Showing papers in "ChemBioChem in 2004"

Fluorine in Medicinal Chemistry

Abstract: Fluorinated compounds are synthesized in pharmaceutical research on a routine basis and many marketed compounds contain fluorine. The present review summarizes some of the most frequently employed strategies for using fluorine substituents in medicinal chemistry. Quite often, fluorine is introduced to improve the metabolic stability by blocking metabolically labile sites. However, fluorine can also be used to modulate the physicochemical properties, such as lipophilicity or basicity. It may exert a substantial effect on the conformation of a molecule. Increasingly, fluorine is used to enhance the binding affinity to the target protein. Recent 3D-structure determinations of protein complexes with bound fluorinated ligands have led to an improved understanding of the nonbonding protein-ligand interactions that involve fluorine.

The Unique Role of Fluorine in the Design of Active Ingredients for Modern Crop Protection

Abstract: The task of inventing and developing active ingredients with useful biological activities requires a search for novel chemical substructures. This process may trigger the discovery of whole classes of chemicals of potential commercial interest. Similar biological effects can often be achieved by completely different compounds. However, compounds within a given structural family may exhibit quite different biological activities depending on their interactions with different intracellular proteins like enzymes or receptors. By varying the functional groups and structural elements of a lead compound, its interaction with the active site of the target protein, as well as its physicochemical, pharmacokinetic, and dynamic properties can be improved. In this context, the introduction of fluorine into active ingredients has become an important concept in the quest for a modern crop protection product with optimal efficacy, environmental safety, user friendliness, and economic viability. Fluorinated organic compounds represent an important and growing family of commercial agrochemicals. A number of recently developed agrochemical candidates represent novel classes of chemical compounds with new modes of action; several of these compounds contain new fluorinated substituents. However, the complex structure-activity relationships associated with biologically active molecules mean that the introduction of fluorine can lead to either an increase or a decrease in the efficacy of a compound depending on its changed mode of action, physicochemical properties, target interaction, or metabolic susceptibility and transformation. Therefore, it is still difficult to predict the sites in a molecule at which fluorine substitution will result in optimal desired effects.

Coiled Coil Domains: Stability, Specificity, and Biological Implications

Abstract: The coiled coil is a common structural motif, formed by approximately 3 ± 5% of all amino acids in proteins. Typically, it consists of two to five -helices wrapped around each other into a left-handed helix to form a supercoil. Whereas regular -helices go through 3.6 residues for each complete turn of the helix, the distortion imposed upon each helix within a left-handed coiled coil lowers this value to around 3.5. Thus a heptad repeat occurs every two turns of the helix. 3] The coiled coil was first described by Crick in 1953. He noted that -helices pack together 20 away from parallel whilst wrapping around each other, with their side chains packing TMin a knobs-into-holes manner∫. The same year, Pauling and Corey put forward a model for -keratin. It was some 20 years later that the sequence of rabbit skeletal tropomyosin was published, and another twenty until the first structure of the leucine zipper motif was solved by Alber and co-workers. These last discoveries pushed the coiled-coil field into the spotlight, as it became apparent that they are found in important structures that are involved in crucial interactions such as transcriptional control. The most commonly observed type of coiled coil is left-handed; here each helix has a periodicity of seven (a heptad repeat), with anywhere from two (in designed coiled coils) to 200 of these repeats in a protein. This repeat is usually denoted (a-b-c-d-e-fg)n in one helix, and (a -b -c -d -e -f -g )n in the other (Figure 1). In this model, a and d are typically nonpolar core residues found at the interface of the two helices, whereas e and g are solventexposed, polar residues that give specificity between the two helices through electrostatic interactions. Similarly in righthanded coiled coils, an eleven-residue repeat is observed (undecatad repeat). 11] The apparent simplicity of the structure with its heptad periodicity has led to extensive studies. Here we aim to outline the importance of individual amino acids in maintaining -helical structure (intramolecular interactions) within individual helices, whilst promoting specific coiled-coil interactions (intermolecular interactions) of correct oligomeric state and orientation. The PV Hypothesis

Chemical biology of the sugar code.

Abstract: A high-density coding system is essential to allow cells to communicate efficiently and swiftly through complex surface interactions. All the structural requirements for forming a wide array of signals with a system of minimal size are met by oligomers of carbohydrates. These molecules surpass amino acids and nucleotides by far in information-storing capacity and serve as ligands in biorecognition processes for the transfer of information. The results of work aiming to reveal the intricate ways in which oligosaccharide determinants of cellular glycoconjugates interact with tissue lectins and thereby trigger multifarious cellular responses (e.g. in adhesion or growth regulation) are teaching amazing lessons about the range of finely tuned activities involved. The ability of enzymes to generate an enormous diversity of biochemical signals is matched by receptor proteins (lectins), which are equally elaborate. The multiformity of lectins ensures accurate signal decoding and transmission. The exquisite refinement of both sides of the protein-carbohydrate recognition system turns the structural complexity of glycans--a demanding but essentially mastered problem for analytical chemistry--into a biochemical virtue. The emerging medical importance of protein-carbohydrate recognition, for example in combating infection and the spread of tumors or in targeting drugs, also explains why this interaction system is no longer below industrial radarscopes. Our review sketches the concept of the sugar code, with a solid description of the historical background. We also place emphasis on a distinctive feature of the code, that is, the potential of a carbohydrate ligand to adopt various defined shapes, each with its own particular ligand properties (differential conformer selection). Proper consideration of the structure and shape of the ligand enables us to envision the chemical design of potent binding partners for a target (in lectin-mediated drug delivery) or ways to block lectins of medical importance (in infection, tumor spread, or inflammation).

