肝素非还原端饱和结构的紫外吸收研究及在肝素寡糖测序中的应用

为了进一步探讨非还原端饱和结构的肝素寡糖在UV 232 nm的吸收情况, 制备了4种饱和结构的肝素二糖, 并用离子对反相液相色谱/离子阱飞行时间质谱(RPIP-LC/MS-IT-TOF)光电二极管阵列检测器分析了它们在UV 232 nm的吸收情况. 分析结果表明, 饱和结构的肝素二糖在UV 232 nm的检出限为9 μg(S/N=10), UV 232 nm/UV 206 nm约为不饱和结构肝素二糖UV 232 nm的7%~40%. 结果还表明, 肝素二糖UV 232 nm的吸收强度受亚硫酸基团(SO₃^2?)影响较大. 另外, 通过比较不饱和结构的肝素/硫酸类肝素(Hep/HS)标样二糖发现, 含N-未取代葡萄糖胺(GlcNH₃⁺)基团的二糖在UV 232 nm的吸收值较低. 最后, 通过简单的UV检测方法, 结合 HNO₂(pH=4.0)裂解法和RPIP-LC/MS-IT-TOF分析, 简化了含GlcNH₃⁺肝素六糖的测序方法. 本研究为以后用 HNO₂(pH=1.5)裂解法对混合组分N-硫酸化的肝素寡糖结构序列分析提供了可能.     

临床肝素类药物酶解分析二糖组成

肝素是一类结构复杂的高分子糖类,以N-硫酸、6-O硫酸氨基葡萄糖和2-O硫酸艾杜糖醛酸为主要成分组成二糖。常见的肝素注射液是以未分级肝素为原料纯化灭菌制备而来,在临床上使用更为广泛的是将肝素降解得到的低分子片段,低分子肝素保留了肝素糖链基本结构,但是不同制备工艺导致其具有不同的还原及非还原端。本研究以肝素类药物为研究对象,使用肝素裂解酶玉,域及芋将肝素注射液、低分子肝素注射液(达肝素、那屈肝素、依诺肝素)、化学合成的类肝素(磺达肝素)进行酶催化降解产生二糖,结合强阴离子交换色谱( Strong anion exchange high performance liquid chromatography,SAX-HPLC)以及紫外检测器在线分析,使用市售肝素二糖标准品确定各种二糖结构。此外,使用反向离子对色谱和电喷雾质谱联用分析磺达肝素中的甲基二糖以及达肝素和那屈肝素中的脱氨二糖结构,为肝素以及类肝素药物的质量控制提供更为精确的结构信息。     

电喷雾质谱在寡糖序列分析中的应用

选取具有不同结构特征的N-糖链、硫酸软骨素寡糖、人乳寡糖以及海洋来源的壳寡糖、褐藻胶寡糖、卡拉胶寡糖和硫酸岩藻寡糖等,对电喷雾质谱在寡糖的主链序列、分支位点、硫酸基取代位置确定、单糖组成和聚合度分析等方面的应用技术及碎片离子的断裂规律进行了总结.根据相邻同类碎片离子之间的质荷比差值可初步判断寡糖的单糖组成类型;通过与色谱分离技术联用或衍生化方法可提高寡糖的分辨率和离子化效率,并测得寡糖的分子量及聚合度;借助串联质谱及对寡糖还原端的特异性标记,可获得寡糖的还原端残基和部分序列信息;根据寡糖产生的特征碎片离子及其丰度大小可判断残基的特定位置和类型.另外,寡糖的分支通常作为一个整体发生糖苷键断裂或产生D离子,据此可判断分支点的位置;根据硫酸寡糖产生的特异性跨环断裂碎片,可以确定硫酸基的连接位置.这些规律和方法的总结为未知寡糖的结构和序列的分析提供了启发和指导.     

