Studies of carbohydrate-carbohydrate-interactions by atomic force microscopy employing functionalized 4-acetylthio-butyl glucopyranosides.

By Fischer glycosylation both anomers of 4-chlorobutyl gluco-as well as galactopyranosides were obtained and transformed into the corresponding 4-acetylthio-butyl glycopyranosides. Dependent on the precursors two straightforward routes were followed to obtain the appropriate 3-O-sulfated derivatives. Unsubstituted and sulfated glucopyranosides were attached to gold surfaces a gold tips. Their interactions were studied using atomic force microscopy for simulations of intercellular glycoside-based interactions and discussed in-depth.

Synthesis of Dideoxy-octonic Acid and Cyclic and Acyclic Derivatives Thereof.

Oxidative cleavage of N-acetyl-neuraminic acid (Neu5Ac, 1) leads to the open chain octonic acid (ADOA, 2), which under various conditions is transformed into an acyclic acid or ester or into a six- or five-membered octonic acid lactone and lactam, respectively. All reactions proceed with high selectivity and in good yields. Strikingly, a simple switch of reaction conditions from basic to acidic catalysis leads to a complete switch in regioselectivity during a crucial cyclization step.

Syntheses of mannopyranoside 6-thiophosphate and 6-deoxy-6-thio-mannopyranoside phosphate as ligands for affinity chromatography.

Direct 6-thiophosphorylation of mannopyranoside gave both the wanted S- as well as the undesired O-phosphates. This required sequential protecting group syntheses to give mannopyranoside6-phosphate and 6-thiophosphate as well as 6-deoxy-6-thio-mannopyranoside6-phosphate, which were transformed into amino-linker compo-nents for affinity chromatography.

Improved synthesis of the Kijanimicin oligodeoxytetrasaccharide.

By sequential synthesis the four 2,6-dideoxy saccharide moieties of the kijanimicin tetrasaccharide could be stereoselectively assembled. For formation of all required 2-deoxy α-glycoside linkages various S-(hexopyranosyl)-phosphorodithioates as donor structures could be convincingly employed. The terminal 2-deoxy ß-glycoside linkage was stereoselectively formed following the dibromomethyl methyl ether approach. The target octadeoxy-tetrasaccha-ride could be obtained via nine subsequent steps in 5% overall yield.

Carbohydrate-based aza-macrocycles by Richman-Atkins cyclization of glucopyranose precursors.

2, 3-Di-ω-halo- as well as 2, 3-di-ω-toluenesulfonamide-alkylated glucopyranoside derivatives were prepared. Their condensation with α,ω-bis-toluenesulfonamide components under varying Richman-Atkins conditions with alkali carbonate in DMF led to carbohydrate-linked aza-macrocycles displaying 14-, 17-, 18-, 21-, 24-, and 25-membered ring structures. Isomeric aza-macrocylic coronands of 20- as well as 30-membered ring size containing two saccharides could be obtained employing Richman-Atkins condensations of two functionalized sugar building units.

In vivo and ex vivo 19-fluorine magnetic resonance imaging and spectroscopy of beta-cells and pancreatic islets using GLUT-2 specific contrast agents.

The assessment of the ß-cell mass in experimental models of diabetes and ultimately in patients is a hallmark to understand the relationship between reduced ß-cell mass/function and the onset of diabetes. It has been shown before that the GLUT-2 transporter is highly expressed in both ß-cells and hepatocytes and that D-mannoheptulose (DMH) has high uptake specificity for the GLUT-2 transporter. As 19-fluorine MRI has emerged as a new alternative method for MRI cell tracking because it provides potential non-invasive localization and quantification of labeled cells, the purpose of this project is to validate ß-cell and pancreatic islet imaging by using fluorinated, GLUT-2 targeting mannoheptulose derivatives (19 FMH) both in vivo and ex vivo. In this study, we confirmed that, similar to DMH, 19 FMHs inhibit insulin secretion and increase the blood glucose level in mice temporarily (approximately two hours). We were able to assess the distribution of 19 FMHs in vivo with a temporal resolution of about 20 minutes, which showed a quick removal of 19 FMH from the circulation (within two hours). Ex vivo MR spectroscopy confirmed a preferential uptake of 19 FMH in tissue with high expression of the GLUT-2 transporter, such as liver, endocrine pancreas and kidney. No indication of further metabolism was found. In summary, 19 FMHs are potentially suitable for visualizing and tracking of GLUT-2 expressed cells. However, current bottlenecks of this technique related to the quick clearance of the compound and relative low sensitivity of 19 F MRI need to be overcome. Copyright © 2016 John Wiley & Sons, Ltd.

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities.

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zemplén.

Trypanosomal Trans-sialidases: Valuable Synthetic Tools and Targets for Medicinal Chemistry.