The Polar Hydrophobicity of Fluorinated Compounds

Abstract: Fluorine substitution is a powerful tool in bioorganic and medicinal chemistry. The chemical inertness and relatively small size of fluorine 6] coupled with the short C F bond length have made C F substitution attractive for the replacement of a number of functional groups, including C OH, C H, and C=O. Fluorine incorporation into biologically active compounds can alter drug metabolism or enzyme substrate recognition. The hydrophobic nature of fluorinated compounds is also cited for improved transport across the blood± brain barrier. Improved oral bioavailability is seen in some systems where fluorine substitution leads to improved hydrolytic stability. 23±26] Furthermore, replacement of sensitive or reactive groups with fluorinated substituents has led to mechanism-based inhibitors for a wide variety of diseases and to chemotherapeutic drugs. 27±31] Review articles appear regularly on these subjects; some recent examples are given in refs. [9, 27, 32±37].

Organic Fluorine: Odd Man Out

Abstract: Linus Pauling had a superb intuitive understanding of chemistry, backed by deep intelligence and a prodigious memory. He seldom made mistakes. The best known is perhaps his ill-fated three-stranded DNA structure, but one of the few other examples concerns hydrogen bonds involving fluorine. This is evident from a comparison between the various editions of his classic TMThe Nature of the Chemical Bond∫. In the early editions he wrote: TMonly the most electronegative atoms should form hydrogen bonds, and the strength of the bond should increase with increase in the electronegativity of the two bonded atomso It is found empirically that fluorine forms very strong hydrogen bonds, oxygen weaker ones, and nitrogen still weaker ones.∫ [1] Pauling went on to discuss the strong hydrogen bond in hydrofluoric acid (HF)n and the very strong one in the hydrogen fluoride ion [HF2] and correctly concluded that the proton in the latter should lie in a single minimum potential well or in a double minimum potential with a very small barrier. It was only in the third edition, published in 1960, some twenty years after the first, that Pauling conceded: TMIt is interesting that in general fluorine atoms attached to carbon do not have significant power to act as proton acceptors in the formation of hydrogen bonds in the way that would be anticipated from the large difference in electronegativity of fluorine and carbon.∫ [2] Over the years, many chemists have followed Pauling's first line of thought, and more or less taken it for granted that organic fluorine acts as a powerful acceptor in the formation of hydrogen bonds. Others have looked at the available structural evidence as collected in the Cambridge Crystallographic Structural Database (CSD) and concluded that organic fluorine is at best only a weak hydrogen-bond acceptor. 4] A further intensive search of the CSD, including detailed inspection of individual crystal structures and backed by ab initio calculations on model systems, confirmed that organic fluorine hardly ever accepts hydrogen bonds, that is, it does so only in the absence of a better acceptor. Even in such compounds like crystalline ammonium trifluoroacetate, in which there are four hydrogen donors per anion, there is no hint of N H¥¥¥F hydrogen bonding; the four N H bonds all point towards oxygen atoms of the trifluoroacetate anions. In the ammonium monofluoro structure there is just a hint of a bifurcated hydrogen bond involving carboxylate O and the syn-planar F atom, but the latter is 0.26 ä more distant from the H atom (Figure 1). Likewise, the evidence for hydrogen bonding to organic fluorine in protein±ligand complexes was examined and found to be unconvincing. Hydrogen bonding involving B F bonds should be stronger than that involving C F bonds. Yet even in crystalline ammonium tetrafluoroborate NH4 BF4 , surely the exemplar of such an expected interaction, no short N H¥¥¥F lengths are observed. The authors commented TMit is believed that hydrogen bonding contributes negligibly to the lattice energy of this crystal∫. A search of the CSD reveals only very few crystal structures showing short intermolecular X H¥¥¥F B lengths; one example is 4,4,8,8-tetrafluoropyrazabole, another is 2,2-difluoro-4,6-dimethyl-3-phenyl1,3,2-difluorodiazaborine (Figure 2). One can hardly deny that these should be classified as genuine hydrogen bonds, but there are few such specimens. It seems clear that with its low polarizability and tightly contracted lone pairs, fluorine is unable to compete with stronger hydrogen-bond acceptors such as oxygen or nitrogen. The few authentic examples of O H¥¥¥F or N H¥¥¥F hydrogen bonding involve systems in which approach of the hydrogen atom to other better acceptor atoms is sterically hindered. Indeed, nowadays the occurrence of a genuine hydrogen bond involving organic fluorine seems to be regarded as sufficiently noteworthy that it deserves special mention in the title of the publication, as, for example, ref. [10] .

Use of Magnetic Nanoparticles as Nanosensors to Probe for Molecular Interactions

Abstract: Biocompatible magnetic nanosensors have been designed to detect molecular interactions in biological media. Upon target binding, these nanosensors cause changes in the spin–spin relaxation times of neighboring water molecules, which can be detected by magnetic resonance (NMR/MRI) techniques. These magnetic nanosensors have been designed to detect specific mRNA, proteins, enzymatic activity, and pathogens (e.g., virus) with sensitivity in the low femtomole range (0.5–30 fmol).