基于MALDI-TOF质谱技术分析人宫颈癌 HeLa细胞N-糖链结构轮廓

以微量HeLa细胞(10⁷个)为对象, 经细胞裂解、还原羧甲基化、胰酶降解和Oasis-HLB柱提取分离得到总糖肽后, 用PNGase F酶解释放N-糖链. 对所得N-糖链用Sep-Pak C₁₈柱纯化后进行完全甲基化衍生, 再采用基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF MS)分析HeLa细胞表面N-糖链的结构轮廓. 结果表明, 在获得的34种N-糖链中, 除高甘露糖型、二天线、三天线、四天线和五天线等N-糖链外, 还出现了在某种程度上与肿瘤发生转移相关的特殊平分型和Lewis结构. 利用MALDI-TOF MS技术可快速分析微量癌细胞表面N-糖链的结构轮廓, 为进一步寻找肿瘤糖链标志物及肿瘤的早期预防诊断提供技术支持.     

痕量完整天然寡糖链的液态二次离子质谱测定

痕量、未衍生、完整的天然寡糖可用质谱法直接获得重要的结构信息。用负离子LSIMS测出了n mol级IgM中的高甘露糖型和杂交型糖链分子量及序列。对麦芽七糖、异麦芽七糖、古洛糖醛酸十聚体及若干天然寡糖谱图中碎片离子进行了研究,从糖链混合物的质谱图中也能分析出有用的分子结构。     

离子肼飞行时间质谱表征硫酸软骨素/硫酸角质素二糖

硫酸软骨素(CS)和硫酸角质素(DS)结构决定它与蛋白的相互作用。因此对CS/DS结构的解析,能够进一步阐明结构与功能之间的关系。采用电喷雾离子阱多级质谱技术(ESI-MSn)在负离子模式(ESI-)下表征8种CS/DS二糖裂解规律。二糖的裂解发生在糖苷键和糖环位置,N-乙酰半乳糖胺上主要断裂方式为0,2X,而糖醛酸主要断裂方式为0,2Z。所含亚硫酸根越多越不稳定。亚硫酸根的位点决定了糖结构稳定性,含4位氧亚硫酸的二糖相对最不稳定。此外糖苷键的位置也决定了糖结构的稳定性。二级质谱表明同分异构体有其特定的裂解规律。该质谱裂解规律有助于CS/DS二糖同分异构体的鉴定。     

肝素类寡糖合成研究进展

肝素(heparin,HP)和硫酸乙酰肝素(heparan sulfate,HS)是糖胺聚糖(glycosaminoglycans,GAGs)家族中的一类线性硫酸化多糖,其结构复杂、牛物活性多样.为了深入研究其结构序列与小同生物活性之间的关系,近年来针对肝素类寡糖的合成与功能研究成为糖化学和糖牛物学研究的一个热点领域,涌现出一些新的合成方法与合成策略.选取了2001年以来国际上一些在肝素类寡糖的化学合成和酶法合成方面的代表性工作予以综述.     

脱水糖苷化方法合成肝素酶底物寡糖

乙酰肝素酶（Heparanase,Hpa）是哺乳动物体内的内切葡萄糖醛酸苷水解酶,通过水解葡萄糖醛酸（GlcA）与己胺糖（GlcN）之间的β-糖苷键,选择性地降解肝素和硫酸肝素糖胺聚糖,从而释放多功能的肝素寡糖.本文报道一条高效的肝素酶底物寡糖的合成路线：采用苯甲酰基保护待硫酸化的羟基,苄基保护羧基和裸露的羟基,叠氮基保护氨基,应用脱水糖苷化方法高效地构建关键的a-GlcN-（1→4）-GlcA糖苷键.然后通过标准化的保护基脱除和硫酸化操作,获得肝素酶底物三糖和四糖1-4.最后五步反应的总收率超过52%.肝素酶底物寡糖的合成为研究肝素酶的底物选择性和活性检测打下了基础.     

含酯糖链皂甙的负离子快原子轰击质谱分析

本实验用负离子快原子轰击(FAB)法测定了8个含酯糖链的双糖链皂甙样品,给出了明显的脱质子分子离子峰,由碎片离子峰可推测糖基的连接顺序;不仅对难挥发、热不稳定和极性大的样品可直接进样分析,而且对在酯糖链皂甙中出现以酯裂解为基峰的碎片,还可初步确定糖链在甙元上的连接位置。     

