亚油酸酰化胰岛素的制备及飞行质谱分析

胰岛素侧链氨基的酰化是胰岛素化学修饰较常用的方法。天然胰岛素分子中有三个裸露的氨基,即G lyA1、PheB1的α-氨基和LysB29的ε-氨基,都可作为胰岛素化学修饰的位点。酰化试剂通常经由N-羟基琥珀酰亚胺酯对胰岛素进行修饰。N-羟基琥珀酰亚胺酯可由羧酸与N-羟基琥珀酰亚胺(HOS     

二亚油酰磷脂酰胆碱分析

通过HPLC/HPLC-MS研究了二亚油酰磷脂酰胆碱的测定方法,磷脂样品首先经过HPLC分离得到磷脂酰胆碱,该磷脂酰胆碱再进一步通过不同的HPLC系统分离二亚油酰磷脂酰胆碱,并通过标准样品和质谱进行二亚油酰磷脂酰胆碱的定性.定量方法通过标准样品外标法定量,由方法的回收率和精密度试验可知,该方法已用来测定一般磷脂样品和磷脂酰胆碱样品中的二亚油酰磷脂酰胆碱含量.     

PEG-醛修饰胰岛素产物的构象、纤维化和抗酶解能力

通过圆二色谱分析研究了PEG-醛修饰胰岛素产物在溶液中的构象变化,采用加速振荡实验研究了PEG化对胰岛素纤维化的影响,以体外酶解实验考察了PEG-醛修饰胰岛素产物的抗酶解能力.结果表明,经PEG-醛修饰可以减少胰岛素单体的自缔合,提高胰岛素在溶液中的稳定性,抑制胰蛋白酶和糜蛋白酶对胰岛素的降解.     

含苯并三唑色酮及1,3,4-噁二唑啉类化合物的合成

研究了一种简便的合成2位含氮杂环色酮类化合物的方法。1H-苯并三唑与ClCH~2COOH反应制得1H-苯并三唑-1-乙酸(1),以此为原料合成了一系列2位含1H-苯并三唑-1-甲基色酮(5)和相关的1,3,4-噁二唑衍生物(8)。     

胰岛素的mPEG修饰及其活性表征

用单甲氧基聚乙二醇-丙醛(mPEG-ALD)、单甲氧基聚乙二醇-丁醛(mPEG-ButyrALD)和单甲氧基聚乙二醇-琥珀酰亚胺碳酸酯(mPEG-SC)对猪胰岛素进行化学修饰. 考察了反应时间、pH、物质的量比及分子量等因素对修饰效果的影响. 通过HPLC, MALDI-TOF-MS(基质辅助激光解吸时间飞行质谱), SDS-PAGE(十二烷基硫酸钠-聚丙烯酰胺凝胶电泳)和TNBS(三硝基苯磺酸钠法)等表征发现mPEG-ButyrALD具有更高的修饰选择性和修饰率. 圆二色光谱(CD)分析修饰产物的二级结构发现, 胰岛素经mPEG衍生物修饰后, 二级结构基本没有改变. 动物实验表明, mPEG-ButyrALD修饰胰岛素后半衰期延长, 因而可以延长降血糖作用时间, 改善胰岛素药效.     

新型双缩硫代对称二氨基脲类衍生物的合成和生物活性测试

探讨了硫代对称二氨基脲与10种含三唑基或苯并三唑基的取代苯乙酮的缩合反应, 制得10种新的1,5-二取代苯乙酮双缩硫代对称二氨基脲类化合物, 产物经IR, ¹H NMR, ¹³C NMR和MS及元素分析确证. 并对这些化合物的抗真菌和抗病毒活性进行了测试.     

1H-苯并三唑及其衍生物的光化学合成

邻苯二胺及其衍生物与N-亚硝基二苯胺在氩气保护、光照条件下,以二氯甲烷为溶剂,发生从N-亚硝基二苯胺到邻苯二胺的亚硝基转移反应,反应最终生成1H-苯并三唑及其衍生物.N-亚硝基二苯胺与N,N'-二甲基邻苯二胺也可以发生亚硝基转移反应,生成N,N'-二甲基-N-亚硝基邻苯二胺,不能环化生成1H-苯并三唑类产物.N-亚硝基...     

