共价有机骨架功能材料及其在糖肽选择性富集中的应用

蛋白质糖基化是生物体中最重要的翻译后修饰手段之一,糖蛋白/糖肽的有效分离和富集成为目前糖蛋白组学研究的首要问题.对于复杂的生物样本,糖蛋白的数量较少,酶解后大量高丰度非糖基化修饰肽的存在,使得低丰度糖肽的检测更加困难.因此,需要一些手段来有效地富集糖肽以提高其检测丰度,发展高选择性的糖肽富集材料及方法就成为在分子水平上...     

人血清Alpha-1-酸性糖蛋白的糖基化分析

获取真实准确的蛋白质糖基化信息是全面了解糖基化修饰生物学功能的前提.针对简单蛋白质的糖基化分析通常采用反相高效液相色谱-串联质谱技术在肽的水平上对糖基化信息进行采集和解析.本文以人血清Alpha-1-酸性糖蛋白(AGP)酶解液为对象,发展了一套简单有效的蛋白质糖基化分析方法.本方法分为三个步骤,第一步是建立糖肽的理论m/z值表;第二步是获取糖蛋白酶解液的LC-MS谱图,并将每一个色谱峰中所包含糖肽的实际m/z值与理论m/z值进行人工匹配;第三步是对每个色谱峰的糖肽结构归属进行LC-MS/MS验证.采用本方法,我们从AGP酶解液中共鉴定出172条糖肽.与单独采用Survey模式的方法相比,本方法能够显著提高糖肽的覆盖率.     

一种弱阳离子交换材料的亲水模式糖肽富集新方法

亲水作用色谱(HILIC)材料在糖肽的富集与分离中得到越来越广泛的应用. 针对目前HILIC材料糖肽选择性不足的缺点, 发展新的糖肽富集方法十分必要. 本工作发展了一种商品化的弱阳离子交换材料(WCX)在亲水模式下对糖肽的富集方法. 首先考察了微固相萃取(SPE)模式下WCX上多肽保留的机理. 结果显示, WCX上糖肽的保留同时受乙腈含量和pH的影响. 将WCX用于牛胎球蛋白(fetuin)胰蛋白酶酶解液中糖肽的富集, 获得了39条糖肽信号. 当fetuin与牛血清白蛋白(BSA)酶解液以物质的量比1:5混合时, 富集到26条糖肽信号. 富集人免疫球蛋白(IgG)胰蛋白酶酶解液时获得25条糖肽信号, 1:5时获得23条糖肽信号. 实验说明WCX对唾液酸化和非唾液酸化糖肽的富集均有高选择性. 该方法拓展了WCX的应用范围, 丰富了HILIC材料的种类.     

聚酰基氨基脲类多孔有机聚合物的制备及其在糖肽富集中的应用

通过均苯三甲酰肼(BTZ)和对苯二异氰酸酯(PDI)的聚合反应制备了一种新型的聚酰基氨基脲类多孔有机聚合物(POP)。采用傅里叶变换红外光谱(FT-IR)、交叉极化魔角自旋碳13核磁共振(CP-MAS13C NMR)、氮气物理吸附/脱附实验和水接触角等手段对POP-1的物理性质进行表征。其中比表面积为166 m~2/g,水接触角为46.4°,表明这种多孔材料具有良好的亲水性。因此,尝试将POP-1应用于标准蛋白质和复杂生物样品中糖肽的亲水色谱富集。通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF/MS)分析,从500 fmol人血清免疫球蛋白G(IgG)酶解液中共鉴定到19条典型的N-连接糖肽;通过毛细管液相色谱-串联质谱(c LC-MS/MS)分析,从100μg鼠肝蛋白酶解液中共鉴定到属于605个糖蛋白的1 919个糖肽的1 350个N-糖基化位点。这些结果表明POP-1在糖蛋白质组学研究中具有较强的应用潜力。     

糖基化肽段库构建和高通量检索新方法

本研究组发展了全新的高通量糖基化肽段检索新方法, 并开发了检索软件GRIP. 新的检索方法突出了糖肽在CID碎裂时糖链发生连续丢失的特性, 同时充分运用了来自糖基化研究中已有的成熟技术所检测的位点及糖链信息, 高通量地自动解析完整糖肽的串级质谱. 我们检测了人血清样品中的糖肽, 在3次实验中一共鉴定得到4116张糖肽的串级谱图, 对应198个N-糖基化位点, 充分体现了糖基化的微观不均一性. 目前国际上关于血清糖肽谱图鉴定报道为62张谱图; 改进后的肼腙富集法鉴定到了36个N-糖基化位点; 本次鉴定结果的规模和质量均未见报道.     

