酸组合而成,在吲哚骨架C2位连有异戊烯基,具有高效的抗肿瘤活性.在已知的全合成中,关键步骤C2位异戊烯基的引入,均是通过多步当量反应实现的.从原子和步骤经济性角度出发,发展高效催化方法实现NH吲哚C2位直接异戊烯基化反应具有重要的意义.但是由于吲哚的氮原子和C3位亲核性都较强,使得挑战很大.在生物体中,吲哚生物碱C2位异戊烯基的引入是通过酶催化实现的.二甲基烯丙基焦磷酸(DMAPP)先在酶作用下,生成异戊烯基碳正离子,然后与吲哚C2位进行傅克反应,引入异戊烯基.受这一生物过程启发,设想通过化学方法能够生成稳定的异戊烯基碳正离子,也可能实现吲哚C2异戊烯基化反应.本文采用廉价易得的1,1-二甲基烯丙醇为异戊烯基前体,对该想法进行了尝试.首先,以色醇为模板底物,对酸催化剂、溶剂及反应温度进行了筛选.在1,2-二氯乙烷(DCE)溶剂中,布朗斯特酸、路易斯酸和固体酸都能促进反应的进行,但会得到吲哚N和C2异戊烯基化两种产物,其中三氯化铝有较好的收率和选择性.通过对不同溶剂考察发现,2-甲基四氢呋喃是最佳溶剂,在80 oC下反应,目标产物收率为75％,产物选择性可达到12:1.随后,对不同类型的3-取代吲哚进行了普适性考察.对于色醇类底物,吲哚苯环上的取代基以及N上的保护基对反应影响不大,都能很顺利地参与反应.在标准条件下,色胺的异戊烯基化反应会发生在C3位,而以氯苯为溶剂时,可以提高C2位选择性,通过该方法可在抗衰老分子褪黑素(melatonin)的C2位引入异戊烯基.3-苯基和烷基取代的吲哚也是合适的底物.肽的后期修饰在生物医药中有着很重要的用途,因此,本文也将该异戊烯基化反应尝试用于修饰色氨酸类衍生物.保护的L-色氨酸酯以及色氨醇都能够顺利发生转化,以中等收率得到目标产物.L-色氨酸与其它各类氨基酸如甘氨酸、丙氨酸、亮氨酸、异亮氨酸、苯丙氨酸、甲硫氨酸、天冬氨酸等形成的肽也可进行C2异戊烯基化反应.此外,该转化过程中,可以完全保持手性,不会发生消旋化.特别是,L-色氨酸与L-脯氨酸酯形成的环二肽brevianamide F,在该条件下,也能发生C2位异戊烯基化反应,快速合成天然吲哚生物碱tryprostatin B.该反应有两种可能的路径,一种是吲哚C2位直接异戊烯基化,另一种是吲哚C3位先异戊烯基化,然后再重排到C2位.为进一步探索机理,对其进行了研究.首先,在标准条件下,3-酯基吲哚可以发生反应,C2位异戊烯基化产物收率为25％.然后,预先将异戊烯基引入C3位,合成了3-酯基-3-异戊烯基-3H吲哚,同样反应条件下,也能检测到目标产物,但收率只有5％.其次,以氘代1,1-二甲基烯丙醇为原料时,3-异戊烯基吲哚也能与其发生反应,并且在C3和C2位都观察到了氘代异戊烯基,两种产物比例为1:2,结果表明两种反应路径都是存在的,但吲哚C2位直接异戊烯基化是主要路径.此外,以固体酸Nafion为催化剂,离子液体[Bmim]Cl为反应介质,在120 oC时,3-取代吲哚与1,1-二甲基烯丙醇的反应选择性会发生改变,得到C2位异戊烯基异构化的产物.总之,以商业可得的1,1-二甲基烯丙醇为前体,首次实现了化学催化NH吲哚C2位直接异戊烯基化反应,获得较好的区域和化学选择性.该方法能够兼容各类官能团,底物适用性广,且可以用于褪黑素以及色氨酸衍生各种肽类化合物的后期修饰.基于该催化方法,可以两步全合成天然吲哚生物碱tryprostatin B,极大提高了合成效率,有助于实现放大生产.在固体酸/离子液体催化体系中,还实现了反应选择性的改变,丰富了产物类型.     