基于电喷雾电离质谱的人肝癌细胞HepG2与正常肝细胞L02的N-糖链的定性定量比较

以培养的原发性肝癌细胞HepG2和正常肝细胞L02为研究对象,采用细胞裂解液提取总蛋白,用PNGase F酶解释放N-糖链,以微晶纤维素柱结合石墨碳柱纯化分离N-糖链,通过电喷雾电离质谱(ESI-MS)和串联质谱(MS/MS)对N-糖链进行序列鉴定,以β-环糊精为内标对2种细胞系的N-糖链进行了定量比较分析.结果表明,在肝癌细胞系HepG2和正常细胞系L02中共检测到26种N-糖链,与L02相比,HepG2的大多数高甘露糖型糖链、唾液酸化糖链和岩藻糖基化糖链的数量都明显升高,其中有15种糖链在数量上具有极显著性差异(p0.01),1种糖链具有显著性差异(p0.05).本研究为进一步探索肝癌中各类N-糖链的表达特点及发现早期肝癌糖链标志物提供了参考.     

二氧化碳和丙烯直接合成甲基丙烯酸的NiPMo/TiO2催化剂的研究

用溶胶-凝胶法以磷钼酸(MPA)的镍盐溶液水解钛酸四丁酯制备了NiPMo/TiO2催化剂.使用ICP、 XRD、 TG-DTA、 IR、 TPD-MS和微反应技术研究了催化剂的化学组成、热稳定性、化学吸附性质和催化反应性能.杂多钼酸盐与TiO2通过O2-在TiO2表面发生了键合.在623 K下,杂多阴离子仍保持原有的Keggin结构.CO2在Lewis酸位Ni(Ⅱ)和Lewis碱位Ni-O-Mo的桥氧协同作用下生成CO2卧式吸附态Ni(Ⅱ)←O-(CO)←(O--Ni).丙烯有多种吸附态在催化剂上吸附.在563 K、 1 MPa和空速1500 h-1的反应条件下,丙烯的摩尔转化率为3.2%,产物MAA选择性为95%.     

Synthesis of 1-benzyloxypyrazin-2(1H)-one derivatives

Different approaches for the synthesis of 1-benzyloxypyrazin-2(1H)-one derivatives from simple amino acids have been investigated. A library of 33 precursors for the preparation of N-hydroxy pyrazinones was obtained in moderate to good yields.     

A general and convenient synthesis of 4-(tosylmethyl)semicarbazones and their use in amidoalkylation of hydrogen,heteroatom, and carbon nucleophiles

A general synthesis of previously unknown semicarbazone-based α-amidoalkylating reagents, 4-(tosylmethyl)semicarbazones, has been developed. The synthesis involved three-component condensation of semicarbazones of aliphatic or aromatic aldehydes with the same or other aldehydes and p-toluenesulfinic acid. The scope and limitations of this reaction were investigated. The compounds obtained were demonstrated to be an efficient α-(4-semicarbazono)alkylating agents. They were reacted with H- (sodium borohydride), O- (sodium methylate), S- (sodium phenylthiolate), N- (pyrrolidine, sodium succinimide), P- (trialkyl phosphites), and C-nucleophiles (sodium diethyl malonate) to give the corresponding products of the tosyl group substitution, 4-substituted semicarbazones, including analogues of nitrofurazone. Among the prepared compounds tested in vitro for antibacterial and antifungal activity, three nitrofuryl-containing semicarbazones exhibited high biological activities with minimum inhibitory concentration (MIC) values of 8–32 μg/mL.     

Synthesis of novel chiral bidentate hydroxyalkyl-N-heterocyclic carbene ligands and their application in palladium-catalyzed Mizoroki–Heck couplings and asymmetric addition of diethylzinc to benzaldehyde

A small library of new chiral bidentate hydroxyalkyl-imidazolium salts 1 is conveniently synthesized on multi-gram scale from inexpensive and commercially available chiral pool amino acids. The corresponding carbenes, generated by deprotonation of imidazolium salts 1, in combination with palladium(II) chloride were tested in the Mizoroki–Heck coupling reaction. The most significant results in terms of yields and reactivities were achieved with low catalyst loading. The catalytic activities of these imidazolium salts were also investigated in the asymmetric addition of diethylzinc to benzaldehyde. The use of MgO nanoparticles as an additive in conjunction with these ligands played a crucial role in increasing the efficiency of these reactions.     