半胱氨酸基麦芽糖修饰亲水硅胶的制备及其对糖肽的富集

糖蛋白与糖肽在复杂生物体系中丰度一般较低,为了在糖蛋白质组学研究中深入和全面分析鉴定糖基化位点与糖链,通常需要进行富集前处理操作。该文设计并合成了一种半胱氨酸基麦芽糖修饰亲水硅胶分离介质(Cys-Mal@SiO_2),将其装填入固相萃取柱中制备新型亲水固相萃取柱。在以人免疫球蛋白G酶解液为样品进行富集鉴定时,Cys-Mal@SiO_2鉴定糖肽的质谱信号强度和信噪比比半胱氨酸修饰硅胶(Cys@SiO_2)、麦芽糖修饰硅胶(Mal@SiO_2)和商品化ZIC-HILIC更高。在复杂的鼠肝蛋白质提取物的糖蛋白质组学分析中,Cys-Mal@SiO_2共鉴定出1 551条糖肽,属于466个糖蛋白的906个N-糖基化位点,比Cys@SiO_2和Mal@SiO_2鉴定糖肽数、蛋白质数和N-糖基化位点数分别多211、67、127个和289、76、193个。将Cys-Mal@SiO_2成功应用于低丰度糖肽的选择性富集与鉴定,在糖蛋白质组学研究中体现出良好的应用潜力。     

4-巯基苯硼酸修饰二维二硫化钼纳米复合材料的制备及其用于N-糖肽特异性富集

蛋白质的N-糖基化修饰在多种生物过程中发挥着至关重要的作用，近年来的许多研究证实异常的蛋白质糖基化与多种疾病的发生发展密切相关，表明糖基化蛋白质具有较大的潜力成为新的生物标志物或者药物靶标。在样本的处理过程中，对N-糖基化肽段进行富集分离后再进行质谱分析已经成为糖蛋白质组学分析前的必要步骤。但是，由于复杂生物样本中N-糖基化肽段的丰度低和离子化效率差等问题，通过质谱鉴定N-糖肽仍然是一项艰巨的任务。研究通过将纳米金线（Au）、4-巯基苯硼酸（4-MPB）与超薄二维二硫化钼（2D-MoS₂）进行反应，成功制备了一种用于富集蛋白质N-糖基化肽段的新型功能纳米复合材料（MoS₂/Au/4-MPB）。二硫化钼纳米材料的层状结构可以为反应提供大量的可修饰位点，便于修饰纳米金线；功能基团4-巯基苯基硼酸对N-糖肽具有高度的选择性，可以对生物样品中N-糖基化肽段进行特异性富集。使用标准蛋白人免疫球蛋白G（IgG）和牛血清白蛋白（BSA）胰蛋白酶酶切产物对新型功能纳米材料的N-糖基化肽段的富集性能进行评估，其灵敏度达到5 fmol，选择性达到1：1000。将其用于生物样品中N-糖基化肽段的富集，从50 μg尿液外泌体蛋白胰蛋白酶酶切产物中共富集鉴定出768个N-糖肽，归属于377个蛋白质。这些结果表明该新型功能纳米复合材料对复杂生物样品中N-糖肽的选择富集有着巨大的应用潜力，为糖蛋白质组的研究提供了一种新方法。     

基于Endo-M N175Q糖链转移反应与丙酮富集糖肽的LC-MS分析糖蛋白N-糖链

基于化学酶标记和丙酮富集糖肽方法,建立了一种可靠、有效、简单的糖蛋白N-糖链分析方法。以唾液酸糖肽(SGP)为模型糖肽,比较了样品中丙酮加入量对SGP的富集效果,最终选择加入样品体积5倍量的丙酮。用丙酮富集经胰蛋白处理的核糖核酸酶B(RNase B)酶解液中的糖肽,以富集分离得到的糖肽(糖基供体)和PDPZ-Boc-Asn-GlcNAc(糖基受体)作为酶反应底物,进行Endo-M N175Q的转糖基反应,得到PDPZBoc-Asn-GlcNAc-N-糖链标记物。采用YMC C18色谱柱为分析柱,10 mmol/L甲酸铵-乙腈为流动相梯度洗脱,经液相色谱-串联质谱(LC-MS)检测得到5种高甘露糖型糖链。结果表明,丙酮可有效地富集大量肽和少量糖肽混合溶液中的糖肽,Endo-M N175Q可将天然糖肽的糖链转移到PDPZ-Boc-Asn-GlcNAc受体上。将该方法应用于胎球蛋白N-糖链分析,检测到5种复杂型N-糖链。该研究为各种糖蛋白N-糖链检测提供了新的分析方法。     