Neem--an omnipotent plant: a retrospection.

Abstract: Neem (Azadirachta indica A. Juss.) has universally been accepted as a wonder tree because of its diverse utility. Multidirectional therapeutic uses of neem have been known in India since the Vedic times. Besides its therapeutic efficacies, neem has already established its potential as a source of naturally occurring insecticide, pesticide and agrochemicals. Safe and economically cheaper uses of different parts of neem in the treatment of various diseases and in agriculture are discussed in this article. It further deals with the active chemical constituents of various neem formulations. Commercially available neem products are also mentioned along with their respective applications. Furthermore, evaluation of safety aspects of different parts of neem and neem compounds along with commercial formulations are also taken into consideration. Systematic scientific knowledge on neem reported so far is thus very useful for the wider interests of the world community.

Chemical strategies for activity-based proteomics.

Abstract: The assignment of molecular and cellular functions to the numerous protein products encoded by prokaryotic and eukaryotic genomes presents a major challenge for the field of proteomics. To address this problem, chemical approaches have been introduced that utilize small-molecule probes to profile dynamics in enzyme activity in complex proteomes. These strategies for activity-based protein profiling enable both the discovery and functional analysis of enzymes associated with human disease.

NF‐κB: A Multifaceted Transcription Factor Regulated at Several Levels

Abstract: NF-kappaB is a generic name for an evolutionarily conserved transcription-factor system that contributes to the mounting of an effective immune response but is also involved in the regulation of cell proliferation, development, and apoptosis The implication of NF-kappaB in central biological processes and its extraordinary connectivity to other signaling pathways raise a need for highly controlled regulation of NF-kappaB activity at several levels While all NF-kappaB activation pathways share a central and critical proteasome-mediated step that leads to the degradation of inhibitory proteins and the release of DNA-binding subunits, there is evidence for a downstream level of NF-kappaB regulation that employs several mechanisms These include promoter-specific exchange of dimers and modification of the transactivating p65 subunit by phosphorylation, acetylation, ubiquitination, or prolyl isomerization The signaling pathways and enzymes controlling this second level of regulation and their potential use as therapeutic targets for the treatment of NF-kappaB-associated pathologies are discussed here

Halogenation of drugs enhances membrane binding and permeation.

Abstract: Halogenation of drugs is commonly used to enhance membrane binding and permeation. We quantify the effect of replacing a hydrogen residue by a chlorine or a trifluoromethyl residue in position C-2 of promazine, perazine, and perphenazine analogues. Moreover, we investigate the influence of the position (C-6 and C-7) of residue CF(3) in benzopyranols. The twelve drugs are characterized by surface activity measurements, which yield the cross-sectional area, the air-water partition coefficient, and the critical micelle concentration. By using the first two parameters (A(D) and K(aw)) and the appropriate membrane packing density, the lipid-water partition coefficients, are calculated in excellent agreement with the lipid-water partition coefficients measured by means of isothermal titration calorimetry for small unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Replacement of a hydrogen residue by a chlorine and a trifluoromethyl residue enhances the free energy of partitioning into the lipid membrane, on average by deltaG(lw) approximately -1.3 or -4.5 kJ mol(-1), respectively, and the permeability coefficient by a factor of approximately 2 or approximately 9, respectively. Despite exhibiting practically identical hydrophobicities, the two benzopyranol analogues differ in their permeability coefficients by almost an order of magnitude; this is due to their different cross-sectional areas at the air-water and lipid-water interfaces.

Tools for glycomics : mapping interactions of carbohydrates in biological systems

Abstract: The emerging field of glycomics has been challenged by difficulties associated with studying complex carbohydrates and glycoconjugates. Advances in the development of synthetic tools for glycobiology are poised to overcome some of these challenges and accelerate progress towards our understanding of the roles of carbohydrates in biology. Carbohydrate microarrays, fluorescent neoglycoconjugate probes, and aminoglycoside antibiotic microarrays are among the many new tools becoming available to glycobiologists.

Fusarin C Biosynthesis in Fusarium moniliforme and Fusarium venenatum

Abstract: Fragments of polyketide synthase (PKS) genes were amplified from complementary DNA (cDNA) of the fusarin C producing filamentous fungi Fusarium moniliforme and Fusarium venenatum by using degenerate oligonucleotides designed to select for fungal PKS C-methyltransferase (CMeT) domains. The PCR products, which were highly homologous to fragments of known fungal PKS CMeT domains, were used to probe cDNA and genomic DNA (gDNA) libraries of F. moniliforme and F. venenatum. A 4.0 kb cDNA clone from F. venenatum was isolated and used to prepare a hygromycin-resistance knockout cassette, which was used to produce a fusarin-deficient strain of F. venenatum (kb = 1000 bp). Similarly, a 26 kb genomic fragment, isolated on two overlapping clones from F. moniliforme, encoded a complete iterative Type I PKS fused to an unusual nonribosomal peptide synthase module. Once again, targeted gene disruption produced a fusarin-deficient strain, thereby proving that this synthase is responsible for the first steps of fusarin biosynthesis.

