Cover image for ChemBioChem

November 7, 2003

Volume 4, Issue 11

Pages 1109–1254

    1. Cover Picture: A Cyclic CCK8 Analogue Selective for the Cholecystokinin Type A Receptor: Design, Synthesis, NMR Structure and Binding Measurements (ChemBioChem 11/2003) (page 1109)

      Stefania De Luca, Raffaele Ragone, Chiara Bracco, Giuseppe Digilio, Luigi Aloj, Diego Tesauro, Michele Saviano, Carlo Pedone and Giancarlo Morelli

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200390114

    2. Real-Time Polymerase Chain Reaction (pages 1120–1128)

      Jochen Wilhelm and Alfred Pingoud

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300662

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      Good timing: Real-time PCR is one of the most important technologies in molecular biology and molecular medicine for quantification of nucleic acids, mutation detection, genotyping and chimerism analysis. The accumulation of products is monitored in real time by measuring specific fluorescence signals. The most common method for signal generation uses TaqMan probes (see picture). Other detection formats such as SYBR Green I or hybridisation probes allow additional product differentiation and mutation analysis by melting curve analyses after PCR.

    3. Vector Systems for the Delivery of Small Interfering RNAs: Managing the RISC (pages 1129–1136)

      Christopher W. Arendt, Guilin Tang and Asher Zilberstein

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300695

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      Silence is golden: Recent breakthroughs in the field of RNA interference have made it feasible to engineer DNA vectors capable of selectively silencing the expression of genes in a wide variety of cell types. For example, lentiviral vectors containing an RNA polymerase III promoter such as U6 have been developed for the expression of small RNA hairpins. These RNA hairpin transcripts serve as substrates for the cytoplasmic ribonuclease Dicer, generating small interfering RNAs (siRNAs) capable of inducing RNA interference in association with the RNA-induced silencing complex (RISC). A variety of vector strategies have been employed to achieve siRNA expression and long-term gene suppression. Recent studies have provided new insights into how these vector strategies might be refined to achieve potent and specific gene suppression, while minimizing the risks associated with these approaches.

    4. New Light on a Long-Known Protein Family (pages 1137–1146)

      Olav M. Andersen and Helle H. Petersen

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300580

      A family affair: The view of the LDLR family has shifted from a family of endocytosis receptors towards a class of membrane proteins participating in complex signaling pathways. The receptors play essential roles in several physiological processes but are also involved in many pathological events, such as atherosclerosis and Alzheimer's disease; this makes them attractive drug targets.

    5. Azide–Alkyne Coupling: A Powerful Reaction for Bioconjugate Chemistry (pages 1147–1149)

      Rolf Breinbauer and Maja Köhn

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300705

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      Tying together biological molecules is a challenge for molecular biologists and chemists. The azide–alkyne [3+2] cycloaddition has recently been introduced as a highly chemoselective ligation reaction in chemical biology. It has found application in the preparation of carbohydrate arrays, in the modification of viruses such as the capsid of the cowpea mosaic virus (CPMV; see scheme), and in the activity-based protein profiling of whole cells.

    6. 4-Fluorophenylglycine as a Label for 19F NMR Structure Analysis of Membrane-Associated Peptides (pages 1151–1163)

      Sergii Afonin, Ralf W. Glaser, Marina Berditchevskaia, Parvesh Wadhwani, Karl-Heinz Gührs, Ute Möllmann, Andrea Perner and Anne S. Ulrich

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300568

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      Pin the label on the peptide: Fluorine-labelling of membrane-associated peptides allows highly sensitive 19F NMR spectroscopy studies of their conformation and interactions with lipid bilayers. 4-Fluorophenylglycine (4F-Phg) was substituted selectively into each hydrophobic position of the fusogenic peptides B18, the antimicrobial peptide PGLa (α helix, see picture) and the antimicrobial peptide gramicidin S (β sheet). L-4F-Phg does not significantly affect the biological activities, whereas D-4F-Phg can be sterically obstructive. L-4F-Phg can be used as a 19F NMR label to characterise the interaction of PGLa with charged and uncharged membranes.

