KelirisMHLSE2012 3 A Keliris I Mamedov GE Hagberg NK Logothetis K Scheffler J Engelmann 2012-09-00 5 7 478–483 Contrast Media and Molecular Imaging Here we report on a dual-modal 19F and 1H MRI paramagnetic probe with a self-immolative linker, Gd–DOMF–Gal. The enzymatic conversion of this probe by β-galactosidase resulted in a simultaneous turning on of the fluorine signal and changed ability of the Gd3+ complex to modulate the 1H MR signal intensity of the surrounding water molecules. A versatile imaging platform for monitoring a variety of enzymes by 19F and 1H MRI using this molecular design is proposed. no notspecified http://www.kyb.tuebingen.mpg.de/ published -478 15017 15421 15017 18821 KelirisZMPSUE2011 3 A Keliris T Ziegler R Mishra R Pohmann M Sauer K Ugurbil J Engelmann 2011-04-00 8 19 2529 2540 Biorganic & Medicinal Chemistry Noninvasive monitoring of intracellular targets such as enzymes, receptors, or mRNA by means of magnetic resonance imaging (MRI) is increasingly gaining relevance in various research areas. A vital prerequisite for their visualization is the development of cell-permeable imaging probes, which can specifically interact with the target that characterizes the cellular or molecular process of interest. Here, we describe a dual-labeled probe, Gd-DOTA-k(FR)-Gal-CPP, designed to report the presence of intracellular β-galactosidase (β-gal) enzyme by MRI. This conjugate consists of a galactose based core serving as cleavable spacer, incorporated between the cell penetrating peptide D-Tat49-57 and reporter moieties (Gd-DOTA, fluorescein (FR)). We employed a facile building block approach to obtain our bimodal probe, Gd-DOTA-k(FR)-Gal-CPP. This strategy involved the preparation of the building blocks and their subsequent assembly using Fmoc mediated solid phase synthesis, followed by the complexation of ligand 14 with GdCl3. Gd-DOTA-k(FR)-Gal-CPP showed a considerably higher relaxivity enhancement (16.8±0.6 mM-1s-1, 123 MHz) relative to the commercial Gd-DOTA (4.0±0.12 mM-1s-1, 123 MHz). The activation of Gd-DOTA-k(FR)-Gal-CPP was based on a cellular retention strategy that required enzymatic cleavage of the delivery vector from galactose moiety following the cell internalization to achieve a prolonged accumulation of the reporter components (Gd-DOTA/FR) in the β-gal expressing cells. Cellular uptake of Gd-DOTA-k(FR)-Gal-CPP in β-gal expressing C6/LacZ and enzyme-deficient parental C6 rat glioma cells was confirmed by fluorescence spectroscopy, MR imaging and ICP-AES measurements. All methods showed higher accumulation of measured reporters in C6/LacZ cells compared to enzyme deficient parental C6 cells. Fluorescence microscopy of cells labeled with Gd-DOTA-k(FR)-Gal-CPP indicated a predominantly vesicular localization of the green fluorescent conjugate around cell nuclei. This cellular distribution was most likely responsible for the observed nonspecific background signal in the enzyme deficient C6 cells. Even though, the specific accumulation of our bimodal probe has to be further improved, it could be already used for cell imaging by MRI and optical modalities. no notspecified http://www.kyb.tuebingen.mpg.de/ published 11 15017 18821 BarbasiewiczBM2007 3 M Barbasiewicz A Brud M Makosza 2007-04-00 8 2007 1209 1213 Synthesis Intramolecular substitution in δ-halocarbanions leading to cyclobutanes is a relatively slow process, thus they readily add to carbonyl groups; the thus-produced anionic adducts cyclize to tetrahydropyran derivatives. A simple mechanistic discussion, optimization of the reaction conditions, and scope of the reaction is presented. no notspecified http://www.kyb.tuebingen.mpg.de/ published 4 GroszekBBSL2006 3 G Groszek S Blazej A Brud D Swierczynski T Lemek 2006-03-00 11 62 2622 2630 Tetrahedron Nucleophilic addition of α-halo-4-tolylsulfonyl methyl anions to quinone methides and subsequent reactions were studied. Three kinds of consecutive reaction products were isolated, depending on the substrate structures and reaction conditions. Two of them were identified as rearrangement products and one as the vicarious nucleophilic substitution (VNS) product. An unexpected 1,2-migration of the tosyl group was observed. The mechanism of the reactions is briefly discussed. no notspecified http://www.kyb.tuebingen.mpg.de/ published 8 BrudMESZU2011 7 A Brud R Mishra J Engelmann W Su T Ziegler K Ugurbil Tübingen, Germany2011-00-00 CMST COST Action D38: Metal-Based Systems for Molecular Imaging Applications no notspecified http://www.kyb.tuebingen.mpg.de/ published 0 15017 18821 5286 7 R Mishra W Su A Brud MG Sauer J Pfeuffer K Ugurbil J Engelmann Helsinki, Finland2008-09-00 154 155 30th European Peptide Symposium (30 EPS) Cell penetrating peptides (CPP) are a special class of peptides that possess the property to traverse the formidable barrier of the plasma membrane and deliver cargos into cells. Using CPP as vectors and DNA, mRNA or proteins/enzymes as potential intracellular targets, a new generation of intracellular contrast agents (CAs) can be developed. These agents have prospective use for molecular imaging (both optical and magnetic resonance imaging) by targeted labeling of cells. Aiming to image the presence of specifi c mRNAs or enzymes, two mRNA targeting (contains a PNA sequence antisense or non-sense to the target mRNA of DsRed) and one enzyme targeted (contains a unit cleavable by -galactosidase) CAs were tested for their activity in the presence and absence of respective targets. The antisense targeting CA, their nonsense derivative and the enzyme targeted CA were taken up effi ciently into cells by an exclusively endosomal mechanism as observed by fl uorescence microscopy. Cell free binding assays proved a specifi c interaction with a synthetic target for