Probing Protein–Carbohydrate Interactions with Microarrays of Synthetic Oligosaccharides

Abstract: Formerly a TMneglected dimension∫ of biochemistry, recent years have seen growing interest in studying the biological function of carbohydrates and glycoconjugates. An emerging understanding of the physiological role of these biomolecules has uncovered their vital participation in a host of fundamental cellular processes. In the form of glycopeptides, glycolipids, glycosaminoglycans, and proteoglyans, glycoconjugates are known to be involved in inflammation, cell ± cell interactions, signal transduction, fertility, and development. 5] Unfortunately, current methods for elucidating the biochemical roles of glycoconjugates are often cumbersome. This demonstrates the need to develop techniques that will satisfy this growing field of study by enabling rapid and facile exploration of biochemical events involving carbohydrates. Inspired by the success of DNA and protein microarrays, 7] the chip-based approach has been put forward as a useful tool in the emerging field of glycomics. Nitrocellulose-coated slides have been employed for the noncovalent immobilization of microbial polysaccharides and neoglycolipid-modified oligosaccharides. 12] Hydrophobic interactions have been utilized to anchor lipid-bearing carbohydrates on polystyrene microtiter plates. Self-assembled monolayers presenting benzoquinone groups enabled the Diels ±Alder-mediated immobilization of cyclopentadiene-derivatized monosaccharides on a gold surface. Another covalent immobilization chemistry involved treating maleimide-functionalized monoand disaccharide glycosylamines with a thiol-derivatized glass slide, or, alternatively, thiol-functionalized carbohydrates with a self-assembled monolayer presenting maleimide groups. Our motivation for developing a system for arraying carbohydrates is based on the need to have microarrays that are fully phosphate buffer (0.1M) and Perfect Hyb hybridization buffer (Sigma, 1:1 v/v) for 5 h to give double-stranded DNA assembly on the surface. The resulting surfaces were rinsed with the hybridization buffer and immersed in a solution of hemin (1.2 M) in buffer (25 mM HEPES, 20 mM KCl, 200 mM NaCl, 0.05% Triton X-100, 1% DMSO; pH 7.4) for 12 h at room temperature. The resulting system was further treated with doxorubicin (5, 5 M) in phosphate buffer (0.1M, pH 7.4) for 1 h at room temperature.

Lighting up biochemiluminescence by the surface self-assembly of DNA-hemin complexes.

Abstract: The discovery of catalytic RNAs (ribozymes) has sparked scientific activities directed to the preparation of new biocatalysts and raised the suggestion that these biomolecules participated in the evolutionary process as preprotein catalysts. 2] Analogously, deoxyribozymes, catalytic DNAzymes, are not found in nature but extensive research efforts have demonstrated the successful synthesis of catalytic deoxyribozymes for many chemical transformations. 4] One interesting example of a catalytic DNA that reveals peroxidase-like activity includes a supramolecular complex between hemin and a single-stranded guanine-rich nucleic acid (aptamer). This complex was reported to catalyze the oxidation of 2,2 -azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS) by H2O2, a common reaction used as an assay for peroxidase activity. It was suggested that the supramolecular docking of the guanine-quadruplex layers facilitates the intercalation of hemin into the complex and the formation of the biocatalytically active hemin center. Enzymes and, specifically, horseradish peroxidase (HRP) 9] are used as biocatalytic labels for the amplified detection of DNA-sensing events. The electrochemical amplified detection of DNA has been accomplished in the presence of different enzymes 8] and the chemiluminescent analysis of DNA in the presence of HRP has been reported. The integration of a DNA biocatalyst into DNA-detection schemes could provide a new method for the detection of nucleic acids that might reveal important advantages: 1) The catalytic DNA may substitute the protein-based biocatalysts, and thus eliminate nonspecific binding phenomena; 2) Tailoring of the DNA biocatalyst as part of the labeled nucleic acid might reduce the number of analytical steps for DNA detection. Here we report that two separated nucleic acids that include the segments A and B–constituting the single-stranded peroxidase deoxyribozyme, which forms a layered G-quadruplex structure (see Scheme 1)–self-assemble in the presence of hemin to form a biocatalyst for the generation of chemiluminescence in the presence of H2O2 and luminol. The effect of hybridization with the DNAzyme compounds on the resulting biochemiluminescence is discussed. We also demonstrate the self-assembly of biocatalytic, supramolecular hemin ±nucleic acid complexes on gold electrodes in monolayer configurations, and describe the biocatalytic and bioelectrocatalytic formation of chemiluminescence at the Acknowledgements

Exploring Recombinant Flavonoid Biosynthesis in Metabolically Engineered Escherichia coli

Abstract: Flavonoids are important plant-specific secondary metabolites synthesized from 4-coumaroyl coenzyme A (CoA), derived from the general phenylpropanoid pathway, and three malonyl-CoAs. The synthesis involves a plant type III polyketide synthase, chalcone synthase. We report the cloning and coexpression in Escherichia coli of phenylalanine ammonia lyase, cinnamate-4-hydroxylase, 4-coumarate:CoA ligase, and chalcone synthase from the model plant Arabidopsis thaliana. Simultaneous expression of all four genes resulted in a blockage after the first enzymatic step caused by the presence of nonfunctional cinnamate-4-hydroxylase. To overcome this problem we fed exogenous 4-coumaric acid to induced cultures. We observed high-level production of the flavanone naringenin as a result. We were also able to produce phloretin by feeding cultures with 3-(4-hydroxyphenyl)propionic acid. Feeding with ferulic or caffeic acid did not yield the corresponding flavanones. We have also cloned and partially characterized a new tyrosine ammonia lyase from Rhodobacter sphaeroides. Tyrosine ammonia lyase was substituted for phenylalanine ammonia lyase and cinnamate-4-hydroxylase in our E. coli clones and three different growth media were tested. After 48 h induction, high-level production (20.8 mg L(-1)) of naringenin in metabolically engineered E. coli was observed for the first time.

