N-Alkylated Iminosugar Based Ligands: Synthesis and Inhibition of Human Lysosomal ß-Glucocerebrosidase.

The scope of a series of N-alkylated iminosugar based inhibitors in the d-gluco as well as d-xylo configuration towards their interaction with human lysosomal ß-glucocerebrosidase has been evaluated. A versatile synthetic toolbox has been developed for the synthesis of N-alkylated iminosugar scaffolds conjugated to a variety of terminal groups via a benzoic acid ester linker. The terminal groups such as nitrile, azide, alkyne, nonafluoro-tert-butyl and amino substituents enable follow-up chemistry as well as visualisation experiments. All compounds showed promising inhibitory properties as well as selectivities for ß-glucosidases, some exhibiting activities in the low nanomolar range for ß-glucocerebrosidase.

Mechanistic Insights into the Chaperoning of Human Lysosomal-Galactosidase Activity: Highly Functionalized Aminocyclopentanes and C-5a-Substituted Derivatives of 4-epi-Isofagomine.

Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid ß-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent ß-d-galactosidase inhibitor 4-epi-isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a "strategic" hydroxyl group. New compounds have revealed highly promising activities with a range of ß-galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.

A new type of pharmacological chaperone for GM1-gangliosidosis related human lysosomal ß-galactosidase: N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols.

N-Functionalized amino(hydroxymethyl)cyclopentanetriols are potent inhibitors of ß-d-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for GM1-gangliosidosis-associated lysosomal acid ß-galactosidase thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.

Synthesis of C-5a-chain extended derivatives of 4-epi-isofagomine: Powerful ß-galactosidase inhibitors and low concentration activators of GM1-gangliosidosis-related human lysosomal ß-galactosidase.

From an easily available partially protected formal derivative of 1-deoxymannojirimycin, by hydroxymethyl chain-branching and further elaboration, lipophilic analogs of the powerful ß-d-galactosidase inhibitor 4-epi-isofagomine have become available. New compounds exhibit improved inhibitory activities comparable to benchmark compound NOEV (N-octyl-epi-valienamine) and may serve as leads towards improved and more selective pharmacological chaperones for GM1-gangliosidosis.

Protocol for inoculating bottled mineral water with bacteria to receive artificial contaminated water samples.

Here, we present a protocol for inoculating drinking water samples with a variety of pathogens or facultative pathogen bacteria. We describe steps for preparing bacterial solutions, inoculating mineral water bottles and other drinking water samples, filtration and incubation of the agar plates, and counting colony-forming unit per mL. We also detail procedures for determining selected chemical properties, such as anions and cations, which can also affect the bacterial growth. For complete details on the use and execution of this protocol, please refer to Schalli et al.1.

The behaviour of Escherichia coli and Pseudomonas aeruginosa in bottled mineral water.

Microbial contamination of bottled water during the filling and capping procedure is a problem which should be avoided. The examination of the influence of carbon dioxide (CO2) on bacterial growth of Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) in bottled mineral water was the aim of this study. Commercially available glass bottles with plastic screw caps filled with natural mineral water (without additional CO2 "still" (StMW) and with CO2 "sparkling" (SpMW) were obtained from a manufacturer in the province of Styria, Austria. The artificial contamination was performed in the lab by opening the bottle with subsequent addition of a bacterial solution with a defined number of bacteria. For each bacterial strain, 12 bottles were prepared. Samples (100 mL) were taken after a specific number of days, filtrated and placed on Endo Agar for cultivation. After incubation for 24 h bacterial colonies were counted. In this study CO2 addition to bottled water reduced colony forming units of the two investigated bacterial strains over time.

High-Speed Dental Instruments: An Investigation of Protein-Contaminated Dental Handpieces with the Bicinchoninic Acid Assay in Dental Offices in Styria, Austria.

Due to permanent contact with bodily secretions such as blood and saliva, the dental workplace poses a high risk of infection for patients as well as for personnel. High-speed dental instruments are still considered one of the major hygienic risks, as the high-speed rotation of the attachments leads to the retraction of infectious material from patients' oral cavities. The aim of this study was to investigate the extent to which dental handpieces are contaminated after use. Spray-water samples were taken from different handpieces used in seven dental offices and protein concentrations were measured photometrically. In the first part of the study, samples were collected from each handpiece before and after the treatment of the patients. Additionally, the changes in protein concentration after consecutive treatments in which the same high-speed dental instrument was used were investigated. The results demonstrated measurable protein concentrations in 91.2% of a total of 398 samples, and 96.4% of the spray-water samples taken after treatment showed a discrepancy from the initial measured protein concentration. In 68.4% an increase in protein concentration was observed, whereas in 27.9% a decrease was measured. In conclusion, the internal contamination of high-speed dental instruments frequently occurs in daily usage and consequently may lead to the transmission of infectious agents by flushing the contaminated water out of the spray water tubes. Moreover, it must be pointed out that internal cleansing of handpieces is insufficient and that a final mechanical disinfection is indispensable.

