Glycoconjugates : synthesis and investigation of carbohydrate-protein interactions

To study the functions of glycoconjugates in biological systems reliable and efficient protocols for glycoconjugate synthesis are needed. To reach this goal we have developed methods for solid-phase synthesis of glycoconjugates that can be monitored with gel-phase 19F spectroscopy using fluorinated linkers, building blocks, and protecting groups. We have developed a new fluorine containing linker suitable for solid-phase synthesis of glycoconjugates. The linker was more acid-labile than similar linkers in order to enable cleavage under mild conditions of the target compound from the linker resin.  A carbamate-based strategy has been applied to attach a spacer carrying an amino group to a fluorinated Wang linker for synthesis of amino-functionalized glycoconjugates using thioglycoside donors with fluorinated protective groups. Cleavage from the solid support was performed with trifluoroacetic acid and subsequent protecting group removal gave the target compound. The terminal amine was conjugated with didecyl squarate and this derivative can be attached to various proteins and solid surfaces carrying primary or secondary amines. To evaluate this methodology we have immobilized glycoconjugates in amino-functionalized microtiter plates and successfully probed them with lectin. In addition, a novel fluorine containing protecting group has been designed, synthesized and evaluated. The protecting group was used for protection of the unreactive 4-OH in a galactose building block that was applied in the synthesis of 6-aminohexyl galabioside and was removed with TBAF in THF. Adenovirus serotype 8 (Ad8), Ad19, and Ad37 cause the severe ocular infection, epidemic keratoconjunctivities (EKC). During infection, the adenoviruses interact with sialic acid containing glycoconjugates on the epithelial cells via fiber structures extending from the viral particles. The virus particle most likely binds to the host cell in a multivalent way by simultaneously using multiple fiber proteins and binding sites. Multivalent sialic acid containing conjugates could efficiently inhibit Ad37 cell attachment and subsequent infection of human corneal epithelial (HCE) cells. Three compact tri- and tetravalent sialic acid conjugates were prepared and evaluated as inhibitors of adenoviral host cell attachment and subsequent infection and all conjugates were potent as anti-adenoviral agents. The conjugates can readily be synthesized from accessible starting materials. A crystal structure of the Ad37 fiber knob protein and the trivalent sialic acid conjugate showed that the three binding sites were all occupied by one sialic acid residue each

Synthesis and Application of a 2-[(4-Fluorophenyl)-sulfonyl]ethoxy Carbonyl(Fsec) Protected Glycosyl Donor in Carbohydrate Chemistry

The 2-[(4-fluorophenyl)sulfonyl]ethoxy carbonyl (Fsec) group for protection of hydroxyl groups has been designed, synthesized, and evaluated. Fsec-Cl was readily prepared in 91% yield over three steps and subsequently used to protect 4-fluorobenzyl alcohol in high yield. The Fsec group was cleaved from the resulting model compound under mild basic conditions e.g., 20% piperidine in DMF and was stable under acidic conditions, e.g., neat acetic acid. The Fsec group was used to protect the unreactive 4-OH in a galactose building block that was later used in the synthesis of 6-aminohexyl galabioside

Identification of small molecules blocking the Pseudomonas aeruginosa type III secretion system protein PcrV

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that employs its type III secretion system (T3SS) during the acute phase of infection to translocate cytotoxins into the host cell cytoplasm to evade the immune system. The PcrV protein is located at the tip of the T3SS, facilitates the integration of pore-forming proteins into the eukaryotic cell membrane, and is required for translocation of cytotoxins into the host cell. In this study, we used surface plasmon resonance screening to identify small molecule binders of PcrV. A follow-up structure-activity relationship analysis resulted in PcrV binders that protect macrophages in a P. aeruginosa cell-based infection assay. Treatment of P. aeruginosa infections is challenging due to acquired, intrinsic, and adaptive resistance in addition to a broad arsenal of virulence systems such as the T3SS. Virulence blocking molecules targeting PcrV constitute valuable starting points for development of next generation antibacterials to treat infections caused by P. aeruginosa. Originally included in thesis in manuscript form.</p

Small molecule screening using a whole cell viral replication reporter gene assay identifies 2-{[2-(benzoylamino)benzoyl]amino}-benzoic acid as a novel anti-adenoviral compound

