Stereoselective synthesis of 1-C-alkyl iminogalactitol derivatives,potential chaperones for galactosidase-linked LSDs: a real challenge

1-C-Alkyl iminogalactitol derivatives are highly desirable target compounds as potential pharmacological chaperones for the treatment of galactosidase-linked lysosomal storage disorders (LSDs). The synthesis of such compounds from d-galactose by a C-1 chain extension process by way of an open-chain 6-amino d-gulo nonulose intermediate was complicated by unexpected deoxygenation reactions leading to 3-deoxy iminogalactitol derivatives and epimers; an alternative, stereocontrolled synthesis from an l-tagatose derivative by C-6 chain-extension was therefore developed, which involved addition of organometallic reagents onto t-butanesulfinylimine intermediates in the key step.     

Effect of High Pressure Treatments on protease and β-Galactosidase Activities of Table Olives

In this work the effect of high pressure treatments (50 and 110 MPa for 15 and 30 min.) on protease and g -galactosidase activities of mature table olives (Portuguese Douro variety) was studied. The influence of the applied treatments was quantified for the soluble and the ionically and covalently bound to cell wall fractions for both enzymes. For g -galactosidase the treatments caused no significant changes in activity, while proteolytic activity was reduced by more than 90% in all cases. For proteolytic activity significantly different baric stabilities were also found for the three fractions studied.     

鹰嘴豆β－半乳糖苷酶的研究──催化水解ONPG酶促反应动力学及其活性位的化学修饰

研究了鹰嘴豆发芽种子的β－半乳糖苷酶Ⅰ和β－半乳糖苷酶Ⅱ在催化水解邻硝基苯酚－β－Ｄ－半乳糖苷酶促反应中的催化性质和反应动力学，求得不同反应温度的米氏常数ＫＭ、最大反应速率ｒｍ和反应活化能Ｅａ等动力学参数，并以各种特征化学修饰剂对β－半乳糖苷酶Ⅰ中的活性基团进行了化学修饰。关联修饰情况与酶活性的关系，推知β－半乳糖苷酶Ⅰ中有色氨酸和巯基存在。它们是酶活性中心的组成基团，而该活性中心至少含有１个色氨酸和２个巯基，探讨了酶的活性位及其催化作用机理。     

Microfabricated electroosmotic pump for capillary-based sequential injection analysis

A microfabricated electroosmotic pump with an integrated serpentine isolation channel was developed on a glass chip and applied to a capillary-based sequential injection analysis (SIA) system for an enzyme inhibition assay. The pump chip contains an anode reservoir, an ion-exchange membrane electric field decoupler (EFD) that also serves as a cathode reservoir, parallel pump channels and an isolation channel. A two-step etching process was adopted to etch the pump channels to a depth of 20 μm and the isolation channel to a depth of 90 μm. The pump flow rate was very stable: the relative standard deviation (RSD) of the pump rate was 1.9% for propulsion and 2.3% for aspiration. The pump chip was successfully applied to a capillary-based sequential injection analysis system with a confocal fluorescence detector. For repetitive analysis of a 13 μM fluorescein solution, an RSD of 0.6% was attained, which demonstrated the flow stability of the EOF pump. The system was then applied to an enzyme inhibition assay, the diethylenetriaminepentaacetic acid (DTPA) inhibition of the β-galactosidase-catalyzed hydrolysis of fluorescein di(β-d-galactopyranoside). Reproducible results (RSD<3.0%) were obtained.     

Scanning Electrochemical Microscopy (SECM) Based Detection of Oligonucleotide Hybridization and Simultaneous Determination of the Surface Concentration of Immobilized Oligonucleotides on Gold

The direct mode of scanning electrochemical microscopy (SECM) was used for the local deposition of oligonucleotide (ODN) patterns on thin gold films and the generation‐collection (GC) mode was applied for the determining the amount of surface‐accessible oligonucleotides. The local deposition was achieved through the micrometer‐sized formation of a conducting polymer bearing 15^mer single‐stranded oligonucleotide strands. After the interaction of the oligonucleotide with its biotin‐labeled complimentary strand, streptavidin was bound. The molecular assembly was completed by linking biotin‐labeled β‐galactosidase from Escherichia coli to the streptavidin. The activity of the linked β‐galactosidase was mapped with SECM in the GC mode by monitoring the oxidation of p‐aminophenol (PAP) formed in the enzyme‐catalyzed hydrolysis of p‐aminophenyl‐β‐D ‐galactopyranoside. The feedback effect due to recycling of the reaction product at the gold surface was analyzed. It was shown experimentally that this effect becomes insignificant at ultramicroelectrode (UME)‐substrate distances larger than 3 UME radii. The flux of formed PAP allowed the determination the surface density of accessible oligonucleotide strands in the functionalized polymer. It was shown that that thicker pyrrole/ODN–Pyrrole polymer films do not lead to a significantly increased accessible ODN surface concentration.