裂殖酵母生物合成L-苯基乙酰基甲醇的研究

用裂殖酵母作为生物催化剂，催化苯甲醛与丙酮酸合成L苯基乙酰基甲醇（L-PAC），后者为合成L-麻黄素等多种药物的前体，转化反应的最佳温度为28℃，pH6．0，时间16h，苯甲醛用量160-200mmol／L，丙酮酸120～180mmol／L，LPAC产量可达15g／L。     

双酶法生物合成L-苯基乙酰基甲醇的研究

双酶偶联体系越来越受到人们重视,广泛应用于医药、食品等领域,是实现高效生物合成的重要方法。本文利用乳酸氧化酶(Lactate Oxidase,LOD)和丙酮酸脱羧酶(Pyruvatedecarboxylase,PDC)偶联构建双酶体系,催化乳酸钠和苯甲醛合成L-苯基乙酰基甲醇(L-Phenylacetylcarbinol,L-PAC),后者是合成L-麻黄素的重要中间体。转化反应的最佳温度为34℃,pH 6.8,转化时间6 h,苯甲醛用量160 mmol/L,乳酸钠用量330 mmol/L,葡萄糖用量3 g/100 mL,L-PAC产量达到8.79 g/L。在L-PAC的制备过程中,以廉价的乳酸钠代替丙酮酸大大降低了生产成本,缩短了反应时间,简化了操作步骤,有利于工业生产。双酶偶联体系为生物合成L-PAC提供了一个新的思路和新方法。     

两相体系中固定化细胞生物合成L-苯基乙酰基甲醇

目的生物合成L-苯基乙酰基甲醇(L-PAC)。方法在10%石油醚/水两相体系中,利用固定化酵母细胞生物合成L-麻黄素的重要中间体L-PAC。结果确定在最佳温度28℃,pH 6.46,活化时间30 min,反应时间10 h,苯甲醛浓度为3 mL/L,添加少量β-环糊精等的条件下,以2%海藻酸钙和10%聚乙烯醇为复合载体,生物合成L-PAC,产量可达8.34 g/L。结论为生物合成L-PAC提供了一种很好的方法。     

响应面法优化微生物萃取转化制备L-苯基乙酰基甲醇的工艺

目的:优化微生物萃取转化制备L-苯基乙酰基甲醇(L-PAC)的条件。方法:以啤酒酵母萃取制备L-PAC。采用高效液相色谱法测定L-PAC浓度。以L-PAC浓度为响应值,通过Box-Behnken响应面法对苯甲醛、曲拉通(Triton)X-100和葡萄糖用量这3个主要因素进行考察,同时进行验证试验。结果:苯甲醛和Triton X-100之间的交互作用最为显著,最优因素组合为苯甲醛1.1%、Triton X-100 0.14 g/m L、葡萄糖0.028 g/m L;验证试验中L-PAC的平均浓度为28.04 mmol/L(RSD=1.35%,n=3),与预测值28.01mmol/L的相对误差为0.11%。结论:利用响应面法对微生物萃取转化制备L-PAC的条件进行了优化,得到了各因素的最优组合,可为大批量转化制备L-PAC提供有利参考。     

水/有机相两相体系中Saccharomyces cerevisiae B5不对称还原制备手性药物中间体R-2'-氯-1-苯乙醇的研究

The process of preparation of chiral pharmaceutical intermediate R-2'-chloro-1-phenyl-ethanol with the method of Saccharomyces cerevisiae B5 asymmetric reduction in water/organic diphasic system was investigated in detail. The result shows that the higher     

利用双水相系统转化雄甾-1,4-二烯-3,17-二酮的研究

利用混合植物甾醇(45％β-谷甾醇，30％豆甾醇，25％菜油甾醇)作为底物筛选适合菌株Mycobacteri-um HCCB006转化生成雄甾-1，4-二烯-3，17-二酮(ADD)的双水相系统，并用表面活性剂对系统进行进一步的优化。经初步研究得到的最佳转化系统为16％ PEG20000／4％Dextran20 000／8％OP，从而使得在底物加量为l％时ADD的产量达到2．858g／L。     