Gold glyconanoparticles as new tools in antiadhesive therapy

Abstract: Gold glyconanoparticles (GNPs) have been prepared as new multivalent tools that mimic glycosphingolipids on the cell surface. GNPs are highly soluble under physiological conditions, stable against enzymatic degradation and nontoxic. Thereby GNPs open up a novel promising multivalent platform for biological applications. It has recently been demonstrated that specific tumor-associated carbohydrate antigens (glycosphingolipids and glycoproteins) are involved in the initial step of tumor spreading. A mouse melanoma model was selected to test glyconanoparticles as possible inhibitors of experimental lung metastasis. A carbohydrate-carbohydrate interaction is proposed as the first recognition step for this process. Glyconanoparticles presenting lactose (lacto-GNPs) have been used successfully to significantly reduce the progression of experimental metastasis. This result shows for the first time a clear biological effect of lacto-GNPs, demonstrating the potential application of this glyconanotechnology in biological processes.

Atropisomerism, Biphenyls, and Fluorine: A Comparison of Rotational Barriers and Twist Angles

Abstract: Axially chiral biaryl compounds are attracting more and more attention. One reason is the growing number of known biologically active natural products that contain the biaryl motif (for example, vancomycin, steganone, and michellamine). Furthermore, the stereogenic axes provide rigid molecular frameworks for highly efficient tools in asymmetric synthesis, such as chiral ligands like BINAP and Meo-BIPHEP, just to mention two of the most prominent ones. The biaryl core is also commonly encountered in the liquid-crystal field, where its derivatives have found commercial application. Moreover, the biphenyl unit belongs to the group of six or seven privileged structures, reputated to be TMsafe bets∫ in pharmaceutical research because they guarantee versatility and high hit rates. The stretched and slim shape of this aromatic unit enables it to intercalate in the empty space between transmembrane-receptor helices and to be recognized as an unnatural ligand. For example, the biaryl unit is a key feature in the sartan family of drugs for high blood pressure: losartan (Merck, Sharpe and Dohme trademarks: Cozaar, Lorzaar), valsartan (Novartis trademark: Diovan), irbesartan (Bristol±Myers Squibb trademark: Aprovel), or candesartan (Astra trademark: Atacand). Similarly, the biaryl unit can dive into deep pockets or long clefts of enzymes. In this way, 3’and 4’-substituted biphenyl derivatives were recognized as extremely potent 17a-hydroxylase-C17,20-lyase (P-45017) inhibitors. [9] They could become promising substances for the treatment of steroidal-hormonedependent cancers, in particular prostate cancer. In view of the increasing importance of axially chiral biaryls, attention has to be paid to the stereoisomerism, called atropisomerism, that arises from the hindered rotation about the sp±sp carbon±carbon single bond. In this article, the influence of fluorine, the only element capable of mimicking hydrogen by virtue of comparable size (TMisosterism∫), will mainly be considered, in particlular how it alters the electronic and geometric properties of the biaryl unit.

mRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure.

Abstract: Approximately 3 000 genes are regulated in a time-, tissue-, and stimulus-dependent manner by degradation or stabilization of their mRNAs. The process is mediated by interaction of AU-rich elements (AREs) in the mRNA's 3'-untranslated regions with trans-acting factors. AU-rich element-controlled genes of fundamentally different functional relevance depend for their activation on one positive regulator, HuR. Here we present a methodology to exploit this central regulatory process for specific manipulation of AU-rich element-controlled gene expression at the mRNA level. With a combination of single-molecule spectroscopy, computational biology, and molecular and cellular biochemistry, we show that mRNA recognition by HuR is dependent on the presentation of the sequence motif NNUUNNUUU in single-stranded conformation. The presentation of the HuR binding site in the mRNA secondary structure appears to act analogously to a regulatory on/off switch that specifically controls HuR access to mRNAs in cis. Based on this finding we present a methodology for manipulating ARE mRNA levels by actuating this conformational switch specifically in a target mRNA. Computationally designed oligonucleotides (openers) enhance the NNUUNNUUU accessibility by rearranging the mRNA conformation. Thereby they increase in vitro and endogenous HuR-mRNA complex formation which leads to specific mRNA stabilization (as demonstrated for TNFalpha and IL-2, respectively). Induced HuR binding both inside and outside the AU-rich element promotes functional IL-2 mRNA stabilization. This opener-induced mRNA stabilization mimics the endogenous IL-2 response to CD28 stimulation in human primary T-cells. We therefore propose that controlled modulation of the AU-rich element conformation by mRNA openers or closers allows message stabilization or destabilization in cis to be specifically triggered. The described methodology might provide a means for studying distinct pathways in a complex cellular network at the node of mRNA stability control. It allows ARE gene expression to be potentially silenced or boosted. This will be of particular value for drug-target validation, allowing the diseased phenotype to ameliorate or deteriorate. Finally, the mRNA openers provide a rational starting point for target-specific mRNA stability assays to screen for low-molecular-weight compounds acting as inhibitors or activators of an mRNA structure rearrangement.

