Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nα‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side‐chain and backbone anchoring strategies,and compatible protection for N‐terminal cysteine*,†

Abstract: Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C‐terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N^α‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N^α‐t‐butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side‐chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N‐terminal cysteine residue have been developed: (i) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐(N‐methyl‐N‐phenylcarbamoyl)sulfenylcysteine [Fmoc‐Cys(Snm)‐OH], allowing creation of an otherwise fully protected resin‐bound intermediate with N‐terminal free Cys; (ii) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐triphenylmethylcysteine [Fmoc‐Cys(Trt)‐OH], generating a stable Fmoc‐Cys(H)‐peptide upon acidolytic cleavage; and (iii) incorporation of N^α‐t‐butyloxycarbonyl‐S‐fluorenylmethylcysteine [Boc‐Cys(Fm)‐OH], generating a stable H‐Cys(Fm)‐peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C‐termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on‐resin, as well as in solution after cleavage/purification.     

Influence of moderate hypothermia on cerebral oxygenation in pigs with intracranial hypertension

BACKGROUND: Moderate hypothermia is one of the effective therapeutic methods for head injury in recent years, there are many mechanisms of moderate hypothermia for brain protection, and its influence on cerebral oxygenation is also one of them. OBJECTIVE: To observe the influence of moderate hypothermia on cerebral oxygenation of animals with acute intracranial hypertension, and further investigate the protective mechanism of moderate hypothermia. DESIGN: A randomized controlled trial. SETTING: Department of Neurosurgery, Renji Hospital affiliated to the Medical College of Shanghai Jiao Tong University. MATERIALS: Twenty healthy little pigs, either male or female, weighing 4.5–5.5 kg, were used. Neurotrend-typed multiparameter monitoring system (Diametrics Company, British); CMA/100 micro-injection pump (Carnegie Company, Sweden). METHODS: The experiment was conducted in the Changzheng Hospital affiliated to the Second Military Medical University of Chinese PLA in November, 2001. The pigs were randomized into two groups: the normothermia group (control group, n =10) and moderate hypothermia group (n =10). ① Bilateral femoral arteries were separated, one was connected to pressometer for monitoring mean arterial pressure (MEP), and the other for analysis of blood gases [including peripheral blood pH value, arterial partial pressure of carbon dioxide (PaCO2), arterial partial pressure of carbon dioxide (PaCO2), HCO3–]. ② Rectal temperature was monitored with mercurial thermometer. ③ Intracranial pressure was monitored using Camino optic ICP probe placed in the subdural space. ④ Neurotrend multiparameter monitoring sensor was inserted into the white matter for about 4 cm to determine cerebral perfusion pressure (CPP, CPP=MAP(ICP), brain tissue partial oxygen pressure (PO2), partial pressure of carbon dioxide (PCO2), HCO3– and brain temperature. The rectal temperature of animals in the moderate hypothermia group was lowered to 34 ℃ using ice bags, and the body temperature was maintained at 33–35 ℃ for 2 hours. The changes of the parameters were observed continuously, and the pigs in the normothermia group were not treated with cooling. MAIN OUTCOME MEASURES: ① MAP, ICP, rectal temperature, CCP; Indexes of cerebral oxygenation detected with Neurotrend-typed multiparameter monitoring system; ② Results of blood gases analysis in the moderate hypothermia group. RESULTS: All the 20 pigs were involved in the analysis of results. ① MAP, ICP, rectal temperature, CCP and indexes of cerebral oxygenation: In the moderate hypothermia group, the ICP after cooling was obviously lower than that before cooling [(3.31±1.19), (5.33±0.95) kPa, P < 0.05], CCP was higher, brain tissue PCO2 [(12.03±1.73), (10.59±2.01) kPa, P < 0.05], and brain tissue pH value was higher [(7.03±1.63), (9.40±1.30) kPa, P < 0.05], whereas the brain temperature was decreased as compared with that before cooling [(34.9±0.3), (37.2±0.2) ℃, P < 0.05]. ② Results of blood gases analysis in the moderate hypothermia group: There were no significant differences in the parameters of peripheral arterial blood gases analysis before and after cooling in the moderate hypothermia group (P > 0.05) CONCLUSION: Moderate hypothermia will not impair the cerebral oxygenation, and it can reduce brain tissue CO2 and decrease brain tissue acidosis.     

芩连四物汤化学物质基础及其抗凝血活性研究

目的:研究芩连四物汤的化学物质基础及其抗凝血生物效应。方法:采用水煎醇沉,醇溶液减压回收乙醇,用适量水溶解后,上D101大孔树脂采用不同浓度的乙醇梯度洗脱,各不同洗脱部位采用硅胶柱层析的方法分离,所得单体通过UV,IR,NMR及MS等方法确定其化学结构;将所得成分配成水溶液(100μmol·L^-1),采用体外法测定对血浆凝血酶时间(TT)的影响。结果:从芩连四物汤中分离并鉴定了5个黄酮类成分、1个生物碱、3个芳香酸、1个倍半萜类成分及2个其它类化合物。黄酮苷元、芳香酸、生物碱及倍半萜类化合物均能显著延长血浆凝血酶时间(TT)。结论:首次对芩连四物汤整方进行化学物质分离和结构分析研究,其中9个化合物,包括千层纸素A、汉黄芩素、6-甲氧基汉黄芩素、小檗碱、黄芩素、阿魏酸、香草酸、咖啡酸、地黄苦苷元显示了一定的体外抗凝血活性。     