α-Amino acid derived enaminones and their application in the synthesis of N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates and other heterocycles

A new and simple synthesis of novel N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates, which exist in equilibrium with their 4-oxo tautomers, has been developed in two steps starting from N-protected α-amino acids. The key intermediates are enaminones, which can also be isolated, characterized, and used for the construction of other functionalized heterocycles, before they spontaneously decompose to pyrrole products. 4-Hydroxypyrroles are prone to partial aerial oxidation but can be efficiently alkylated or reduced to stable polysubstituted pyrrolidine derivatives.     

Specific intramolecular aromatic CH insertion of diazosulfonamides

The chemoselectivity in the intramolecular CH insertion of various diazosulfonamides has been experimentally studied. The results reveal that the aliphatic 1,4-, 1,5-, or 1,6-C(sp³)?H insertions of diazosulfonamides are not accessible, while the aromatic 1,5-C(sp²)?H insertion can be realized specifically by adjusting the diazo-adjacent group. In addition, the general chemoselectivities in the intramolecular CH insertions of diazosulfonyl compounds are summarized. Generally, diazosulfones undergo both aromatic 1,5-C(sp²)?H and aliphatic 1,5- and 1,6-C(sp³)?H insertions, while diazosulfonates undergo aliphatic 1,5- and 1,6-C(sp³)?H insertions. However, diazosulfonamides only undergo aromatic 1,5-C(sp²)?H insertion.     

CoFe2O4自形成磁性液体场致结构化对磁化的影响

The Langevin paramagnetic theory can’t describe the relation between magnetization of ferrofluids and applied magnetic field. The structuralization of ferrofluids, which is considered the main influence factor of the magnetization, is regarded. The part of magnetization works is deposited when the structure is forming. This action influences the magnetization of ferrofluids directly or indirectly. On the base of the “compressing” model, the Langevin function that usually describes the magnetization of ferrofluid is modified, and a well-fitted curve is obtained. An equation of the relation between the equivalent volume fraction after being “compressed” and the intensity of magnetic field is discovered, which approximately describes the process of magnetization. The relation between the approximate initial susceptibility and the volume fraction can be obtained from modified formula.     

Microwave-assisted copper-catalyzed stereoselective ring expansion of three-membered heterocycles with α-diazo-β-dicarbonyl compounds

Microwave-assisted copper-catalyzed ring expansions of three-membered heterocycles with α-diazo-β-dicarbonyl compounds were investigated. Thiiranes generated 3-acyl-5,6-dihydro-1,4-oxathiines in the presence of copper sulfate and trans-3-acyl-5,6-dihydro-1,4-oxathiines as stereospecific products for 1,2-disubstituted cis-thiiranes through an intramolecular S_N2 process. Oxiranes gave rise to 2-acyl-5,6-dihydro-1,4-dioxines under the catalysis of copper hexafluoroacetylacetonate and cis-3-acyl-5,6-dihydro-1,4-dioxines as stereospecific products for 1,2-disubstituted cis-oxiranes via an intimate ion-pair mechanism. The current method provides a direct and simple strategy in efficient preparation of 3-acyl-5,6-dihydro-1,4-oxathiines and 2-acyl-5,6-dihydro-1,4-dioxines, important agents in medicinal and agricultural chemistry, from readily available thiiranes and oxiranes, respectively.     

Highly regioselective Buchwald–Hartwig amination at C-2 of 2,4-dichloropyridine enabling a novel approach to 2,4-bisanilinopyridine (BAPyd) libraries

The highly regioselective Buchwald–Hartwig amination at C-2 of the cheap and readily accessible reagent, 2,4-dichloropyridine with a range of anilines and heterocyclic amines is described. This new methodology is robust and provides a facile access to 4-chloro-N-phenylpyridin-2-amines on 0.25 mol scale. These intermediates undergo a further Buchwald–Hartwig amination at higher temperature to enable rapid exploration of the chemical space at C-4 and to provide a library of 2,4-bisaminopyridines.     

N-Heterocyclic carbene-palladacyclic complexes: synthesis,characterization and their applications in the C-N coupling and α-arylation of ketones using aryl chlorides

N-Heterocyclic carbene-palladacyclic complexes 3 were successfully achieved in a one-pot procedure under mild conditions. The structure of 3a was unambiguously confirmed by X-ray single crystal diffraction and it was an active catalyst in the Buchwald-Hartwig amination and α-arylation of ketones even at very low catalyst loadings (0.01?mol%).