D—,L—色氨酸与牛血清白蛋白立体专一性结合研究

对血清白蛋白(SA)的结合内源性化合物及许多药物的性质已进行了广泛研究,有关氨基酸与SA的结合还很少报道。氨基酸吸收路径几乎完全是血液,它们与SA的结合性质,无疑与其在体内的运输和代谢密切相关。色氨酸含有亲水性的NH_3~+及COO~-和疏水性的吲哚环,研究它与SA的结合性质对于全面阐明SA结合各种配体的规律和机理都有重要意义。本文采用离心超过滤法测定了25℃、pH 7.40时D-、L-、DL-色氨酸(Trp)与牛血清白蛋     

石墨炉原子吸收光谱法测定L-色氨酸中的铅

采用干灰法消化L-色氨酸样品,建立了测定L-色氨酸中重金属(以铅计)含量的石墨炉原子吸收光谱法。对测定L-色氨酸的仪器工作条件进行了探讨。铅的浓度在0~32μg/L内与吸光度成良好的线性关系,线性回归方程为A=0.023 170c 0.003 600,相关系数r=0.999 8,测定结果的相对标准偏差为1.1%(n=5),回收率为97.7%~100.6%。     

β-环糊精增敏荧光猝灭法对粮食样品中L-色氨酸的分析研究

研究了Se(Ⅳ)-丁基罗丹明B与L-色氨酸结合物在β-环糊精(β-CD)介质中的荧光光谱行为.加入L-色氨酸可使Se(Ⅳ)-丁基罗丹明B体系发生荧光猝灭,β-CD的加入导致溶液中荧光猝灭值ΔIF增加,且L-色氨酸的质量浓度与ΔIF呈良好的线性关系,据此建立了荧光猝灭测定L-色氨酸的新方法.优化了实验条件,结果表明:β-CD对测定具有增敏作用,色氨酸的质量浓度在1.0 ～30.0 mg/L范围内与荧光强度呈线性关系,回归方程为ΔIF =399.1ρ(mg/L)+258.9,相关系数r=0.999 3,检出限为0.21 mg/L.将方法用于粮食样品中L-色氨酸的测定,结果令人满意.     

L-色氨酸分子印迹壳聚糖膜的制备及透过选择性

以L-色氨酸为印迹分子,选取富含羟基和氨基的多功能团聚合物壳聚糖（CS）为基本成膜材料,通过相转化法结合碱液处理与硫酸交联两种后处理方式制备了L-色氨酸分子印迹CS膜（MIM）,采用红外光谱（FT-IR）对膜的组成和结构进行表征,通过渗透实验考察了分子印迹壳聚糖膜对L-色氨酸的分子识别性能,考察了碱液处理与硫酸交联两种后处理方式对膜的溶胀性以及MIM内物质传递的影响．     

线扫伏安法同时测定5-羟基色氨酸和色氨酸

研究了色氨酸和5-羟基色氨酸在玻璃碳电极上的电化学行为,研究了不同pH、静置时间、扫描速度以及表面活性剂等的影响,探讨了色氨酸和5-羟基色氨酸在玻璃碳电极上的氧化机理,建立了线扫伏安法同时测定色氨酸和5-羟基色氨酸的方法.实验发现,在含3.33×10-4 mol/L十二烷基磺酸钠的0.1mol/L柠檬酸(pH=4.50)介质中,5-羟基色氨酸和色氨酸分别在 0.675V和 1.070V产生一灵敏的氧化峰.对5-羟基色氨酸,相应的氧化峰的峰高与浓度在2.40×10-5 mol/L～8.00×10-4 mol/L范围内呈良好的线性关系,线性相关系数为0.9994;对色氨酸,其氧化峰的峰高与浓度在2.40×10-5 mol/L～6.40×10-4 mol/L浓度范围内呈良好的线性关系,线性相关系数为0.9929.该方法测量2.00×10-4 mol/L色氨酸和5-羟基色氨酸的相对标准偏差分别为3.2%和3.8%(n=10).     

穿心莲内酯的溶剂浮选

应用溶剂浮选法对穿心莲乙醇提取液中的穿心莲内酯进行分离富集,优化了穿心莲内酯的浮选条件.实验表明:浮选溶剂为乙酸乙酯,水相与有机相体积比为2: 1,加入水相体积4%的乙醇,溶液pH 7,氮气流速200 mL/min,浮选时间40 min为最佳浮选条件.在最佳实验条件下,穿心莲内酯平均收率为94.1%; RSD为1.1%,平均标准加入回收率95.1%; 富集倍数为9.7倍.溶剂浮选法分离穿心莲内酯不仅能得到满意的回收率和富集倍数,并能有效地除去水溶性杂质,提高目标物纯度.与溶剂萃取法相比, 溶剂浮选法能节约有机溶剂和操作时间.     