Adenovirus infections are widespread in society and are occasionally associated with severe, but rarely with life-threatening, disease in otherwise healthy individuals. In contrast, adenovirus infections present a real threat to immunocompromised individuals and can result in disseminated and fatal disease. The number of patients undergoing immunosuppressive therapy for solid organ or hematopoietic stem cell transplantation is steadily increasing, as is the number of AIDS patients, and this makes the problem of adenovirus infections even more urgent to solve. There is no formally approved treatment of adenovirus infections today, and existing antiviral agents evaluated for their anti-adenoviral effect give inconsistent results. We have developed a whole cell-based assay for high-throughput screening of potential anti-adenoviral compounds. The assay is unique in that it is based on a replication competent adenovirus type 11p GFP-expressing vector (RCAd11pGFP). This allows measurement of fluorescence changes as a direct result of RCAd11pGFP genome expression. Using this assay, we have screened 9,800 commercially available small organic compounds. Initially, we observed approximately 400 compounds that inhibited adenovirus expression in vitro by &gt;/= 80% but only 24 were later confirmed as dose-dependent inhibitors of adenovirus. One compound in particular, 2-[[2-(benzoylamino)benzoyl]amino]-benzoic acid, turned out to be a potent inhibitor of adenovirus replication

Towards small molecule inhibitors of mono-ADP-ribosyltransferases

Protein ADP-ribosylation is a post-translational modification involved in DNA repair, protein degradation, transcription regulation, and epigenetic events. Intracellular ADP-ribosylation is catalyzed predominantly by ADP-ribosyltransferases with diphtheria toxin homology (ARTDs). The most prominent member of the ARTD family, poly(ADP-ribose) polymerase-1 (ARTD1/PARP1) has been a target for cancer drug development for decades. Current PARP inhibitors are generally non-selective, and inhibit the mono-ADP-ribosyltransferases with low potency. Here we describe the synthesis of acylated amino benzamides and screening against the mono-ADP-ribosyltransferases ARTD7/PARP15, ARTD8/PARP14, ARTD10/PARP10, and the poly-ADP-ribosyltransferase ARTD1/PARP1. The most potent compound inhibits ARTD10 with sub-micromolar IC50

Towards small molecule inhibitors of mono-ADP-ribosyltransferases

Protein ADP-ribosylation is a post-translational modification involved in DNA repair, protein degradation, transcription regulation, and epigenetic events. Intracellular ADP-ribosylation is catalyzed predominantly by ADP-ribosyltransferases with diphtheria toxin homology (ARTDs). The most prominent member of the ARTD family, poly(ADP-ribose) polymerase-1 (ARTD1/PARP1) has been a target for cancer drug development for decades. Current PARP inhibitors are generally non-selective, and inhibit the mono-ADP-ribosyltransferases with low potency. Here we describe the synthesis of acylated amino benzamides and screening against the mono-ADP-ribosyltransferases ARTD7/PARP15, ARTD8/PARP14, ARTD10/PARP10, and the poly-ADP-ribosyltransferase ARTD1/PARP1. The most potent compound inhibits ARTD10 with sub-micromolar IC50

PARP inhibitor with selectivity toward ADP-ribosyltransferase ARTD3/PARP3

Inhibiting ADP-ribosyl transferases with PARP-inhibitors is considered a promising strategy for the treatment of many cancers and ischemia, but most of the cellular targets are poorly characterized. Here, we describe an inhibitor of ADP-ribosyltransferase-3/poly(ADP-ribose) polymerase-3 (ARTD3), a regulator of DNA repair and mitotic progression. In vitro profiling against 12 members of the enzyme family suggests selectivity for ARTD3, and crystal structures illustrate the molecular basis for inhibitor selectivity. The compound is active in cells, where it elicits ARTD3-specific effects at submicromolar concentration. Our results show that by targeting the nicotinamide binding site, selective inhibition can be achieved among the closest relatives of the validated clinical target, ADP-ribosyltransferase-1/poly(ADP-ribose) polymerase-1

Chemical Probes to Study ADP-Ribosylation: Synthesis and Biochemical Evaluation of Inhibitors of the Human ADP-Ribosyltransferase ARTD3/PARP3

The racemic 3-(4-oxo-3,4-dihydroquinazolin-2-yl)-<i>N</i>-[1-(pyridin-2-yl)­ethyl]­propanamide, <b>1</b>, has previously been identified as a potent but unselective inhibitor of diphtheria toxin-like ADP-ribosyltransferase 3 (ARTD3). Herein we describe synthesis and evaluation of 55 compounds in this class. It was found that the stereochemistry is of great importance for both selectivity and potency and that substituents on the phenyl ring resulted in poor solubility. Certain variations at the meso position were tolerated and caused a large shift in the binding pose. Changes to the ethylene linker that connects the quinazolinone to the amide were also investigated but proved detrimental to binding. By combination of synthetic organic chemistry and structure-based design, two selective inhibitors of ARTD3 were discovered