Biotransformation of trans-1,1,1,3-tetrafluoropropene (HFO-1234ze)

trans-1,1,1,3-Tetrafluoropropene (HFO-1234ze) is a non-ozone-depleting fluorocarbon replacement with a low global warming potential and is developed as foam blowing agent. The biotransformation of HFO-1234ze was investigated after inhalation exposure. Male Sprague-Dawley rats were exposed to air containing 2000; 10,000; or 50,000 ppm (n = 5/concentration) HFO-1234ze. Male B6C3F1 mice were only exposed to 50,000 ppm HFO-1234ze. All inhalation exposures were conducted for 6 h in a dynamic exposure chamber. After the end of the exposures, animals were individually housed in metabolic cages and urines were collected at 6 or 12 h intervals for 48 h. For metabolite identification, urine samples were analyzed by ¹H-coupled and ¹H-decoupled ¹⁹F-NMR and by LC/MS-MS or GC/MS. Metabolites were identified by ¹⁹F-NMR chemical shifts, signal multiplicity, ¹H-¹⁹F coupling constants and by comparison with synthetic reference compounds. In urine samples of rats exposed to 50,000 ppm HFO-1234ze, the predominant metabolite was S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid and accounted for 66% of all integrated ¹⁹F-NMR signals in urines. No ¹⁹F-NMR signals were found in spectra of rat urine samples collected after inhalation exposure to 2000 or 10,000 ppm HFO-1234ze likely due to insufficient sensitivity. S-(3,3,3-Trifluoro-trans-propenyl)-l-cysteine, N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-l-cysteine and 3,3,3-trifluoropropionic acid were also present as metabolites in urine samples of rats and mice. A presumed amino acid conjugate of 3,3,3-trifluoropropionic acid was the major metabolite of HFO-1234ze in urine samples of mice exposed to 50,000 ppm and related to 18% of total integrated ¹⁹F-NMR signals. Quantification of three metabolites in urines of rats and mice was performed, using LC/MS-MS and GC/MS. The quantified amounts of the metabolites excreted with urine in both mice and rats, suggest only a low extent (< 1% of dose received) of biotransformation of HFO-1234ze and 95% of all metabolites were excreted within 18 h after the end of the exposures (t_1/2 app. 6 h). The obtained results suggest that HFO-1234ze is likely subjected to an addition-elimination reaction with glutathione and to a CYP 450 mediated epoxidation at low rates.     

Biotransformation of 2,3,3,3-tetrafluoropropene (HFO-1234yf) in rabbits

2,3,3,3-Tetrafluoropropene (HFO-1234yf) is a non-ozone-depleting fluorocarbon replacement with a low global warming potential and is developed as refrigerant. Due to lethality observed after high concentration inhalation exposures of HFO-1234yf in a developmental toxicity study with rabbits, the biotransformation of HFO-1234yf was investigated in this species. Female New Zealand White rabbits were exposed to air containing 2000; 10,000; or 50,000 ppm (n = 3/concentration) HFO234yf. All inhalation exposures were conducted for 6 h in a dynamic exposure chamber. Animals were individually housed in metabolic cages after the end of the exposures and urines were collected at 12 h intervals for 60 h. For metabolite identification, urine samples were analyzed by ¹H-coupled and ¹H-decoupled ¹⁹F-NMR and by LC/MS-MS or GC/MS. Metabolites were identified by ¹⁹F-NMR chemical shifts, signal multiplicity, ¹H-¹⁹F coupling constants and by comparison with synthetic reference compounds. In urine samples of rabbits exposed to 2000; 10,000; or 50,000 ppm HFO-1234yf, the predominant metabolite was N-acetyl-S-(3,3,3-trifluoro-2-hydroxypropanyl)-l-cysteine and accounted for app. 48% of total ¹⁹F-NMR signal intensities. S-(3,3,3-Trifluoro-2-hydroxypropanyl)mercaptolactic acid, 3,3,3-trifluoro-1,2-dihydroxypropane, 3,3,3-trifluoro-2-propanol and inorganic fluoride were also present as urinary metabolites. In incubations of rabbit liver S9 fractions containing glutathione, NADPH and HFO-1234yf, 3,3,3-trifluoro-1,2-dihydroxypropane, S-(3,3,3-trifluoro-2-hydroxypropanyl)glutathione, 3,3,3-trifluoro-2-propanol and inorganic fluoride were identified as metabolites of HFO-1234yf by ¹⁹F-NMR. The quantity of recovered metabolites in urine suggest a low extent (< 0.1% of dose received) of biotransformation of HFO-1234yf in rabbits, and 95% of all metabolites were excreted within 12 h after the end of the exposures (t_1/2 app. 9.5 h). The obtained results indicate that HFO-1234yf is metabolized in rabbits by a CYP450-mediated epoxidation at low rates and glutathione conjugation of the epoxide. The differences in urinary metabolite patterns between rats and rabbits seen with HFO-1234yf are likely due to species-specific processing of glutathione S-conjugates. Rabbits also show a larger extent of biotransformation of HFO-1234yf.     