Fluorine Interactions at the Thrombin Active Site: Protein Backbone Fragments H ? Cα ? CO Comprise a Favorable C ? F Environment and Interactions of C ? F with Electrophiles

Abstract: In a systematic fluorine scan of a rigid inhibitor to map the fluorophilicity/fluorophobicity of the active site in thrombin, one or more F substituents were introduced into the benzyl ring reaching into the D pocket. The 4-fluorobenzyl inhibitor showed a five to tenfold higher affinity than ligands with other fluorination patterns. X-ray crystal-structure analysis of the protein-ligand complex revealed favorable C-F...H-C(alpha)-C=O and C-F...C=O interactions of the 4-F substituent of the inhibitor with the backbone H-C(alpha)-C=O unit of Asn98. The importance of these interactions was further corroborated by the analysis of small-molecule X-ray crystal-structure searches in the Protein Data Base (PDB) and the Cambridge Structural Database (CSD). In the C--F...C=O interactions that are observed for both aromatic and aliphatic C-F units and a variety of carbonyl and carboxyl derivatives, the F atom approaches the C=O C atom preferentially along the pseudotrigonal axis of the carbonyl system. Similar orientational preferences are also seen in the dipolar interactions C--F.C[triple chemical bond]N, C-F.C-F, and C-F...NO(2), in which the F atoms interact at sub-van der Waals distances with the electrophilic centers.

A Miniature Membrane‐less Biofuel Cell Operating at +0.60 V under Physiological Conditions

Abstract: The engineering objective of our work is the design of a power source enabling further miniaturization of autonomous implanted electronic devices, the size of which is usually limited by their battery. As the size of microelectronic circuits and of sensors shrinks, the size of the low-power sensor-transmitter package (of potential value in physiological research and in medicine) becomes increasingly dependent on the size of its power source. If stabilized for operation in vivo, a miniature glucose/O2 biofuel cell could power an implanted sensor-transmitter that would broadcast, for a few weeks, the local glucose concentration, relevant to diabetes management; or the local temperature, indicative of infection after surgery; or a pressure difference, indicating blockage of the flow of fluid in the central nervous system. Contrary to conventional cells, which contain at least nine components (anode, cathode, case, case seal, membrane, membrane seal, ion-conducting electrolyte, plumbing to the anode compartment, and plumbing to the cathode compartment), the miniature biofuel cell that we are developing contains only two, an anode and a cathode. These consist only of two 7 mm diameter “wired” enzyme bioelectrocatalystcoated carbon fibers. Underlying the simplicity and the unprecedented miniaturization of the cells are the selectivities of the thick, immobilized, glucose-electro-oxidation and O2-electroreduction catalyzing “wired” enzyme coatings of the carbon-fiber electrodes. Their immobilization and selectivity enables the operation of a single compartment cell, containing both glucose and O2 in the same compartment. Because the anode is selective for glucose, oxygen is not rapidly electroreduced on it, even though the anode is poised at a highly reducing potential. Similarly, because the cathode is selective for O2, glucose is not electrooxidized on it, even though the cathode is poised at a highly oxidizing potential. In classical fuel cells, H2/O2 or methanol/O2, H2 or methanol would be oxidized at the cathode and O2 would be reduced at the anode if the reactants were allowed to mix. As a result, the power output would approach nil. As illustrated in Figure 1, in the compartment-less biofuel cell, glucose electrons reduce glucose oxidase (GOx), glucose being electro-oxidized to d-glucono-1,5-lactone [Eq. (1)] . The electrons are collected and transported to the anode by an

Spatial and temporal sequence of events in cell adhesion: From molecular recognition to focal adhesion assembly

Abstract: A new concept that attributes a pivotal role to the pericellular coat in the regulation of the early stages of cell adhesion is presented. Quick, adaptable, and transient adhesion through multiple cooperative weak interactions provides the cell with an additional level of modulation in the decision-making process that precedes the commitment to adhesion at a particular site. Hyaluronan emerges as a modulator of cell adhesion in certain cells, mediating binding or repulsion through its polyelectrolyte character, in addition to its chirality and molecular-recognition properties. The biophysical properties of hyaluronan as well as its ultrastructural organization are analyzed in relation to this proposed function.

Two-dimensional multiarray formation of hepatocyte spheroids on a microfabricated PEG-brush surface.

Abstract: A two-dimensional microarray of ten thousand (100 x 100) hepatocyte heterospheroids, underlaid with endothelial cells, was successfully constructed with 100 microm spacing in an active area of 20 x 20 mm on microfabricated glass substrates that were coated with poly(ethylene glycol) brushes. Cocultivation of hepatocytes with endothelial cells was essential to stabilize hepatocyte viability and liver-specific functions, allowing us to obtain hepatocyte spheroids with a diameter of 100 microm, functioning as a miniaturized liver to secret albumin for at least one month. The most important feature of this study is that these substrates are defined to provide an unprecedented control of substrate properties for modulating cell behavior, employing both surface engineering and synthetic polymer chemistry. The spheroid array constructed here is highly useful as a platform of tissue and cell-based biosensors and detects a wide variety of clinically, pharmacologically, and toxicologically active compounds through a cellular physiological response.