油酰丝裂霉素及其脂质体的合成与抗肿瘤活性

用油酸与丝裂霉素Ｃ在缩合剂存在合成了油酰丝裂霉素Ｃ，利用元素分析，红外光谱及核磁共振等确定其结构，用改良的逆相蒸发法制成了油酰丝裂霉素Ｃ脂质体，动物实验表明该脂质体抗肿瘤效果为游离丝裂霉素Ｃ的２．２倍，而毒性仅为游离丝裂霉素的１／２０。     

α,ω-二(1-H-苯并三唑基)烷烃的合成和生物活性的研究

在相转移催化剂存在下,以苯并三唑为原料与二卤代烷反应合成了4个新的α,ω-二(1-H-苯并三唑基)烷烃,它们的结构经元素分析,IR和1H NMR证实.初步考查了它们的生物活性.     

乙基二苯甲酰甲烷的合成及表征

乙基二苯甲酰甲烷是一种光吸收性能良好的化合物。该法以苯甲酸乙酯和苯乙酮为起始原料,经克莱森缩合生成二苯甲酰甲烷,再与溴乙烷进行烷基化反应,最后生成乙基二苯甲酰甲烷,并对合成产物进行结构分析及紫外光吸收性能表征。     

The proteolytic activity of insulin‐degrading enzyme: a mass spectrometry study

The prominent role that insulin‐degrading enzyme (IDE) has on amyloidogenic peptides degradation has recently boosted a lot of attention toward this enzyme. Although many substrates are known to be degraded by IDE, little is known about the changes in the proteolytic activity of the enzyme upon modification of environmental factors. In a previous work we have already shown the great potentiality of atmospheric pressure/laser desorption ionization‐mass spectrometry (AP/MALDI‐MS) for studying the interaction between IDE and insulin. Here, the activity of IDE was investigated regarding cleavage sites' preferentiality upon modification of environmental factors by AP/MALDI‐MS. The roles that IDE/insulin concentration ratio, reaction time, adenosine 5′‐triphosphate (ATP) and metal ions (Zn and Cu) have on the insulin cleavage pattern produced by IDE are investigated and a plausible interpretation involving the proteolytic action of the different IDE oligomeric forms is proposed. Copyright © 2009 John Wiley & Sons, Ltd.     

Zn Nanoparticles Modified Screen Printed Carbon Electrode as a Promising Sensor for Insulin Determination

Screen printed carbon electrodes (SPCEs) modified by a combination of chitosan, multi walled carbon nanotubes (MWCNTs) and zinc nanoparticles (ZnNPs) were studied for the first time as a suitable candidate for non-enzymatic insulin determination. In an effort to find the most suitable modification for electrochemical insulin determination, the stability, analytical characteristics, and selectivity were determined. The results confirmed that the ZnNPs/chitosan-MWCNTs prepared with the Zn deposition time of 45 s displayed the best electrocatalytic activity towards insulin oxidation in a wide linear concentration range (0.5 μM to 5 μM), with low limit of detection and high sensitivity.     

Simultaneous quantitative determination of insulin aspart and insulin degludec in human plasma using simple shoot LC method

Combining short-acting and long-acting insulin analogs was a real challenge that was overcome by NovoNordisk through the co-formulation of insulin aspart and insulin degludec in single-dosage form. The proposed study provides a simple, short, and reliable HPLC method with diode array detection that is developed and validated for the simultaneous determination of insulin aspart and insulin degludec in human plasma. The proposed method achieved good separation between the two analytes utilizing a C8 column at 35°C in a very short run time (6 min), with a simple, low-cost, and reliable extraction method through precipitation of plasma protein. Gradient elution was applied using a mobile phase consisting of 0.1 M sodium sulfate (pH 3.4) and acetonitrile. The method was validated according to EMA Guideline on Bioanalytical Validation. The proposed method had a linear range from 3.0 to 300 μg/mL for insulin aspart and from 3.5 to 300 μg/mL for insulin degludec. The intra- and inter-day precision of insulin aspart were 0.36–3.33% and 1.59–8.84%, respectively, and accuracy was between 10.06 and 3.09% The intra- and inter-day precision of insulin degludec were 0.29–1.93% and 0.89–5.14%, respectively, and accuracy was between −5.29 and 3.91%.     

The ABC of Insulin: The Organic Chemistry of a Small Protein

Insulin is a small protein crucial for regulating the blood glucose level in all animals. Since 1922 it has been used for the treatment of patients with diabetes. Despite consisting of just 51 amino acids, insulin contains 17 of the proteinogenic amino acids, A- and B-chains, three disulfide bridges, and it folds with 3 α-helices and a short β-sheet segment. Insulin associates into dimers and further into hexamers with stabilization by Zn²⁺ and phenolic ligands. Selective chemical modification of proteins is at the forefront of developments in chemical biology and biopharmaceuticals. Insulin's structure has made it amenable to organic and inorganic chemical reactions. This Review provides a synthetic organic chemistry perspective on this small protein. It gives an overview of key chemical and physico-chemical aspects of the insulin molecule, with a focus on chemoselective reactions. This includes N-acylations at the N-termini or at Lys^B29 by pH control, introduction of protecting groups on insulin, binding of metal ions, ligands to control the nano-scale assembly of insulin, and more.     