Biotransformation of 2,3,3,3-tetrafluoropropene (HFO-1234yf) in male, pregnant and non-pregnant female rabbits after single high dose inhalation exposure

2,3,3,3-Tetrafluoropropene (HFO-1234yf) is a novel refrigerant intended for use in mobile air conditioning. It showed a low potential for toxicity in rodents studies with most NOAELs well above 10,000 ppm in guideline compliant toxicity studies. However, a developmental toxicity study in rabbits showed mortality at exposure levels of 5,500 ppm and above. No lethality was observed at exposure levels of 2,500 and 4,000 ppm. Nevertheless, increased subacute inflammatory heart lesions were observed in rabbits at all exposure levels. Since the lethality in pregnant animals may be due to altered biotransformation of HFO-1234yf and to evaluate the potential risk to pregnant women facing a car crash, this study compared the acute toxicity and biotransformation of HFO-1234yf in male, female and pregnant female rabbits. Animals were exposed to 50,000 ppm and 100,000 ppm for 1 h. For metabolite identification by ¹⁹F NMR and LC/MS-MS, urine was collected for 48 h after inhalation exposure. In all samples, the predominant metabolites were S-(3,3,3-trifluoro-2-hydroxypropanyl)-mercaptolactic acid and N-acetyl-S-(3,3,3-trifluoro-2-hydroxypropanyl)-L-cysteine. Since no major differences in urinary metabolite pattern were observed between the groups, only N-acetyl-S-(3,3,3-trifluoro-2-hydroxypropanyl)-L-cysteine excretion was quantified. No significant differences in recovery between non-pregnant (43.10 ± 22.35 μmol) and pregnant female (50.47 ± 19.72 μmol) rabbits were observed, male rabbits exposed to 100,000 ppm for one hour excreted 86.40 ± 38.87 μmol. Lethality and clinical signs of toxicity were not observed in any group. The results suggest that the lethality of HFO-1234yf in pregnant rabbits unlikely is due to changes in biotransformation patterns or capacity in pregnant rabbits.     

头孢菌素类抗生素在乙腈/NaCl双相体系中的分配

目的研究几种头孢菌素类抗生素在亲水有机溶剂、无机盐和水组成的双相体系(乙腈/NaCl体系)中的分配规律。方法用滴定法制作体系的相图,HPLC测定头孢菌素在两相中的浓度。结果头孢菌素浓度和温度对头孢菌素的分配影响不大;NaCl浓度和乙腈浓度则有一定影响,两者浓度的增加都使头孢菌素向下相(盐相)转移;pH对头孢菌素的分配有较大影响,pH很低时头孢菌素主要分配在上相(乙腈相),pH升高到3.8左右大部分头孢菌素即转移到下相。结论通过调节pH,用亲水有机溶剂/盐双相体系萃取分离头孢菌素类抗生素是可行的。     

双时相^18F-氟代脱氧葡萄糖PET／CT在鼻咽癌诊断中的应用价值

目的初步探讨双时相^18F-氟代脱氧葡萄糖（^18F—FDG）PET／CT在鼻咽癌诊断中的应用价值。方法36例经病理明确诊断的鼻咽癌患者及39例鼻咽炎症或淋巴增生患者行双时相^18F—FDGPET／CT全身检查,测定病灶部位初始及延迟标准放射性摄取值（SUVmax）并行半定量分析,观察分析相应CT、PET形态。结果36例晚期鼻咽癌患者病灶区初始SUVmax、延迟SUVmax值分别为：10．6±4．1、11．7±4．6。延迟SUVmax明显高于初始扫描,病灶呈不规则形态;39例鼻咽部炎症或淋巴增生患者病灶区SUVmax、延迟SUVmax值分别为：4．0±1．2、3．8±1．3,延迟SUVmax值低于初始扫描,病灶形态多表现为双侧对称的条状,呈“八”字形。结论双时相PET／CT有助于鼻咽癌的诊断,也有利于鼻咽癌与鼻咽炎症或淋巴增生的鉴别诊断,对鼻咽疾病诊治中有良好的医学前景。     