Probing the Proteolytic Stability of β‐Peptides Containing α‐Fluoro‐ and α‐Hydroxy‐β‐Amino Acids

Abstract: One of the benefits of beta-peptides as potential candidates for biological applications is their stability against common peptidases. Attempts have been made to rationalize this stability by altering the electron availability of a given amide carbonyl bond through the introduction of polar substituents at the alpha-position of a single beta-amino acid. Such beta-amino acids (beta-homoglycine, beta-homoalanine), containing one or two fluorine atoms or a hydroxy group in the alpha-position, were prepared in enantiopure form. A versatile method for preparing these alpha-fluoro-beta-amino acids by the homologation of appropriate alpha-amino acids and C-OH->C-F or C=O-->CF(2) substitution with DAST, is described. Consequently, a series of beta-peptides possessing an electronically modified residue at the N terminus or embedded within the chain was synthesized, and their proteolytic stability was investigated against a selection of enzymes. All ten beta-peptides tested were resilient to proteolysis. Introducing a polar, sterically undemanding group, into the alpha-position of beta-amino acids in a beta-peptide chain does not appear to facilitate localized or general enzymatic degradation.

Expanding the diversity of unnatural cell-surface sialic acids.

Abstract: Novel chemical reactivity can be introduced onto cell surfaces through metabolic oligosaccharide engineering. This technique exploits the substrate promiscuity of cellular biosynthetic enzymes to deliver unnatural monosaccharides bearing bioorthogonal functional groups into cellular glycans. For example, derivatives of N-acetylmannosamine (ManNAc) are converted by the cellular biosynthetic machinery into the corresponding sialic acids and subsequently delivered to the cell surface in the form of sialoglycoconjugates. Analogs of N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) are also metabolized and incorporated into cell surface glycans, likely through the sialic acid and GalNAc salvage pathways, respectively. Furthermore, GlcNAc analogs can be incorporated into nucleocytoplasmic proteins in place of {beta}-O-GlcNAc residues. These pathways have been exploited to integrate unique electrophiles such as ketones and azides into the target glycoconjugate class. These functional groups can be further elaborated in a chemoselective fashion by condensation with hydrazides and by Staudinger ligation, respectively, thereby introducing detectable probes onto the cell. In conclusion, sialic acid derivatives are efficient vehicles for delivery of bulky functional groups to cell surfaces and masking of their hydroxyl groups improves their cellular uptake and utilization. Furthermore, the successful introduction of photoactivatable aryl azides into cell surface glycans opens up new avenues for studying sialic acid-binding proteins and elucidating the role of sialic acid in essential processes such as signaling and cell adhesion.

Type II Thioesterase Restores Activity of a NRPS Module Stalled with an Aminoacyl-S-enzyme that Cannot Be Elongated

Abstract: Nonribosomal peptide synthetases (NRPSs) carry out the biosynthesis of numerous peptide natural products, including many with important clinical applications. The NRPS, organized into a series of modules, is an efficient, high-fidelity assembly line for the production of a particular peptide. Each module consists of domains, whose activities contribute to the accuracy of these assembly-line systems. The activation (A) domain uses ATP to selectively load an amino acid onto the module through formation of a thioester bond to the pantetheine arm of the thiolation (T) domain. Peptide-bond formation, catalyzed by the condensation (C) domain, is stringent for both sidechain identity and stereochemistry. The C domain accepts an aminoacylor peptidylthioester from the preceding module for nucleophilic addition by the amine of the loaded amino acid; this generates the elongated peptide attached to the downstream module. The peptide product is synthesized one amino acid at a time until it reaches the final module. There, the fully synthesized chain is released by a type I thioesterase (TEI), the terminal domain of the NRPS assembly. Despite the high fidelity of this process, an error in any step of the assembly-line synthesis severely impacts the efficiency of the system and creates a bottleneck that results in a buildup of unprocessed intermediates. For example, an error by the A domain, which can load amino acids other than that normally accepted by the C domain, would prevent peptide-bond formation. The loaded module would be blocked until the incorrect amino acid was hydrolyzed (Figure 1). A type II thioesterase (TEII), whose gene is associated with the gene cluster of many NRPSs and related polyketide synthases (PKSs), improves the efficiency of product formation in these systems and has been proposed to edit modules through hydrolysis of acyl groups. In the surfactin NRPS, TEII was shown to regenerate misacylated modules resulting from priming of the apomodule with acyl-CoA groups. In this study we provide evidence to expand the editing function of TEIIs to include restoring the activity of modules stalled by loaded amino acids that cannot be processed. N-acetylcysteamine (SNAC) thioesters have been used previously to assay NRPS domain activities. 13–15] Hydrolysis of SNAC substrates was used here to explore the specificity of the TEII from the tyrocidine biosynthetic operon, TycF. TycF accepted a broad variety of aminoacyl-SNACs of different sidechain identity and stereochemistry with a 20-fold kcat/Km range between the mostand least-active substrate (Table 1). A series of peptidyl-SNACs derived from the tyrocidine sequence was

Membrane protein-lipid interactions in mixed micelles studied by NMR spectroscopy with the use of paramagnetic reagents.