家兔颈后肌受长期应力作用的实验研究

目的 建立颈后肌受长期应力作用动物模型并进行相关研究.方法 30只家兔随机分为模型组和对照组,每组15只.模型组家兔置于特制低头位框架中,低头角度为45度,每天1次,每次5小时;对照组除不低头外余条件相同.12周后分别进行颈椎X线、颈后部红外热像图、血清肌酸激酶和乳酸脱氢酶含量以及颈后肌超氧化物歧化酶和丙二醛含量观察.结果 模型组颈椎X线改变积分值实验后明显高于实验前和对照组,差异具有显著性;模型组与对照组颈后部红外热像图有明显差异;模型组血清肌酸激酶、乳酸脱氢酶的含量均明显高于对照组;模型组丙二醛含量明显高于对照组而超氧化物歧化酶含量明显低于对照组.结论 家兔颈后肌受长期应力作用后出现颈部的生物力学和生物化学失衡.     

谷胱甘肽、自由基在砷剂诱导卵巢癌细胞凋亡中的作用

目的　观察谷胱甘肽、自由基在砷剂诱导卵巢癌细胞凋亡中的作用。方法　砷剂孵育卵巢癌细胞 4 8h后 ,琼脂糖凝胶电泳及流式细胞仪检测细胞凋亡 ,分光光度法检测细胞内谷胱甘肽、活性氧及丙二醛含量。结果　砷剂具有诱导卵巢癌细胞凋亡作用 ,砷剂诱导后细胞内谷胱甘肽含量减少 (P <0 .0 1)、活性氧及丙二醛含量增加(P <0 .0 5 )。结论　氧化还原系统的改变是砷剂诱导卵巢癌细胞凋亡的重要途径之一。     

活性氧簇在皮炎湿疹发病中的作用

活性氧簇是重要的氧的衍生物,氧化还原反应状态的平衡对维持机体的内环境起着重要的作用.皮肤是氧应激损害的主要靶器官,过多活性氧造成组织损伤,其主要通过影响核因子-κB、NO、蛋白激酶介导的磷酸化和基因异常表达发挥作用.多种炎症性皮肤疾病与活性氧簇相关,活性氧簇在皮炎、湿疹的发病中起着重要作用,抗氧化剂有望成为治疗这类疾病的新途径.     

Kv2.1钾通道在神经元缺氧损伤中的潜在药理学作用

 目的观察钾通道阻断剂四乙铵(TEA)对缺氧缺血损伤整体、离体模型的保护作用,阐明Kv2.1电压依赖性钾通道在缺氧缺血细胞损伤中发挥的作用。方法应用大鼠暂时性大脑中动脉阻塞(tMCAO)脑缺血模型验证TEA对脑缺血损伤的保护作用。采用膜片钳技术观察氧糖剥夺(OGD)对稳定转染Kv2.1钾通道的人胚胎肾293(HEK293)细胞膜电位以及TEA对OGDKv2.1-HEK293细胞钾电流的影响。MTT法观察OGD对Kv2.1-HEK293的损伤及TEA的保护作用。结果TEA(5μg·kg^-1)侧脑室注射可以显著减小tMCAO大鼠的脑梗死体积;应用转基因细胞的研究证实Kv2.1-HEK293对OGD损伤的敏感性明显提高;OGD可以降低Kv2.1-HEK293细胞膜电位;TEA(10mmol·L^-1)能显著抑制OGDKv2.1-HEK293钾电流,同时使其所受的细胞损伤降低。结论钾通道阻断剂TEA对整体和离体缺氧缺血损伤模型均发挥细胞保护作用,这种保护作用与对Kv2.1的阻断密切相关;提示Kv2.1可能是抗脑缺血药物开发的潜在靶点。     

版纳藤黄化学成分的研究

目的:研究版纳藤黄Garcinia xipshuanbannaensis枝叶的化学成分.方法:利用正相硅胶、反相RP-18柱色谱及葡聚糖凝胶Sephadex LH-20等手段进行分离纯化,根据波谱数据鉴定化合物的结构.结果:分离鉴定了15个化合物,分别为bannaxanthone E(1),大叶藤黄醇(xanthochymol,2),异大叶藤黄醇(isoxanthochymol,3),环大叶藤黄醇(cycloxanthochymol,4),osajaxanthone(5),gentisein(6),mangostinone(7),山柰酚(8),槲皮素(9),牡荆素(10),2"-O-acetylvitexin(11),3-乙酰齐墩果酸(12),(-)-表儿茶素[(-)-epicatechin,13],β-谷甾醇(14),胡萝卜苷(15).结论:化合物4～9,11～13为首次从该种植物分离得到,化合物11～13为首次从该属植物分离得到.     

化学实验进行开放式教学的研究

通过与传统的化学实验教学模式的比较，详述了开放式教学模式的特点，并且针对实施过程中所遇到的问题提出了详尽的解决方案。