大鼠心肌多胺代谢限速酶ODC、SSAT活性分析

目的建立大鼠心肌多胺代谢限速酶鸟氨酸脱羧酶(ODC)及精脒/精胺乙酰转移酶(SSAT)活性分析方法。方法以Langendorff离体灌流心肌为实验材料,制备心肌组织匀浆;分别以dl[114C]Ornithine及[114C]acetylCoenzymeA为底物,以液体闪烁计数仪记录生成的14CO2及[14C]acetylspermidine的放射活度,并以其代表ODC,SSAT的活性;计算大鼠心肌ODC、SSAT的酶促反应动力学参数,筛选出适宜的底物浓度;同时观察一氧化氮(NO)供体硝普钠(SNP)对酶活性的影响。结果①大鼠心肌ODC、SSAT基础活性分别为:(9.67±3.09)nmol·mg-1Pro·h-1;(3.59±0.91)nmol·mg-1Pro·min-1。②ODC催化LOrnithine的酶促反应动力学参数Km=(54.95±8.14)μmol·L-1;Vmax=(2.364±0.37)nmol·mg-1·h-1;SSAT催化AcetylCoenzymeA的酶促反应动力学参数Km=(12.87±1.88)μmol·L-1;Vmax=(0.50±0.07)nmol·mg-1·min-1。③大鼠心肌ODC、SSAT活性检测的底物浓度分别为:90μmol·L-1(18.5kBq)DL[114C]Ornithine及36μmol·L-1(2.96kBq)[114C]acetylCoA。④SNP呈浓度依赖性地抑制ODC的活性、诱导SSAT的活性。结论建立了大鼠心肌多胺代谢限速酶鸟氨酸脱羧酶(ODC)及精脒/精胺乙酰转移酶(SSAT)活性的分析方法,该方法简便易行;根据Km值确定测定大鼠心肌ODC及SSAT?     

Kinetics of 3-(4-methylbenzylidene)camphor in rats and humans after dermal application

The toxicokinetics of 4-MBC after dermal administration were investigated in human subjects and in rats. Humans (3 male and 3 female subjects) were exposed to 4-MBC by topical application of a commercial sunscreen formulation containing 4% 4-MBC (w/w), covering 90% of the body surface and resulting in a mean dermal 4-MBC dose of 22 mg/kg bw. In rats, dermal 4-MBC doses of 400 and 2000 mg/kg bw were applied in a formulation using an occlusive patch for 24 h. Concentrations of 4-MBC and its metabolites were monitored over 96 h in plasma (rats and humans) and urine (humans). In human subjects, plasma levels of 4-MBC peaked at 200 pmol/ml in males and 100 pmol/ml in females 6 h after application and then decreased to reach the limit of detection after 24 h (females), respectively, 36 h (males). After dermal application of 4-MBC, peak plasma concentrations of 3-(4-carboxybenzylidene)-6-hydroxycamphor were 50-80 pmol/ml at 12 h and of 3-(4-carboxybenzylidene)camphor were 100-200 pmol/ml at 24 h. In male and female rats, peak plasma levels of 4-MBC were 200 (dose of 400 mg/kg bw) and 1 200 pmol/ml (dose of 2000 mg/kg bw). These levels remained constant for up to 24-48 h after dermal application. Peak plasma concentrations of 3-(4-carboxybenzylidene)-6-hydroxycamphor were 18,000 pmol/ml (males) and of 3-(4-carboxybenzylidene)camphor were 55,000 pmol/ml (females) between 48 and 72 h after application of the high dose of 4-MBC. In human subjects, only a small percentage of the dermally applied dose of 4-MBC was recovered in the form of metabolites in urine, partly as glucuronides. The obtained results suggest a more intensive biotransformation of 4-MBC in rats as compared to humans after dermal application and a poor absorption of 4-MBC through human skin.