Abstract: For solution NMR studies of the structure and function of membrane proteins, these macromolecules have to be reconstituted and solubilized in detergent micelles. Detailed characterization of the mixed detergent/protein micelles is then of key importance to validate the results from such studies, and to evaluate how faithfully the natural environment of the protein in the biological membrane is mimicked by the micelle. In this paper, a selection of paramagnetic probes with different physicochemical properties are used to characterize the 60 kDa mixed micelles consisting of about 90 molecules of the detergent dihexanoylphosphatidylcholine (DHPC) and one molecule of the Escherichia coli outer-membrane protein X (OmpX), which had previously been extensively studied by solution NMR techniques. The observation of highly selective relaxation effects on the NMR spectra of OmpX and DHPC from a water-soluble relaxation agent and from nitroxide spin labels attached to lipophilic molecules, confirmed data obtained previously with more complex NMR studies of the diamagnetic OmpX/DHPC system, and yielded additional novel insights into the protein-detergent interactions in the mixed micelles. The application of paramagnetic probes to the well-characterized OmpX/DHPC system indicates that such probes should be widely applicable as an efficient support of NMR studies of the topology of mixed membrane protein-detergent micelles.

Biosynthesis of volatiles by the myxobacterium Myxococcus xanthus.

Abstract: The volatiles emitted from cell cultures of myxobacterium Myxococcus xanthus were collected by use of a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Two new natural products, (S)-9-methyldecan-3-ol ((S)-1) and 9-methyldecan-3-one (2), were identified and synthesized, together with other aliphatic ketones and alcohols, and terpenes. Biosynthesis of the two main components (S)-1 and 2 was examined in feeding experiments carried out with the wild-type strain DK1622 and two mutant strains JD300 and DK11017, which are impaired in the degradation pathway from leucine to isovaleryl-SCoA. Isovaleryl-SCoA is used as a starter, followed by chain elongation with two malonate units. Subsequent use of methyl malonate and decarboxylation leads to (S)-1 and 2. Furthermore, 3,3-dimethylacrylic acid (DMAA) can be used by the mutant strain to form isovaleryl-SCoA, which corroborates recent data on the detection of a novel variety of the mevalonate pathway giving rise to isovaleryl-SCoA from HMGCoA.

Dendritic Polyamines: Simple Access to New Materials with Defined Treelike Structures for Application in Nonviral Gene Delivery

Abstract: Polycationic dendrimers are interesting nonviral vectors for in vitro DNA delivery. We describe a simple approach to the synthesis of dendritic polyamines with different molecular weights and adjustable flexibility (degrees of branching; DB). Both parameters influence the transfection efficiency and the cell toxicity of the polymer. Functionalization of hyperbranched polyethylenimine (PEI) by a two-step procedure generated fully branched pseudodendrimers (analogues of polypropylenimine (PPI) and polyamidoamine (PAMAM) dendrimers). The DNA transfection efficiencies observed for these polymers depended on the cell line investigated. The highest efficiencies were observed for polymers whose unfunctionalized PEI cores had molecular weights in the range M(w)=6000-25 000 g mol(-1). The cytotoxicity of the dendrimers generally rises with increasing core size. The data collected for NIH/3T3 and COS-7 cells indicate a maximum transfection efficiency at around 60 % branching for the PPI analogues, and at a PEI-core molecular weight of M(w)=25 000 g mol(-1). PAMAM functionalization of PEI (M(w)=5000 and 21 000 g mol(-1)) leads to polymers with little or no cytotoxity in the cell lines investigated.

DDI‐μFIA—A Readily Configurable Microarray‐Fluorescence Immunoassay Based on DNA‐Directed Immobilization of Proteins

Abstract: We describe a chip-based immunoassay for multiplex antigen detection, based on the self-assembly of semi-synthetic DNA-protein conjugates to generate an easily configurable protein microarray. The general principle of this microarray-fluorescence immunoassay (microFIA) is similar to that of a two-sided (sandwich) immunoassay. However, covalent single-stranded DNA-streptavidin conjugates are employed for the efficient immobilization of biotinylated capture antibodies through hybridization to complementary surface-bound DNA oligomers. In a model system, we use the DNA-directed immobilization (DDI) of antibodies to generate an antibody microarray for the parallel detection of the tumor marker human carcinoembryonic antigen (CEA), recombinant mistletoe lectin rViscumin (rVis), ceruloplasmin (CEP), and complement-1-inactivator (C1A) in human blood serum samples. Detection limits down to 400 pg mL(-1) are reached. In addition, we describe a method for the internal standardization of protein microarray analyses, based on the simultaneous measurement of constant amounts of the blood proteins CEP and C1 A, intrinsically present in human serum, to compensate for interexperimental variations usually occurring in microarray analyses. The standardization leads to a significantly higher data reliability and reproducibility in intra- and interassay measurements. We further demonstrate that the DDI-microFIA can also be carried out in a single step by tagging of the analyte simultaneously with both capture and detection antibody and subsequent immobilization of the immunocomplex formed, on the DNA microarray capture matrix. This protocol significantly reduces handling time and costs of analysis.