In contrast to the general hydrolases, trans-sialidase from Trypanosoma cruzi (TcTS) shows excellent regio- and stereoselectivity as well as high yields in transfer reactions. Discussed are the occurrence of trans-sialidases and studies on the transfer mechanism. In detail, the preparative use by chemoenzymatic syntheses with TcTS are outlined with emphasis on the design of modified donor and acceptor substrates. Another section focuses on attempts to develop inhibitors for TcTS, and these endeavors are based on donor- and acceptor-inspired modifications as well as on some completely different structures.

Transglycosylations employing recombinant α- and ß-galactosidases and novel donor substrates.

Recombinant α- and ß-galactosidases could be prepared in larger amounts for chemoenzymatic syntheses of glycosylated oligosaccharides relevant in nutrition approaches. α-Galactosidase RafA from Escherichia coli, another thermophilic α-galactosidase AgaB from Geobacillus stearothermophilus KVE39, and also a thermophilic ß-galactosidase BglT from Thermus thermophilus TH 125 could be employed in α- and in ß-glycosylations, respectively. With model structures as well as sucrose, isomaltitol, and isomaltulose the stereo- and regiospecificities were studied. Further, a number of modified donor structures with structural variation and different leaving groups were synthesized, employed, and compared to classical donors for these transglycosylations.

Some findings in transgalactosylations employing modified donor substrates.

The scope of transgalactosylation with ß-galactosidase (bovine testis) was studied employing a series of modified donor substrates based on p-nitrophenyl ß-D-galactopyranoside and as uniform acceptor allyl 2-N-acetamido-2-deoxy-α-D-galactopyranoside. Structurally diverse donor molecules were recognized by the enzyme and led to novel disaccharide components, yet an excessive structural distortion was not accepted.

Fluorescently labeled substrates for monitoring α1,3-fucosyltransferase IX activity.

Fucosylation is often the final process in glycan biosynthesis. The resulting glycans are involved in a variety of biological processes, such as cell adhesion, inflammation, or tumor metastasis. Fucosyltransferases catalyze the transfer of fucose residues from the activated donor molecule GDP-ß-L-fucose to various acceptor molecules. However, detailed information about the reaction processes is still lacking for most fucosyltransferases. In this work we have monitored α1,3-fucosyltransferase activity. For both donor and acceptor substrates, the introduction of a fluorescent ATTO dye was the last step in the synthesis. The subsequent conversion of these substrates into fluorescently labeled products by α1,3-fucosyltransferases was examined by high-performance thin-layer chromatography coupled with mass spectrometry as well as dual-color fluorescence cross-correlation spectroscopy, which revealed that both fluorescently labeled donor GDP-ß-L-fucose-ATTO 550 and acceptor N-acetyllactosamine-ATTO 647N were accepted by recombinant human fucosyltransferase IX and Helicobacter pylori α1,3-fucosyltransferase, respectively. Analysis by fluorescence cross-correlation spectroscopy allowed a quick and versatile estimation of the progress of the enzymatic reaction and therefore this method can be used as an alternative method for investigating fucosyltransferase reactions.

Expedient and versatile formation of novel amino-deoxy-ketoheptuloses.

Novel monoketoheptuloses have been synthesized employing an amination step in a pre- and/or post-C1 chain elongation using a Petasis reagent by starting from aldohexoses or aldohexosamines. A series of gluco and manno configured 1-/3-deoxy-1-/3-amino-ketohept-2-uloses could be obtained.

Glycoconjugated amphiphilic polymers via click-chemistry for the encapsulation of quantum dots.

Herein, we present a strategy for the glycoconjugation of nanoparticles (NPs), with a special focus on fluorescent quantum dots (QDs), recently described by us as "preassembly" approach. Therein, prior to the encapsulation of diverse nanoparticles by an amphiphilic poly(isoprene)-b-poly(ethylene glycol) diblock copolymer (PI-b-PEG), the terminal PEG appendage was modified by covalently attaching a carbohydrate moiety using Huisgen-type click-chemistry. Successful functionalization was proven by NMR spectroscopy. The terminally glycoconjugated polymers were subsequently used for the encapsulation of QDs in a phase transfer process, which fully preserved fluorescence properties. Binding of these nanoconstructs to the lectin Concanavalin A (Con A) was studied via surface plasmon resonance (SPR). Depending on the carbohydrate moiety, namely, D-manno-heptulose, D-glucose, D-galactose, 2-deoxy-2-{[methylamino)carbonyl]amino}-D-glucopyranose ("des(nitroso)-streptozotocin"), or D-maltose, the glycoconjugated QDs showed enhanced affinity constants due to multivalent binding effects. None of the constructs showed toxicity from 0.001 to 1 µM (particle concentration) using standard WST and LDH assays on A549 cells.

Novel efficient routes to indoxyl glycosides for monitoring glycosidase activities.

