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Synthetic strategies for the fluorescent labeling of epichlorohydrin-branched cyclodextrin polymers.

ABSTRACT: The fluorescent tagging of cyclodextrin derivatives enlarges their spectroscopic properties thus generating chemosensors, biological tools for visualization and sophisticated photoresponsive devices. Cyclodextrin polymers, due to the cooperative interactions, exhibit additional properties compared to their monomeric counterpart. These macromolecules can be prepared either in well water-soluble form or as gels of high swelling. Two versatile synthetic strategies for introducing a fluorescent tag (rhodamine, fluorescein, nitrobenzofuran or coumarin) into the water-soluble epichlorohydrin branched cyclodextrin polymers were worked out and compared. The fluorescent labeling was realized in three steps: 1) building in azido moieties, 2) transforming the azido groups into amino groups and 3) coupling the proper fluorescent compound to the amino groups. The other strategy started by functionalization of the monomer prior to the branching. Either the fluorescent-labeled monomer or the intermediate azido derivative of the monomer was branched. Further tuning of the properties of the polymer was achieved via branching of the methylated cyclodextrin derivative. The key intermediates and the fluorescent final products were characterized by various spectroscopic techniques and capillary electrophoresis. The applied synthetic routes were evaluated based on the molecular weight, cyclodextrin content of the products and the efficiency of labeling.

SUBMITTER: Malanga M

PROVIDER: S-EPMC4311710 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Synthetic strategies for the fluorescent labeling of epichlorohydrin-branched cyclodextrin polymers.

Malanga Milo M Bálint Mihály M Puskás István I Tuza Kata K Sohajda Tamás T Jicsinszky László L Szente Lajos L Fenyvesi Éva É

Beilstein journal of organic chemistry 20141216

The fluorescent tagging of cyclodextrin derivatives enlarges their spectroscopic properties thus generating chemosensors, biological tools for visualization and sophisticated photoresponsive devices. Cyclodextrin polymers, due to the cooperative interactions, exhibit additional properties compared to their monomeric counterpart. These macromolecules can be prepared either in well water-soluble form or as gels of high swelling. Two versatile synthetic strategies for introducing a fluorescent tag ...[more]

PMID: 25670971

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Project description:The simultaneous measurement of gene expression for thousands of genes in a single analysis by the microarray technology allows researchers to describe transcriptomes in various samples of interest. Problems with variation in data quality derived from microarray experiments are well known and might result from poor RNA quality, background problems, or sub optimal signal strength. To assess variation due to the fluorescent dye chosen, three different dye pairs were tested for labelling of cDNA in gene expression analysis experiments on a porcine immune focused oligonucleotide microarray (POM3). This in-house oligonucleotide microarray allowed a direct comparison of background fluorescence, Median signal intensities, numbers of spots detected, and resistance to photobleaching between different dye pairs. We tested Alexa Flour 546/647, Cy3/Cy5 as well as Oyster 550/650 all from Genisphere Inc., Hatfield, PA, USA. Keywords: Comparison of fluorescent dyes Each dye pairs were hybridized on two slides. The same two samples were compared on all the slides used in the present study. The liver sample from a healthy animal was labeled with the high wavelength dyes (Oyster 650/Alexa 647/Cy5) and the liver sample from the sick animal was labeled with the low wavelength dyes (Oyster 550/Alexa 546/Cy3). Each slide was scanned 3 times, immediately after hybridization (first round), after 1 month of storage in darkness and after 24 hours on the bench (12 hours of daylight and 12 hours of artificial light). Total RNA from the infected and control liver samples were extracted using RNeasy midi kit (Qiagen, Denmark) and DNase treated using RNase-Free DNase Set (Qiagen). 3DNATM Array 900 expression array detection kits (Genisphere Inc.) were used for the labeling and cDNA synthesis reaction of the RNA in the present study. Three different pairs of fluorescent dyes Oyster 550/650 (Genisphere Inc.), Alexa 546/647 (Genisphere Inc.), and Cy3/Cy5 (Genisphere Inc.) were used in separate labeling reactions. Labeling was done according to the manufacturers protocol for large-scale cDNA synthesis. For the cDNA synthesis 9.2Âµg total RNA from each liver sample was used and mixed with 20U AmpliQ RTenzyme and 10x first strand buffer (Bie og Berntsen, RÃ¸dovre, Denmark). Hybridization and washing were performed according to the manufacturers instructions (Genisphere) using Corning hybridization chambers. For one cDNA hybridization reaction; 6.5Âµl cDNA from each synthesis, 0.5Âµl Salmon sperm (10Âµg/Âµl) and 13.5Âµl 2 x Formamide-Based Hybridization Buffer (3DNA Array 900, Genisphere) was used. A total hybridization mix of 29Âµl was applied under a 22I x 25 mm LifterSlip (Erie Scientific, Portsmouth, NH, USA) carefully avoiding air bubbles. Slides were incubated at 44oC in a water bath over night. Wash buffer 1 (3DNA Array 900, Genisphere) was preheated to 44oC for the post cDNA hybridization wash. For two 3DNA hybridization reaction mixes; 2.5Âµl of each Capture reagent and 26Âµl SDS-Based hybridization buffer was mixed to a final volume of 52Âµl. 26Âµl 3DNA hybridization mix was applied to each slide, and incubated in darkness in a water bath at 50oC for 4 hours. Wash buffer 1 was preheated to 60oC for the post 3DNA hybridization wash. Slides were scanned on a CCD ArrayWoRxe auto (Applied Precision, Issaquah, WA, USA) using different exposure times (0.3 for 595 nm and 1.2 for 685 nm).

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Synthetic strategies for the fluorescent labeling of epichlorohydrin-branched cyclodextrin polymers.

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Synthetic strategies for the fluorescent labeling of epichlorohydrin-branched cyclodextrin polymers.

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