Glycosylidene Carbenes. Part 6. Synthesis of alkyl and fluoroalkyl glycosides

The syntheses of glycosides from the diazirine 1 and a range of alcohols under thermal and/or photolytic conditions are described. Yields and diastereoselectivities depend upon the pK_HA values of the alcohols, the solvent, and the reaction temperature. The glycosidation of weakly acidic alcohols (MeOH, EtOH, i-PrOH, and t-BuOH, 1 equiv. each) in CH₂Cl₂ at room temperature leads to the glycosides 2–5 in yields between 60 and 34% (Scheme 1 and Table 1). At ?70 to ?60°, yields are markedly higher. In CH₂Cl₂, diastereoselectivities are very low. In THF, at ?70 to ?60°, however, glycosidation of i-PrOH leads to α-D -/β-D - 4 in a ratio of 8:92. More strongly acidic alcohols, such as CF₃CH₂OH, (CF₃)₂ CHOH, and (CF₃)₂C(Me)OH, and the highly fluorinated long-chain alcohols CF₃(CF₂)₅(CH₂)₂OH ( 11 ) and CHF₂(CF₂)₉CH₂OH ( 13 ) react (CH₂Cl₂, r.t.) in yields between 73 and 85% and lead mainly to the β-D -glucosides β-D - 6 to β-D - 8 , β-D - 12 , and β-D - 14 (d.e. 14–68%). Yields and diastereoselectivities are markedly improved, when toluene, dioxane, 1,2-dimetoxyethane, or THF are used, as examined for the glycosidation of (CF₃)₂C(Me)OH, yielding (1,2-dimethoxyethane, 25°) 80% of α-D -/ β-D - 8 in a ratio of 2:98 (d.e. 96%; Table 4). In EtCN, (CF₃)₂C(Me)OH yields up to 55% of the imidate 10 . Glycosidation of di-O-isopropylideneglucose 15 leads to 16 (CH₂Cl₂, r.t.; 65%, α-D / β-D = 33:67). That glycosidation occurs by initial protonation of the intermediate glycosylidene carbene is evidenced, for strongly acidic alcohols, by the formation of 10 , derived from the attack of (CF₃)₂MeCO^? on an intermediate nitrilium ion (Scheme 4), and for weakly acidic alcohols, by the formation of α-D - 9 and β-D - 9 , derived by attack of i-PrO^? on intermediate tetrahydrofuranylium ions. A working hypothesis is presented (Scheme 3). The diastereoselectivities are rationalized on the basis of a protonation in the σ plane of the intermediate carbene, the stabilization of the thereby generated ion pair by interaction with the BnO? C(2) group, with the solvent, and/or with the alcohol, and the final nucleophilic attack by RO^? in the π plane of the (solvated) oxonium ion.     

UVA-INDUCED AUTOCRINE STIMULATION OF FIBROBLAST-DERIVED COLLAGENASE/MMP-1 BY INTERRELATED LOOPS OFINTERLEUKIN–1 andINTERLEUKIN–6

Abstract— Previous work has shown that fibroblast-derived collagenase/matrix-metalloproteinase-1(MMP–1), responsible for the breakdown of dermal interstitial collagen, was dose-dependently induced in vitro and in vivo by UVA irradiation and this induction was at least partly mediated byinterleukin–6(IL–6). We here provide evidence that UVA-inducedIL–1α andIL–1β play a central role in the induction of the synthesis both ofIL–6 and collagenase/MMP–1. In contrast to the late increase ofIL–1α andIL–1β mRNA levels at 6 h postirradiation, bioactivity ofIL–1 is already detectable at 1 h postirradiation. This early peak ofIL–1 bioactivity appears to be responsible for the induction ofIL–6 synthesis and together withIL–6 lead to an increase of the steady-state mRNA level of collagenase/MMP–1 as deduced from studies usingIL–1α andIL–1β antisense oligonucleotides or neutralizing antibodies againstIL–1α andIL–1β Besides the early posttranslationally controlled release of intracellularIL–1, a latter pretranslationally controlled synthesis and release ofIL–1 perpetuates the UV response. From these data we suggest a UV-induced cytokine network consisting ofIL–1α,IL–1β andIL–6, which via interrelated autocrine loops induce collagenase/MMP–1 and thus may contribute to the loss of interstitial collagen in cutaneous photoaging.     

Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening

The yolk protein precursor vitellogenin (Vtg) in plasma has proved to be a simple and sensitive biomarker for assessing exposure of fish to environmental estrogens. Within international bodies such as the Organization for Economic Cooperation and Development (OECD) work is ongoing to develop screening and testing programmes for endocrine disrupting effects of new chemicals, and in the focus of this development are the fish test species common carp (Cyprinus carpio), fathead minnow (Pimephales promelas), zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes). In this study we have developed quantitative enzyme linked immunosorbent assays (ELISAs) for Vtg in common carp/fathead minnow, zebrafish and Japanese medaka. The assays were developed using a combination of monoclonal and polyclonal fish Vtg antibodies in a sandwich format, using stabilized Vtg from the test species as a standard. The carp Vtg ELISA has a working range of 1–63 ng/mL, a minimal detection limit of 0.6 ng/mL, and may also be used for quantification of Vtg in fathead minnow. In fathead minnow whole-body homogenate samples, the practical detection limit is 400 ng/mL due to the matrix effect. The zebrafish Vtg ELISA has a working range of 0.5–63 ng/mL, a minimal detection limit of 0.4 ng/mL, and a practical detection limit of 200 ng/mL in whole-body homogenate samples. The medaka Vtg ELISA has a working range of 0.25–16 ng/mL, a minimal detection limit of 0.1 ng/mL, and a practical detection limit of 125 ng/mL in whole-body homogenate samples. The intra- and inter-assay variations were below 20% for all assays. The assays were evaluated with sets of representative samples spanning the wide dynamic range of Vtg-levels found in fish exposed to environmental estrogens, and all three assays are currently undergoing international inter-laboratory validation.     

Selective enzyme amplification of NAD/NADH using coimmobilized glycerol dehydrogenase and diaphorase with amperometric detection

A flow system for substrate recycling of NAD⁺/NADH was set up with an enzyme reactor containing coimmobilized glycerol dehydrogenase (GDH) and diaphorase. The product from the diaphorase catalysis, hexacyanoferrate(II), aws detected amperometrically at a glassy carbon electrode. The amplification factor was 150 for a reactor volume of 100 μ l at a flow-rate of 0.5 ml/min. With a stopped flow of four minutes, the signal increased another 88 times, resulting in a signal amplification of 13 300 times. Equations are derived for the amplification factor and used for a discussion of the optimization of amplification systems. The K_m for GDH with glycerol as a substrate was found to be 5 × 10⁻³ M at pH 8.0. GDH from Cellulomonas sp. was purified on a gel filtration column and the purified enzyme showed a specificity toward NAD⁺, compared to NADP⁺, that was higher than 99.9%. Due to the NAD⁺ specificity of the purified GDH, the enzyme amplification system reported here could be used in detection systems for enzyme immunoassays when using alkaline phosphatase as a label and NADP⁺ as a substrate. The stability of immobilized GDH and diaphorase is several orders of magnitude better than that of alcohol dehydrogenase, which is the enzyme commonly used for NAD⁺-specific detection in these applications.     

Studies on the Chemistry of Thienoannelated O , N - and S , N -Containing Heterocycles, 28 [1]. Synthesis of Imidazo[1,5- d ]thieno[2,3- b ][1,4]thiazine Derivatives as GABA -Receptor Ligands

Summary.  GABA-receptor-ligands are still very interesting in drug-development. Usually benzodiazepines are used in the treatment but they have serious side-effects. Thus, a recently synthesized quioxaline derivative which showed reduced side-effects in an animal model was used as a model-substance. The cyclus was modified to optimize the pharmacological profile. Accordingly, a series of imidazo-thieno-thiazines was synthesized starting from 5-acetyl-2-chloro-3-nitrothiophene to yield 6-ethyl-2,3-dihydro-1H-thieno[2,3-b][1,4]thiazine-2-one. Reaction with potassium tert-butoxide and diethylchlorophosphate gave an intermediate, which resulted in the desired ring system after adding the corresponding isocyanides and potassium tert-butoxide. Corresponding author. E-mail: thomas.erker@univie.ac.at Received August 6, 2002; accepted August 13, 2002     

A chemo- and stereoselective reduction of cycloalkynes to (E)-cycloalkenes

A stereoselective entry into (E)-cycloalkenes is described, comprising the ring closing alkyne metathesis (RCAM) of suitable diynes, a ruthenium-catalyzed trans-selective hydrosilylation of the cycloalkynes thus formed, followed by a desilylation of the resulting vinylsilanes mediated by AgF.     