布洛芬-扑热息痛孪药的合成

以布洛芬（IPF）和扑热息痛（PCM）为原料,无水丙酮和乙酸乙酯为溶剂,二环己基碳二亚胺（DCC）为脱水剂,4-二甲氨基吡啶（DMAP）为催化剂,合成了布洛芬-扑热息痛孪药（PHI）,以重结晶法对目标产物进行纯化,产物结构经IR和1H NMR确定。 探讨了原料比例、脱水剂用量、催化剂用量、反应时间和溶媒用量对目标物产率的影响,并采用正交试验筛选最优合成工艺,最终确定的最优反应条件为:n(PCM)∶n(IPF)=1∶1.5,催化剂的最佳用量为布洛芬质量的10%,反应时间为8 h,溶媒体积为70 mL（V(乙酸乙酯)∶V(丙酮)=1∶1）,在该条件下,目标物PHI的产率为66.53%。     

固定化细胞酶法拆分N-乙酰-D,L-3-甲氧基丙氨酸

利用氨基酰化酶固定化细胞酶法拆分了N-乙酰-D,L-3-甲氧基丙氨酸. 考察了温度、pH值、底物浓度、金属离子和拆分时间对酶促反应的影响. 确定了氨基酰化酶固定化细胞手性拆分N-乙酰-D,L-3-甲氧基丙氨酸的最佳工艺条件为pH=7.0, 反应温度50 ℃及底物浓度500 mmol/L. 10^-4 mol/L的Co²⁺和Mg²⁺对氨基酰化酶有显著激活作用, Cu²⁺和Zn²⁺对酶促反应有明显抑制作用. 在最佳条件下, 氨基酰化酶固定化细胞对N-乙酰-L-3-甲氧基丙氨酸的摩尔转化率达96%.     

Optimization of enzyme catalyzed peptide synthesis in a “water - water-immiscible organic solvent” biphasic system

Optimal conditions were found, for enzymatic synthesis of the dipeptide, N-acetyl-L-tryptophanyl-L-leucine amide in the biphasic system water - ethyl acetate. The synthesis was carried out using both free and. immobilized α-chymotrypsin. Optimization was performed, by such parameters as the “organic phase/aqueous phase” volume ratio, the pH of aqueous phase, and the concentration of starting reactants. Under most favourable condition the dipeptide was synthesized. on the preparative scale in ca. 100% yield. As a result of immobilization (adsorption on the Sorsilen terephtalate support) the enzyme practically did not inactivate and may be used repeatedly.     

A catalyst-free novel synthesis of diethyl carbonate from ethyl carbamate in supercritical ethanol

Diethyl carbonate has been synthesized via the alcoholysis of ethyl carbamate in supercritical ethanol under catalyst-free conditions.The influences of various parameters such as reaction temperature,reaction time,reaction pressure,ethanol/ethyl molar ratios and reaction loading volume on the yield of DEC were studied systematically.The experimental results indicated that the alcoholysis of ethyl carbamate was greatly improved in supercritical ethanol.The optimal reaction conditions were as follows：a reaction temperature of 573 K,a reaction time of 30 min,a reaction pressure of 13.2 MPa,an ethanol/ethyl carbamate molar ratio of 10 and a reactor loading volume of 285 μL respectively.The optimal yield of DEC was 22.9%.     

有机溶剂/缓冲液两相体系中全细胞催化拆分环氧氯丙烷

研究了有机溶剂/缓冲液两相体系中重组大肠杆菌E.coli BL21 (DE3)全细胞水解动力学拆分外消旋环氧氯丙烷制备(R)-环氧氯丙烷的过程.结果表明,最适反应条件为:最适有机溶剂异辛烷与缓冲液的体积比7:3,最适缓冲液pH 8.0,底物浓度574mmol/L,全细胞加入量0.07g湿菌体/ml溶液,温度30℃.在此条件下于1L反应器中反应45min,(R)-环氧氯丙烷的摩尔产率、光学纯度和时空产率分别达到37.5%,99.3%ee和0.286mol/(L·h).与单一水相体系相比,异辛烷/缓冲液两相体系中底物浓度和(R)-环氧氯丙烷时空产率分别提高了55.2%和98.6%.     

pH‐zone‐refining elution–extrusion countercurrent chromatography: Separation of hydroxyanthraquinones from Cassiae semen