Dissolved Carbon Dioxide: The Lifespan of Staphylococcus aureus and Enterococcus faecalis in Bottled Carbonated Mineral Water.

During the process of mineral water production, many possible contamination settings can influence the quality of bottled water. Microbial contamination can originate from different sources, for example, the ambient air, the bottles, the caps, and from the bottling machine itself. The aim of this study was to investigate the influence of three different carbon dioxide (CO2) concentrations (3.0 g/L, 5.5 g/L, and 7.0 g/L; 20 bottles each) in bottled mineral water on the bacterial growth of Staphylococcus aureus (S. aureus) and Enterococcus faecalis (Ent. faecalis). The examined mineral water was artificially contaminated before capping the bottles inside the factory. After a specific number of days, water samples were taken from freshly opened bottles and after filtration (100 mL), filters were placed on Columbia Agar with 5% Sheep blood to cultivate S. aureus and Slanetz and Bartley Agar to cultivate Ent. faecalis. The respective colony-forming units (CFU) were counted after incubation times ranging from 24 to 120 h. Colony-forming units of S. aureus were not detectable after the 16th and 27th day, whereas Ent. faecalis was not cultivable after the 5th and 13th day when stored inside the bottles. The investigation of the bottles that were stored open for a certain amount of time with CO2 bubbling out showed only single colonies for S. aureus after the 5th day and no CFUs for Ent. faecalis after the 17th day. A reduction in the two investigated bacterial strains during storage in carbonated mineral water bottles means that a proper standardized disinfection and cleaning procedure, according to valid hygiene standards of industrial bottling machines, cannot be replaced by carbonation.

Background concentrations of airborne, culturable fungi and dust particles in urban, rural and mountain regions.

Geographic location and meteorological factors can affect the content of bioaerosol concentrations. This study was conducted to determine the natural background concentrations of culturable fungal spores and dust particles in three different geographical areas. Focus was given to the dominant airborne genera Cladosporium, Penicillium, Aspergillus and the species Aspergillus fumigatus. The influence of weather conditions on the microorganism concentrations in urban, rural and mountain regions were examined. Possible correlations between particle counts and culturable fungal spore concentrations were investigated. 125 measurements of the air were conducted using the air sampler MAS-100NT® and the particle counter Alphasense OPC-N3. The analyses of the collected samples were based on culture methods using different media. The highest median of fungal spore concentrations was detected in the urban region and was of 2.0 × 103 CFU/m3 for xerophilic fungi and 1.7 × 103 CFU/m3 for the genus Cladosporium. The concentrations of fine and coarse particles in rural and urban regions were the highest of 1.9 × 107 pa/m3 and 1.3 × 107 pa/m3, respectively. Little cloud cover and slight wind had a positive influence on the concentration of fungal spores. Furthermore, correlations were observed between air temperature and the concentrations of xerophilic fungi as well as the genera Cladosporium. In contrast, relative humidity correlated negatively with total fungi and Cladosporium and no correlation was found with the other fungi. For the region of Styria in summer and early autumn, the natural background concentration for xerophilic fungi ranged between 3.5 × 102 and 4.7 × 103 CFU/m3 air. No significant differences were detected between the fungal spore concentrations in urban, rural and mountainous regions. The data of this study could be used as a reference to compare the natural background concentrations of airborne culturable fungi in further studies concerning air quality assessment.

Cefsulodin and Vancomycin: A Supplement for Chromogenic Coliform Agar for Detection of Escherichia coli and Coliform Bacteria from Different Water Sources.

Background microorganism growth on Chromogenic Coliform Agar (CCA) can be challenging. For this reason, a new alternative method with a Cefsulodin/Vancomycin (CV)-supplemented CCA should be developed in this study. CCA supplemented with CV was validated according to ÖNORM EN ISO 16140-4:2021 using water from natural sources in Styria, Austria. Results show that the alternative method using the supplemented CCA has similar values in relation to sensitivity (82.2%), specificity (98.6%) and higher selectivity (59%) compared to the reference method. Repeatability and reproducibility were acceptable for the alternative method and showed similar results with the reference method. The alternative method shows a very low false positive rate and a low false negative rate paired with good performance regarding the inclusion study. The exclusion study shows the advantage of our method by suppressing background microorganisms and facilitating the process of enumeration of Escherichia coli and other coliform bacteria on CCA plates. Aeromonas hydrophila and Pseudomonas aeruginosa growth was inhibited using the supplement. To conclude, the coliform CV selective supplement combined with CCA is an appropriate tool for coliform bacteria detection in water samples.

Sila-Peterson Reaction of Cyclic Silanides.