Development of combinatorial chemistry methods for coatings: high-throughput adhesion evaluation and scale-up of combinatorial leads

Coupling of combinatorial chemistry methods with high-throughput (HT) performance testing and measurements of resulting properties has provided a powerful set of tools for the 10-fold accelerated discovery of new high-performance coating materials for automotive applications. Our approach replaces labor-intensive steps with automated systems for evaluation of adhesion of 8 x 6 arrays of coating elements that are discretely deposited on a single 9 x 12 cm plastic substrate. Performance of coatings is evaluated with respect to their resistance to adhesion loss, because this parameter is one of the primary considerations in end-use automotive applications. Our HT adhesion evaluation provides previously unavailable capabilities of high speed and reproducibility of testing by using a robotic automation, an expanded range of types of tested coatings by using the coating tagging strategy, and an improved quantitation by using high signal-to-noise automatic imaging. Upon testing, the coatings undergo changes that are impossible to quantitatively predict using existing knowledge. Using our HT methodology, we have developed several coatings leads. These HT screening results for the best coating compositions have been validated on the traditional scales of coating formulation and adhesion loss testing. These validation results have confirmed the superb performance of combinatorially developed coatings over conventional coatings on the traditional scale.     

A Heuristic Decomposition Algorithm for Scheduling Problems on Mixed Graphs

We consider a scheduling problem where a set of n jobs has to be processed on a set of m machines and arbitrary precedence constraints between operations are given. Moreover, for any two operations i and j values a _ij >0 and a _ji >0 may be given where a _ij is the minimal difference between the starting times of operations i and j when operation i is processed first. Often, the objective is to minimize the makespan but we consider also arbitrary regular criteria. Even the special cases of the classical job shop problem J//C_max belong to the set of NP-hard problems. Therefore, approximation or heuristic algorithms are necessary to handle large-dimension problems. Based on the mixed graph model we give a heuristic decomposition algorithm for such a problem, i.e. the initial problem is partitioned into subproblems that can be solved exactly or approximately with a small error bound. These subproblems are obtained by a relaxation of a subset of the set of undirected edges of the mixed graph. The subproblems are successively solved and a proportion of the results obtained for one subproblem is kept for further subproblem definitions. Numerical results of the algorithm presented here are given.     

7-Deazaadenosine: Oligoribonucleotide building block synthesis and autocatalytic hydrolysis of base-modified hammerhead ribozymes

A 7-deazaadenosine ( = tubercidin; c⁷A; 1 ) building block for solid-phase oligoribonucleotide synthesis was prepared. The amino group of 1 was protected with the (dimethylamino)methylidene residue (→ 3 ), and the monomethoxytrityl group was introduced at OH? C(5′) (→ 4 ). Protection of OH? C(2′) was carried out by silylation, showing that use of the (i-Pr)₃Si group resulted in high 2′-O-selectivity (→ 5b , 80%). Reaction of 5b with PCl₃ afforded the phosphonate 7 which was used in solid-phase oligoribonucleotide synthesis. The autocatalytic hydrolysis of hammerhead ribozymes using pG-G-G-A-G-U-C-A-G-U-C-C-C-U-U-C-G-G-G-G-A-C-U-C-U-G-A-A-G-A-G-G-C-G-C as substrate strand (S) and modified G-C-G-C-C-G-A-A-A-C-U-C-C-C as enzyme strand (E) was studied. When c⁷A replaced A¹³ or A¹⁴, a small decrease of catalytic activity was observed, while modification in position A¹⁵ enhanced the autocatalytic hydrolysis. The results demonstrate, that the atom N(7) of adenosine in any of these positions is not crucial for ribozyme action.     

Syntheses and structures of magnesium pyridine thiolates--model compounds for magnesium binding in photosystem I

Novel magnesium pyridine-2-thiolates were prepared by using alkane elimination chemistry. The resulting complexes display a metal coordination environment composed of sulfur/nitrogen bonding from the intramolecularly stabilized mercaptopyridine ligand, in addition to coordination by the oxygen centers from two THF donors. The compounds are well-suited model compounds for the magnesium centers in Photosystem I, in which magnesium, situated in the central chlorophyll ligand, is bound to sulfur from a nearby methionine residue. All compounds were characterized by (1)H, (13)C NMR, and IR spectroscopy, in addition to Xray crystallography.