新型核壳型亲水聚合物-硅胶杂化填料的合成及其在蛋白质N-糖链富集中的应用

蛋白质的N-糖基化修饰与多种重要的生理、病理进程密切相关,是多种重大疾病诊断标志物研究的热点。由于糖蛋白本身多是低丰度表达的蛋白质,且糖链结构具有高度微不均一性,这使得蛋白质糖基化修饰的分析具有一定的挑战。本研究利用表面引发-原子转移自由基聚合(SI-ATRP)法,以带有双键的氨基葡萄糖为单体(GMAG),成功制备了新型核壳型亲水聚合物-硅胶杂化填料(pGMAG-SiO₂)。由于在硅胶表面引入致密的亲水聚合物层,该填料不仅保持了硅胶良好的机械强度,而且显著提高了其亲水性,因此非常适合作为亲水填料用于蛋白质的N-糖链富集。以麦芽七糖和鸡卵清蛋白的N-糖链为研究对象,考察了该填料对N-糖链的富集效果,并将该杂化填料成功用于人血浆中糖蛋白N-糖链的富集检测,共鉴定了47种糖型。以上结果表明,pGMAG-SiO₂填料对N-糖链具有较高的亲和性,可以用于N-糖链的高覆盖率鉴定。     

利用亲水富集策略分析人血浆中N-糖基化蛋白及N-糖类型

蛋白质的N-糖基化是最重要的翻译后修饰之一,许多已知的血浆肿瘤诊断标志物及治疗靶标都是N-糖基化蛋白。针对血浆的糖蛋白质组研究有利于发现新的蛋白标志物。然而,血浆蛋白质浓度分布的动态范围非常宽,且同一位点上的糖链存在微观不均一性,影响了血浆中糖蛋白的鉴定效率。本文利用亲水材料ZIC-HILIC制备亲水富集柱分别对人血浆中的N-糖链和N-糖肽进行富集,并结合碱性反相色谱进行肽段的预分离和高准确度质谱分析,最终在健康人的血浆中鉴定到了299个糖基化蛋白、637个糖基化位点,并识别出31种不同的糖型。在这些鉴定到的糖基化位点中,新发现有107个N-糖基化位点(占总位点数的16.8%)。本方法操作简单,可以有效富集N-糖肽和N-糖,为在血浆中寻找糖蛋白和糖链生物标志物提供了可靠的手段。     

固相萃取净化及超高效液相色谱-串联质谱法测定橡胶制品中的13种N-亚硝胺

建立了固相萃取(SPE)净化、超高效液相色谱-串联质谱(UHPLC-MS/MS)测定橡胶制品中13种N-亚硝胺的方法。样品于密闭萃取瓶中于60 ℃下用甲醇超声萃取30 min,C₁₈固相萃取小柱对萃取液进行净化,经C₁₈色谱柱分离,最后用电喷雾正离子(ESI⁺)和多重反应监测模式(MRM)对13种N-亚硝胺进行定性、定量测定。实验中对样品前处理、色谱分离条件和质谱检测条件进行了优化。在优化的实验条件下,橡胶样品中添加N-亚硝基二甲胺(NDMA)与N-亚硝基-二乙基胺(NDEA)为500 μg/kg、其他组分均为50 μg/kg时,各组分的相对标准偏差(RSD,n=7)小于10%;在实际样品中的加标回收率为70.7%~117.0%;方法的检出限(LOD,以10倍标准偏差计)为0.5~500 μg/kg。方法可应用于橡胶制品中13种N-亚硝胺的测定。     

Pronase E酶解释放糖蛋白N-糖链的方法及荧光标记衍生物的LC-MS分析

建立了一种用非特异性酶链酶蛋白酶 E(Pronase E)从糖蛋白上释放N-糖链的方法. 以牛胰核糖核酸酶 B(Ribo B)和鸡白蛋白(Chicken Albumin)为材料, 用Pronase E代替N-糖苷酶 F(PNGase F)释放N-糖链. 当蛋白酶质量与糖蛋白质量比为1∶1时, 得到只带一个天冬氨酸(Asn)的闭环N-糖链, 称其为糖氨酸(glycan-Asn), 这样既为糖链引入了天然的-NH2活性基团, 同时还保持了糖链原有的还原端闭环结构. 以9-氯甲酸芴甲酯(Fmoc-Cl)为衍生试剂对解离后的糖氨酸进行衍生, 采用高效液相色谱-电喷雾质谱联用技术(HPLC-ESI/MS)对Fmoc-Cl糖氨酸衍生物进行分析, 建立了糖蛋白的Pronase E酶解、微量糖氨酸的Fmoc-Cl衍生以及糖氨酸衍生物的HPLC-ESI/MS分析方法, 该方法保持了N-糖链的天然结构, 便于以-NH2为功能基团进一步进行荧光标记、分离制备以及糖链与蛋白质的相互作用研究.     