Seven hydroxyanthraquinones were successfully separated from the traditional Chinese medicinal herb Cassiae semen by conventional and pH‐zone‐refining countercurrent chromatography with an environmentally friendly biphasic solvent system, in which elution–extrusion mode was investigated for pH‐zone‐refining countercurrent chromatography for the first time. A two‐phase solvent system composed of n‐hexane/ethyl acetate/ethanol/water (5:3:4:4, v/v/v/v) was used for the conventional countercurrent chromatography while the same system with a different volume ratio n‐hexane/ethyl acetate/ethanol/water (3:5:2:6, v/v/v/v) was used for pH‐zone‐refining countercurrent chromatography, in which 20 mmol/L of trifluoroacetic acid was added in the organic phase as a retainer and 15 mmol/L of ammonia was added to the aqueous phase as an eluter. A 400 mg crude sample could be well separated by pH‐zone‐refining countercurrent chromatography, yielding 53 mg of aurantio‐obtusin, 40 mg of chryso‐obtusin, 18 mg of obtusin, 24 mg of obtusifolin, 10 mg of emodin, and 105 mg of the mixture of chrysophanol and physcion with a purity of over 95.8, 95.7, 96.9, 93.5, 97.4, 77.1, and 19.8%, as determined by high‐performance liquid chromatography. Furthermore, the difference in elution sequence between conventional and pH‐zone‐refining mode was observed and discussed.     

Enzymatic synthesis of sorbitan methacrylate according to acyl donors

Recently, sugar polymers have been considered for use as biomaterials in medical applications. These biomaterials are already used extensively in burn dressings, artificial membranes, and contact lenses. In this study, we investigated the optimum conditions under which the enzymatic synthesis of sorbitan methacrylate can be affected using Novozym 435 in t-butanol from sorbitan and several acyl donors (ethyl methacrylate, methyl methacrylate, and vinyl methacrylate). The enzymatic synthesis of sorbitan methacrylate, catalyzed by Novozym 435 in t-butanol, reached an approx 68% conversion yield at 50 g/L of 1,4-sorbitan, 5% (w/v) of enzyme content, and a 1∶5 molar ratio of sorbitan to ethyl methacrylate, with a reaction time of 36 h. Using methyl methacrylate as the acyl donor, we achieved a conversion yield of approx 78% at 50 g/L of 1,4-sorbitan, 7% (w/v) of enzyme content, at a 1∶5 molar ratio, with a reaction time of 36 h. Sorbitan methacrylate synthesis using vinyl methacrylate as the acyl donor was expected to result in a superior conversion yield at 3% (w/v) of enzyme content, and at a molar ratio greater than 1∶2.5. Higher molar ratios of acyl donor resulted in more rapid conversion rates. Vinyl methacrylate can be applied to obtain higher yields than are realized when using ethyl methacrylate or methyl methacrylate as acyl donors in esterification reactions catalyzed by Novozym 435 in organic solvents. Enzyme recycling resulted in a drastic reduction in conversion yields.     

Synthesis of Rapeseed Biodiesel Using Short-Chained Alkyl Acetates as Acyl Acceptor

In this study, we conducted experiments using a response surface methodology to determine the optimal reaction conditions for the enzymatic synthesis of biodiesel from rapeseed oil and short-chained alkyl acetates, such as methyl acetate or ethyl acetate, as the acyl acceptor at 40 °C. Based on our response surface methodology experiments, the optimal reaction conditions for the synthesis of biodiesel were as follows: methyl acetate as acyl acceptor, catalyst concentration of 16.50%, oil-to-methyl acetate molar ratio of 1:12.44, and reaction time of 19.70 h; ethyl acetate as acyl acceptor, catalyst concentration of 16.95%, oil-to-ethyl acetate molar ratio of 1:12.56, and reaction time of 19.73 h. The fatty acid ester content under the above conditions when methyl acetate and ethyl acetate were used as the acyl acceptor was 58.0% and 62.6%, respectively. The statistical method described in this study can be applied to effectively optimize the enzymatic conditions required for biodiesel production with short-chained alkyl acetates.     

Protease-catalyzed Synthesis of Bz-Arg-Gly-Asp-OMe in Full Aqueous Medium

Synthesis of N-benzoyl-argininylglycylasparagine methyl ester(Bz-Arg-Gly-Asp-OMe),a precursor tripeptide of Arg-Gly-Asp)was catalyzed by papain under kinetic control,at alkaline pH,in a full aqueous medium.The substrates were N-benzoyl-argininylglycine ethyl ester and asparagine dimethyl ester.An aqueous solution of 0.1 mol/L KCl/NaOH containing 8 mmol/L EDTA and 2 mmol/L DTT was selected as the reaction medium.The synthesized hydrophilic tripeptide was soluble in the reaction medium during the reaction process,however,the secondary hydro-lysis of the tripeptide product was not considerable.The effects of different factors,including water content,temperature,reaction time,and molar ratio of the substrates,on the yield of Bz-Arg-Gly-Asp-OMe were examined.The optimal reaction conditions were 0.05 mol/L Bz-Arg-Gly-OEt and 0.15 mol/L Asp(-OMe)2·HCl in 0.1 mol/L KCl/NaOH solution(pH 8.5),at 40 ℃,and a reaction time of 60 min,with a maximum conversion yield of 62.4%.