基于叠氮基-氰基点击化学的苯硼酸亲和硅胶的制备及其在糖蛋白/糖肽选择性富集中的应用

硼亲和色谱法在糖肽/糖蛋白选择性富集中的应用趋于成熟。硼酸亲和材料的选择性，生物相容性，制备过程是否简便均是开发新型苯硼酸功能化材料需要考虑的问题。该研究立足硼酸亲和材料开发的关键问题，设计并开发了一种新型苯硼酸亲和硅胶（TCNBA）。该材料采用基于叠氮基-氰基的无铜催化点击化学方法进行合成，生物相容性好，制备方法简便。红外光谱和X射线光电子能谱图表征结果证明材料合成成功。TCNBA的糖肽富集选择性利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行评价，结果表明，TCNBA能够分别从辣根过氧化物酶（HRP）和免疫球蛋白G（IgG）酶解液中鉴定出13个和11个糖肽；以HRP和牛血清白蛋白（BSA）酶解液混合物（物质的量比1：10）作为研究对象，富集后能够鉴定出5个糖肽。TCNBA的糖蛋白富集选择性利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法（SDS-PAGE）进行评价，以HRP、IgG、核糖核酸酶B（RNaseB）作为考察对象，结果表明，TCNBA对糖蛋白具有较好的富集选择性。以实际样品人血清为测试对象验证TCNBA在实际生物样品中的应用价值。结果显示，富集后非糖蛋白得到较大程度去除，糖蛋白得以富集。所制备的材料和方法具有大规模实际蛋白质样品分离处理的应用前景。     

新型四肽亲水材料用于糖肽的高效富集

本研究采用原子转移自由聚合(Atom-transfer radical-polymerization, ATRP)法合成了一种新型四肽亲水作用色谱材料 (Poly-DAPD),用于糖肽的选择性富集.通过氮气吸脱附、热重分析和X射线光电子能谱等技术进行表征,结果表明,四肽已成功接枝到硅球上.固相萃取富集实验表明,合成的亲水材料对牛胎球蛋白(Fetuin)糖肽富集选择性高;与商品化ZIC-HILIC材料相比,Poly-DAPD材料富集掺有5摩尔倍数牛血清蛋白(BSA)的Fetuin样品时,在获得的糖肽数目及抗干扰性能方面都更具优势.此Poly-DAPD材料可进一步用于不同糖蛋白的糖基化分析研究.     

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.     

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.     

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.     

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.     

KMnO4-mediated oxidative CN bond cleavage of tertiary amines: Synthesis of amides and sulfonamides

KMnO₄-mediated oxidative CN bond cleavage of tertiary amines producing secondary amine was introduced, which was trapped by electrophiles (acyl chloride and sulfonyl chloride) to form amides and sulfonamides. The reaction could take place at mild condition, tolerating a wide range of function groups and affording products in moderate to excellent yields.     

Chemistry of heterocyclic compounds at the A. E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences, over 50 years (Review)

The review contains a concise historical account and information on the most significant researches undertaken by the staff at the A. E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences on the Chemistry of Heterocyclic Compounds. Dedicated to Academician of the Russian Academy of Sciences B. A. Trofimov on his 70th jubilee. Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 10, pp. 1443–1502, October, 2008.     

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.     

Sulfur-directed metal-free and regiospecific methyl C(sp3)–H imidation of thioanisoles

Regiospecific and direct imidation of the methyl C(sp³)–H bond of thioanisoles is realized under mild and metal-free conditions with N-fluorobis(benzenesulfonyl)imide as an oxidant and nitrogen source. Proposed mechanism suggests that thionium ion intermediates and a Pummerer-type reaction are involved. The imidation has advantages such as high step-economy, excellent functionality tolerance, and regiospecificity, giving structurally diverse imidation products.     

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.