A new efficient synthesis for broad access to indoxyl glycosides was developed. Indoxylic acid allyl ester linked to a sugar structure served as the key intermediate in this route. Selective ester cleavage and mild decarboxylation led to the corresponding indoxyl glycosides in good yields. This synthesis was applied for preparation of indoxyl glycosides of fucose, sialic acid, and 6'-sialyl lactose.

Immunocytochemistry of GLUT2, uptake of fluorescent desnitroso-streptozotocin analogs and phosphorylation of D-glucose in INS-1E cells.

The non-invasive imaging of GLUT2-expressing cells remains a challenge. As streptozotocin, and similarly alloxan, may be transported into cells by GLUT2, the major aim of the present study was to assess the possible use of fluorescent desnitroso-streptozotocin analogs for in vitro labeling of GLUT2-expressing cells. INS-1E cells, human embryonic kidney (HEK) cells, rat isolated pancreatic islets, rat hepatic cells, rat exocrine pancreatic cells and tumoral insulin-producing BRIN-BD11 cells were incubated in the presence of two distinct fluorescent desnitroso-streptozotocin analogs, probes A and B. The immunocytochemistry of GLUT2 in INS-1E cells and the phosphorylation of D-glucose by INS-1E cell homogenates were also examined. The uptake of probes A and B (12.0 µM) by INS-1E cells yielded apparent intracellular concentrations approximately one order of magnitude higher than the extracellular concentration. The two probes differed from one another by the absolute values for their respective uptake and time course, but not so by the pattern of their concentration dependency. Comparable results were recorded in HEK cells, rat isolated pancreatic islets and hepatocytes. Vastly different findings were recorded, however, in rat exocrine pancreatic cells, which do not express GLUT2. Moreover, an unusual concentration dependency for the uptake of each probe was observed in tumoral BRIN-BD11 cells. It is proposed that suitable fluorescent desnitroso-streptozotocin analogs may be used to label GLUT2-expressing cells.

Acceptor-influenced and donor-tuned base-promoted glycosylation.

Base-promoted glycosylation is a recently established stereoselective and regioselective approach for the assembly of di- and oligosaccharides by using partially protected acceptors and glycosyl halide donors. Initial studies were performed on partially methylated acceptor and donor moieties as a model system in order to analyze the key principles of oxyanion reactivities. In this work, extended studies on base-promoted glycosylation are presented by using benzyl protective groups in view of preparative applications. Emphases are placed on the influence of the acceptor anomeric configuration and donor reactivities.

(19)F-heptuloses as tools for the non-invasive imaging of GLUT2-expressing cells.

Suitable analogs of d-mannoheptulose are currently considered as possible tools for the non-invasive imaging of pancreatic islet insulin-producing cells. Here, we examined whether (19)F-heptuloses could be used for non-invasive imaging of GLUT2-expressing cells. After 20 min incubation, the uptake of (19)F-heptuloses (25 mM) by rat hepatocytes, as assessed by (19)F NMR spectroscopy, ranged from 0.50 (1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose) to 0.25 (1,3-dideoxy-1,3-difluoro-d-mannoheptulose) and 0.13 (1-deoxy-1-fluoro-d-glucoheptulose, 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose) µmol per 3×10(6)cells. (19)F MRI experiments also allowed the detection of 1-deoxy-1-fluoro-d-mannoheptulose in rat hepatocytes. All three (19)F-mannoheptuloses cited above, as well as 7-deoxy-7-fluoro-d-mannoheptulose and 1-deoxy-1-fluoro-d-glucoheptulose inhibited insulin release evoked in rat isolated pancreatic islets by 10mM d-glucose to the same extent as that observed with an equivalent concentration (10mM) of d-mannoheptulose, while 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose (also 10mM) were less potent than d-mannoheptulose in inhibiting insulin release. The 1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose only marginally affected INS-1 cell viability. These findings are compatible with the view that selected (19)F-heptuloses may represent suitable tools for the non-invasive imaging of hepatocytes and insulin-producing cells by (19)F MRI.

Synthesis and hydrolysis studies of novel glyco-functionalized platinum complexes.

Cisplatin and some of its derivatives are among the most active cytostatics for cancer treatment. Unfortunately, application of platinum complexes always indicates side effects, and frequently primary or developed resistance of tumour cells appear. Therefore, development of novel analogues especially with natural ligands is expedited. Glyco-functionalized ligands were obtained via ether synthesis with ω-halo ethers, Finkelstein reaction, with further treatment with malonate and final deprotection followed by preparation of the disodium salts. Subsequent complexation led to novel platinum derivatives, the stabilities of which in aqueous solution media were studied.

Defining oxyanion reactivities in base-promoted glycosylations.

Saccharide oxyanions obtained by base treatment could be employed in glycosylation to give oligosaccharides with high stereo- and regioselectivities.