the antisense but not for non-sense CA. Magnetic Resonance studies showed a higher uptake in transgenic DsRed expressing cells than the parent cells. However, no difference was observable for antisense versus non-sense CA in DsRed cells, due to the vesicular entrapment which is preventing the specifi c interaction between CA and cytosolic target. Since a comparable cellular distribution was visible for the enzyme targeted agent, a specifi c accumulation in -galactosidase containing cells is also unlikely. The results show that even though the designed CAs were effi ciently taken up into cells, they can interact specifi cally with the target only if colocalization is achieved. However, a lack of specifi city is caused by the endosomal entrapment. Further modifi cations are required to achieve the release from endosomes or a direct uptake into the cytosol. no notspecified http://www.kyb.tuebingen.mpg.de//fileadmin/user_upload/files/publications/Ritu%20Poster_EPS2008_5286[0].pdf published 1 15017 18821 5795 7 AJ Brud R Mishra K Ugurbil T Ziegler J Engelmann Nice, France2008-09-00 2008 World Molecular Imaging Congress (WMIC) Molecular imaging of cells or cellular processes can be obtained by targeting e.g. specific enzymes, receptors or mRNA. Aiming to track specific enzymes present in the cytosol an intracellular MR contrast agents (CA) targeting the enzyme b-galactosidase (b-gal) was synthesized as a model system. The conjugate was composed of four domains: a gadolinium loaded ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-acetate (DOTA), fluorescent dye FITC for optical imaging, an enzymatically cleavable “sensor unit” based on the enzyme targeting sugar moiety (b-D-galactopyranose), and a cell penetrating peptide (d-Tat57-49) as delivery vector. In the presented approach the MR detectable part should be selectively accumulated in the target cells containing b-gal, due to the enzymatic cleavage of internalization vector (CPP) from the enzyme targeting sugar moiety. Cellular uptake of CA was confirmed by the fluorescence microscopy and spectroscopy in C6 glioma cells as well as in the transgenic C6/LacZ7 cell line containing the targeted b-gal. Fluorescence studies have shown that the obtained CA was efficiently internalized into both cell lines in a concentration dependent manner from 5µM to 30µM without inducing significant toxicity. However, no selective accumulation of CA was observed in the b-gal expressing cells. The vesicular localization of CA around the nucleus indicates to a predominantly endosomal uptake mechanism without any detectable release into the cytosol (Fig.1). Thus, this endosomal entrapment of CA might explain, that there is no selective accumulation of CA in targeted cells, because of the inability of the contrast agent inside the vesicles to interact with the targeted enzyme located in the cytosol. Nonetheless, the synthesized CA showed an excellent ability for intracellular delivery and might prove to be useful in the cellular labeling and tracking studies. no notspecified http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/WMIC-2008-Brud.pdf published 0 15017 18821 EngelmannSBMPU2008 7 J Engelmann W Su A Brud R Mishra J Pfeuffer K Ugurbil Tübingen, Germany2008-00-00 CMST COST Action D38: Metal-Based Systems for Molecular Imaging Applications no notspecified http://www.kyb.tuebingen.mpg.de/ published 0 15017 18821 BrudEPZU2006 7 A Brud J Engelmann J Pfeuffer T Ziegler K Ugurbil Orléans, France2006-04-00 COST Chemistry Action D18: Lanthanide Chemistry for Diagnosis and Therapy no notspecified http://www.kyb.tuebingen.mpg.de/ published 0 15017 18821 BrudEPZU2005 7 A Brud J Engelmann J Pfeuffer T Ziegler K Ugurbil Köln, Germany2005-09-00 4th Annual Meeting of the Society for Molecular Imaging (SMI 2005) Reporter genes are widely applied to study gene expression and regulation in biological systems. Bacterial LacZ gene, which encodes enzyme beta-galactosidase, represents one of the most commonly used reporter genes, among others such as green fluorescent protein and luciferase. Together with fluorogenic and chromogenic substrates of the enzyme beta-galactosidase, LacZ has been utilized as a standard method of assaying clonal insertion, transcriptional activation, protein expression and protein interaction as well. The use of MR detectable substrates provides a new non-invasive tool for detection of gene expression [1]. beta-Galactosidase catalyzes the hydrolysis of the glycosidic bond between the anomeric carbon at position C1 of the beta-D-galactopyranose and an aglycone part. The replacement of hydroxyl groups at positions C2 - C6 of galactopyranose may induce the loss of enzyme activity. However, it offers a possibility to develop new marker molecules for bi-modal detection by MR/optical imaging. Here we report the syntheses of a series of compounds containing a MR detectable part (Gd(III)-DO3A) attached to beta-galactopyranose moiety by different types of linkers, and a chromophore (p-nitrophenol or 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO) [2]) at the anomeric carbon for testing enzyme activity. The tests are based on colorimetric detection of released yellow p-nitrophenol or the absorbance/fluorescence shift of released DDAO upon beta-galactosidase hydrolysis. These compounds can prove helpful for the development of new gene expression markers offering the possibility of detection by MR and optical imaging. Coupling to molecules which facilitate internalization into cells can provide a new class of intracellular contrast agents. no notspecified http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/4th-Soc-Mol-Imag-Brud.pdf published 0 15017 18821 Keliris2011 10 A Keliris Brud2010 10 A Brud 5292 10 A Brud R Mishra K Ugurbil W Su T Ziegler J Engelmann