微量生物样品中糖蛋白N-糖链电喷雾电离质谱分析前处理方法的建立

基于电喷雾电离质谱检测技术,建立了一种可靠、高效、简单,适合于微量糖蛋白N-糖链解离、富集纯化的方法.以糖蛋白牛胰核糖核酸酶(Rib B)和卵清白蛋白(OVA)为模型蛋白直接酶解,比较了4种方法纯化酶解样品的效果.比较了直接酶解和经过聚偏氟乙烯(PVDF)膜富集后酶解微量复杂生物来源样品胎牛血清的酶解效果,最终建立了微量生物样品中糖蛋白N-糖链的质谱分析前处理方法.采用PVDF膜吸附复杂生物样品中的糖蛋白,N-糖苷酶F(PNGase F)酶直接在膜上完成糖链释放(37℃,24 h),采用微晶纤维素柱结合石墨碳柱对糖链进行富集纯化,用于微克级胎牛血清和健康人血清中N-糖链质谱分析的前处理.本方法通用性好,在微量生物样品糖链质谱分析检测的前处理方面具有一定应用价值.     

基于固定化Pronase E酶与质谱的糖链结构分析方法

位点特异性糖链结构的解析,是糖蛋白质结构分析面临的巨大挑战。Pronase E蛋白水解酶,能够降解糖蛋白或糖肽中大部分的氨基酸序列,而保留糖链与少量氨基酸的序列,与色谱、质谱分析等联用,可以实现糖链结构的鉴定,同时,保留的氨基酸序列可以辅助实现修饰位点的识别,两者结合,可以获取位点特异性糖链结构的信息。但Pronase E酶解的缺点是,酶解效率较低,常需要较高浓度的蛋白酶。本实验将Pronase E固定化在基质上,固定化后的Pronase E具备较高的局部浓度,从而实现目标糖蛋白的快速高效酶解。采用核糖核酸酶B作为标准糖蛋白,优化了Pronase E酶切的方案,包括酶切时蛋白与酶的用量比、酶切时间、固定化Pronase E酶的有效贮存时间等;同时优化选择了糖链的富集方法,并对于基质辅助激光解吸飞行时间质谱分析中,糖链适合的基质进行对比选择,从而获得更好的糖链谱图及更为丰富的糖链结构信息。     

基于电喷雾电离质谱(ESI-MS)的苯胺稳定同位素标记对还原性寡糖(链)的定性定量分析方法

建立了一种基于电喷雾电离质谱的苯胺稳定同位素标记对还原性寡糖链进行定性及相对定量分析的研究方法. 用苯胺标记乳糖标准品, 优化了影响标记效率的各种因素, 在弱酸性环境下, 选择糖链/苯胺/硼氢氰化钠的摩尔比为1∶1.2∶10, 于70 ℃反应15 min即可标记完全; 同时考察了4对d0/d5苯胺标记的麦芽糊精寡糖在电喷雾电离质谱中的线性、动态范围以及重现性. 结果表明, 在15倍动态范围内, 相对定量方法呈良好的线性关系(R=0.9986)和重现性(CV=10.20%). 为进一步验证定量方法的可靠性, 将其应用于人奶中游离寡糖(HMOs)和牛奶中游离寡糖(BMOs)的分析. 研究结果表明, 人奶中的乳糖含量高于牛奶, 人奶游离寡糖比牛奶游离寡糖种类复杂, 且岩藻糖基化程度高. 该方法成本低廉, 标记效率高且后处理方法简单方便, 适于微量样品通量化分析, 对差异糖组的研究有重要意义.     

固相萃取-超高效液相色谱-电喷雾串联质谱法同时检测尿样中的麻黄碱和N-甲基麻黄碱

建立了固相萃取-超高效液相色谱-电喷雾串联质谱(SPE-UPLC-ESI MS/MS)联用方法,定量测定尿样中的麻黄碱和N-甲基麻黄碱。样品经Oasis MCX柱提取、纯化和富集后,采用电喷雾(ESI)离子源电离,正离子多反应监测(MRM)模式质谱进行定性和定量分析。麻黄碱和N-甲基麻黄碱在0.0250~2.50 μg/L质量浓度范围内线性关系良好,线性相关系数分别为0.9998和0.9992,提取回收率高于80%,提取效率的RSD小于5.0%,检出限均达到0.01 μg/L,可大大延长尿样检材中麻黄碱和N-甲基麻黄碱的检测周期。结果表明,该方法快速、准确,为尿液中痕量麻黄碱和N-甲基麻黄碱的分析提供了灵敏的分析方法。     