Sila-Peterson type reactions of the 1,4,4-tris(trimethylsilyl)-1-metallooctamethylcyclohexasilanes (Me3Si)2Si6Me8(SiMe3)M (2a, M = Li; 2b, M = K) with various ketones were investigated. The obtained products strongly depend on the nature of the ketone component. With 2-adamantanone 2a,b afforded the moderately stable silene 3. 3 is the first example of an Apeloig-Ishikawa-Oehme-type silene with the tricoordinate silicon atom incorporated into a cyclopolysilane framework and could be characterized by NMR and UV spectroscopy as well as by trapping reactions with water, methanol, and MeLi. The reaction of 2b with aromatic ketones also follows a sila-Peterson type mechanism with formation of carbanionic species. With 1,2-diphenylcyclopropenone 2b reacted by conjugate 1,4-addition to give a spirocyclic carbanion. In most cases the underlying reaction mechanism could be elucidated by the isolation and characterization of unstable intermediates and final products after proper derivatization.

Synthesis of modified 1,5-imino-d-xylitols as ligands for lysosomal ß-glucocerebrosidase.

ABSTRACT: Modified 1,5-dideoxy-1,5-imino-d-xylitol analogues with different substitution patterns involving position C-1 and/or the ring nitrogen were prepared, which were designed to serve as precursors for the preparation of iminoxylitol-based ligands and tools for the elucidation and modulation of human lysosomal ß-glucocerebrosidase. Biological evaluation of the synthesized glycomimetics with a series of glycoside hydrolases revealed that these substitution patterns elicit excellent ß-glucosidase selectivities.

N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols: A new family of activity promotors for a GM1-gangliosidosis related human lysosomal ß-galactosidase mutant.

From 1,2;3,4-di-O-isopropylidene-α-D-galactopyranose, a series of highly functionalized (hydroxymethyl)cyclopentanes was easily available. In line with reports by Reymond and Jäger on similar structures, these amine containing basic carbasugars are potent inhibitors of ß-D-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for GM1-gangliosidosis-associated lysosomal acid ß-galactosidase mutant R201C, thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.

A Morita-Baylis-Hillman based route to C-5a-chain-extended 4-epi-isofagomine type glycosidase inhibitors.

By Morita-Baylis-Hillman reaction of 2,3-O-isopropylidene-D-glyceraldehyde with α,ß-unsaturated carbonyl as well as hetero analogous carbonyl compounds such as acrylonitrile, suitable precursors of isofagomine and of 4-epi-isofagomine are available. Elaboration of the structures by amine introduction, followed by intramolecular ring closure and subsequent hydroboration of the double bond provides 4-epi-isofagomine derivatives featuring chain extensions at C-5a which are determined by the structures of the carbonyl compounds employed. As an example, the synthesis of C-(5aR)- and C-(5aS)-5a-C-pentyl-4-epi-isofagomines, powerful inhibitors of ß-galactosidases, is outlined. In line with reported data, the (C-5aR) epimer was found a highly potent experimental pharmacological chaperone for GM1-associated human lysosomal ß-galactosidase mutant R201C.

C-5a-substituted validamine type glycosidase inhibitors.

A series of N-alkyl derivatives of the D-galactosidase inhibitor 1,4-di-epi-validamine featuring lipophilic substituents at position C-5a was prepared and screened for their glycosidase inhibitory properties. Products turned out selective for ß-galactosidases as well as ß-glucosidases.

From secondary alcohols to tertiary fluoro substituents: A simple route to hydroxymethyl branched sugars with a fluorine substituent at the branching point.

From a secondary hydroxyl group, by the simple sequence of oxidation, Wittig reaction of the obtained ulose with methoxymethylene triphenyl phosphorane, exposure of the resulting exocyclic enol ether to Selectfluor and subsequent reduction of the α-fluoro aldehyde thus obtained, tertiary fluoro substituents can be introduced into carbohydrate and carbohydrate-related scaffolds at a branching point now bearing a new hydroxymethyl group.

5-Fluoro derivatives of 4-epi-isofagomine as D-galactosidase inhibitors and potential pharmacological chaperones for GM1-gangliosidosis as well as Fabry's disease.

Electrophilic fluorination of an exocyclic methoxymethylene enol ether derived from N-tert-butyloxycarbonyl-1,5-dideoxy-1,5-imino-3,4-O-isopropylidene-D-erythro-pent-2-ulose (11) provided the 5-fluoro derivative of the powerful ß-galactosidase inhibitor 4-epi-isofagomine (8). This structural alteration, in combination with N-alkylation, led to considerably improved α-galactosidase selectivity. New compounds may serve as leads en route to new pharmacological chaperones for Fabry's disease.

Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable ß-galactosidase inhibitors and activity promotors of GM1-gangliosidosis related human lysosomal ß-galactosidase mutant R201C.

From an easily available partially protected analog of 1-deoxy-L-gulo-nojirimycin, by chain-branching at C-4 and suitable modification, lipophilic analogs of the powerful ß-D-galactosidase inhibitor 4-epi-isofagomine have been prepared. New compounds exhibit considerably improved inhibitory activities when compared with the unsubstituted parent compound and may serve as leads toward new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.