THE CRYSTAL STRUCTURE OF SILVER CARP INSULIN AT MEDIUM RESOLUTION

It is an important way of surveying the structure-functionrelationship of insulin tostudy insulins from different species. Based on the structure model of an orthorhombic crys-tal obtained by the molecular replacement method, the crystallographic refinement of a hex-amer of silver carp insulin in an asymmetric unit has been carried out with 2.8A resolutiondata using the restrained least- squares method. The comparisons of insulin structures haveshown that the six silver carp insulin molecules have very similar but not identical three- di-mensional structures which are similar to the known 2Zn pig insulin structure but remarka-bly different in some local conformations.     

THE CHARACTERISTICS AND MOTION MODEL OF INSULIN MONOMER

The extensive conformational comparisons among the determined structures of the differeat species and crystal forms of insulin and the varied insulin derivatives were performed by using the least-squares superimposition technique and the graphics technique. The results of the investigation showed that the structure of molecule I in 2Zn insulin was closer to that of the natural monomer; the conformational difference between two molecules of a dimer came out during dimerization and it was further improved and stabilized during the hexamerization and packing of hexamers in crystal; through the hinge peptides, such as A10, B4, B8, B24, B20 and B23, there was a flexible relative motion among the structural segments in the insulin molecule, and the residues at the B-chain C-terminal might have a shift of more than 10; the mobility for each residue side-chain was very different due to the different surroundings.     

STUDIES ON THE CRYSTAL STRUCTURE OF Al-(D-TRYPTOPHAN) INSULIN

We have determined the crystal structure of Al-(D-Trp) insulin and discovered that it belongs to the trigonal system with space group R3. The parameters of the unit cell are a=b=78.6, c=50.0. A set of data for half a sphere reciprocal space to a spacing of 2.2 were collected. The model was adjusted and refined by using a step-by-step approach and a stereochemically-restrained least squares program, assisted by manual revision based on the difference Fourier maps, to a final R-factor of 0.218. The main and side chains of both Al-D-Trp residues in the asymmetric unit are well ordered. The packing of Al-(D-Trp) insulin in the unit cell, the conformational differences with other insulin structures and its structure and function relationship bave also been discussed.     

Separation of human,bovine, and porcine insulins,three very closely related proteins,by micellar electrokinetic chromatography

Human, bovine, and porcine insulins are small proteins with very closely related amino acid sequences, which makes their separation challenging. In this study, we took advantage of the high‐resolution power of CE, and more particularly of micellar electrokinetic chromatography, to separate those biomolecules. Among several surfactants, perfluorooctanoic acid ammonium salt was selected. Then, using a design of experiments approach, the optimal BGE composition was found to consist of 50 mM ammonium acetate pH 9.0, 65 mM perfluorooctanoic acid ammonium salt, and 4% MeOH. The three insulins could be separated within 12 min with a satisfactory resolution. This method could be useful to detect possible counterfeit pharmaceutical formulations. Indeed, it would be easy to determine if human insulin was replaced by bovine or porcine insulin.     

新颖的双胰岛素取代钴(III)卟啉的制备和表征

A novel insulin-disubstituted Co(Ⅲ)protoporphyrin IX,CoPI,where I is insulin and CoP is Co(Ⅲ)protopor-phyrin IX,was prepared by covalently coupling the propionate groups on the porphyrin ring to the ε-amino groupsof B29-Lys on insulin via amide linkages.The FTIR spectra in the amide I region(1600—1700 cm~(-1))and circulardichroism study show that CoPI has a conformation similar to the insulin at pH 6.9,whereas it exhibits significantconformational changes in structure as compared with the insulin self at pH 8.2.The pH absorption titration indi-cates that the alkaline conditions(pH≥8.0)are required for the formation of complexes between the free CoP andthe insulin.The thermodynamic and kinetic data reveal that free CoP is bound to either the zinc-insulin or free insu-lin with a dissociation constant of(2.0±0.3)×10~(-5)or(2.2±0.3)×10~(-5)mol/L.