气相色谱-串联质谱法测定肉制品中10种挥发性N-亚硝胺类化合物

建立了气相色谱-串联质谱（GC-MS/MS）检测肉制品中10种挥发性N-亚硝胺类化合物残留量的方法。肉制品样品经同时蒸馏萃取法（SDE）萃取，采用冷冻去脂净化法，在多反应监测模式下分析，外标法定量。结果表明，采用优化后的条件，10种挥发性N-亚硝胺类化合物在1.00~1000 μg/L范围内线性关系良好，相关系数均在0.99以上。方法的检出限（LOD，S/N=3）和定量限（LOQ，S/N=10）分别为0.01~0.02 μg/kg和0.04~0.07 μg/kg。选取3种不同类型的肉制品（火腿肠、中式香肠和腌渍腊肉），在空白样品添加水平为LOQ水平、1.0、2.0 μg/kg时，10种挥发性N-亚硝胺类化合物的平均回收率为74.8%~94.3%，相对标准偏差小于8.3%。市售3类肉制品中6种挥发性N-亚硝胺类化合物（N-亚硝基二甲胺、N-亚硝基二乙胺、N-亚硝基吡咯烷、N-亚硝基哌啶、N-亚硝基二丁胺、N-亚硝基二丙胺）均有不同程度检出，且腌渍腊肉中每种挥发性N-亚硝胺类化合物的检出值最高。该方法操作简单，萃取充分，灵敏度高，试剂用量少，可满足实验室大量样品的日常检测需求。     

配体结构对含2-甲基咪唑及N-乙基咪唑的NAMI 衍生物水解及溶液稳定性的影响

制备并用UV、循环伏安(CV)和NMR 法研究了NAMI(新抗肿瘤转移抑制剂, trans-[RuCl₄(DMSO)(imidazole)]Na·2DMSO)衍生物trans-[RuCl₄(DMSO)(2-MeIm)]Na·2DMSO (2-MeIm＝2-甲基咪唑, 化合物1)和trans-[RuCl₄(DMSO)-(N-EtIm)]Na·2DMSO (N-EtIm＝N-乙基咪唑, 化合物2)的水解机理-动力学、溶液稳定性和电化学性质. 化合物1 和化合物2 与NAMI 相似, 在pH 7.40 的缓冲溶液中发生两步脱氯水解反应(I 氯水解及II 氯水解) (分步反应); 在酸性溶液(pH 5.00)中脱DMSO 水解. 通过线性拟合得到各水解反应速率常数k_obs 及半衰期t_1/2. 结果表明化合物在酸性溶液中的稳定性相对较高. 在NAMI 衍生物咪唑环的N 位引入乙基比在2 位引入甲基生成的化合物稳定. 含氮配体相同时,NAMI-A(新抗肿瘤转移抑制剂, A: 该系列中的第一个化合物, trans-[RuCl₄(DMSO)(imidazole)][Himidazole])衍生物略比相应的NAMI 衍生物稳定.     

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.     

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.     

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.     

Toward new camptothecins. Part 6: Synthesis of crucial ketones and their use in Friedländer reaction

In the context of the preparation of camptothecin and luotonin A analogs, the synthesis of some key keto-precursors and their use in Friedländer condensation are described. This paper also focuses on the stability of these keto intermediates and emphasizes the major differences between indolizinones and pyrroloquinazolinones series. Noteworthy is also the report of some original structures isolated as by-products of some experiments.     

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.     

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%).     

Iodine-mediated oxidative Pictet-Spengler reaction using terminal alkyne as the 2-oxoaldehyde surrogate for the synthesis of 1-aroyl-β-carbolines and fused-nitrogen heterocycles

An efficient iodine-mediated oxidative Pictet-Spengler reaction in dimethyl sulphoxide (DMSO) using terminal alkynes as the 2-oxoaldehyde surrogate for the synthesis of aryl (9H-pyrido[3,4-b]indol-1-yl)methanones is described. The scope of the protocol includes the total synthesis of Fascaplysin, Eudistomins Y₁ and Y₂. The methodology is extended for preparing pyrrolo[1,2-a]-quinoxaline and indolo[1,5-a]quinoxaline derivatives. The utility of 1-aroyl-β-carbolines was demonstrated by performing palladium-catalyzed β-carboline directed ortho-C(sp2)-H functionalization of the phenyl ring with thiomethyl (SMe) group using DMSO as source and for accessing 4-aryl-canthin-6-ones.     

Facile synthesis of bicyclic 1-arylpyrazol-5-ones

In this Letter, we described a facile method for constructing fused bicyclic 1-arylpyrazol-5-one ring system. We employed various methylene-containing carboxylic acids as the substrates and proved that the pyrazolone ring closure requires activated methylene group in intermediate II. Accordingly, a series of structurally diversified, fused bicyclic 1-arylpyrazol-5-ones was prepared in moderate to high yields using the requisite substrates.