    7. Mutagenic Stabilization of the Photocycle Intermediate of Green Fluorescent Protein (GFP) (pages 1164–1171)

      Jens Wiehler, Gregor Jung, Christian Seebacher, Andreas Zumbusch and Boris Steipe

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300595

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      Stuck in the middle: Site-directed mutagenesis was used to produce a new phenotype of the green fluorescent protein. In the new mutant, the chromophore exists in a state that has hitherto only been known as a short-lived photocycle intermediate (see scheme). The spectra of this mutant were used for the first determination of the dimerization constant of wild-type GFP.

    8. Gene Expression within Cell-Sized Lipid Vesicles (pages 1172–1175)

      Shin-ichiro M. Nomura, Kanta Tsumoto, Tsutomu Hamada, Kazunari Akiyoshi, Yoichi Nakatani and Kenichi Yoshikawa

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300630

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      Cross-sectional images of a giant vesicle containing transcription/translation reaction solution have been obtained by confocal microscopy. The green signal indicates the expression of rsGFP within the vesicular space, while the red (hydrophobic fluorochrome) indicates the spherical vesicular membrane shape. In the early stage of the reaction, the expression efficiency inside the vesicle was remarkably higher than that in the solution outside.

    9. A Cyclic CCK8 Analogue Selective for the Cholecystokinin Type A Receptor: Design, Synthesis, NMR Structure and Binding Measurements (pages 1176–1187)

      Stefania De Luca, Raffaele Ragone, Chiara Bracco, Giuseppe Digilio, Luigi Aloj, Diego Tesauro, Michele Saviano, Carlo Pedone and Giancarlo Morelli

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300635

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      Designer peptides: A cyclic CCK8 analogue, cyclo29,34[Dpr29,Lys34]-CCK8 (Dpr=L-2,3-diaminopropionic acid), has been designed on the basis of the complex between the N-terminal fragment of the CCKA receptor and its natural ligand CCK8. The conformational features of cyclo29,34[Dpr29,Lys34]-CCK8 have been determined in aqueous solution and in water containing DPC-d38 micelles. The binding properties of cyclo29,34[Dpr29,Lys34]-CCK8 to the N-terminal receptor fragment (see model) have been investigated in a micellar environment. Estimates of the apparent dissociation constant were in the range of 70–150 nM. Preliminary in vivo studies have been performed with cell lines transfected with the CCKA receptor. Picture = structure of analogue (stick/space filled) and receptor (ribbon).

    10. NMR Structure of the (+)-CPI-indole/d(GACTAATTGAC)-d(GTCAATTAGTC) Covalent Complex (pages 1188–1193)

      Carla Bassarello, Paola Cimino, Giuseppe Bifulco, Dale L. Boger, Jarrod A. Smith, Walter J. Chazin and Luigi Gomez-Paloma

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300642

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      Exciting 3D effects! The three-dimensional NMR structure of the covalent (+)-CPI-indole–DNA adduct (pink) in comparison with various other duocarmycin agents is described ((+)-duocarmycin SA, green; (+)-DSI, blue). The (+)-CPI-indole belongs to the CC-1065/duocarmycin family of antitumor compounds. This study may provide the structural basis for the rational design of novel antitumor agents with DNA alkylating properties.

    11. Stabilisation of RNA Bulges by Oligonucleotide Complements Containing an Adenosine Analogue (pages 1194–1200)

      Annemieke Madder, Robert Ehrl and Roger Strömberg

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300531

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      RNA-bulge stabilisation can be achieved by the incorporation of the modified nucleoside 2′-deoxy-2′-β-(1-naphthylmethyl) tubercidine (see scheme) into oligonucleotides. This stabilisation is specific to RNA and does not occur in DNA complements.

    12. Evidence for the Combined Participation of a C10 and a C15 Precursor in the Biosynthesis of Moenocinol, the Lipid Part of the Moenomycin Antibiotics (pages 1201–1205)

      Ines Neundorf, Christian Köhler, Lothar Hennig, Matthias Findeisen, Duilio Arigoni and Peter Welzel

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300622

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      Biosynthesis of an unusual C25terpenoid: Upon feeding of [2-13C,4-2H]-1-deoxy-D-xylulose to S. ghanaensis H2 the deuterium label was retained exclusively at positions C-7 and C-17 in the moenocinol part of the moenomycins. This result confirms the hypothesis that the C25 structure of moenocinol is assembled from a C10 and a C15 precursor, each of which requires for its formation the involvement of a dimethylallyl diphosphate starter unit (see diagram).

    13. Chemical and Enzymatic Synthesis of Fluorinated-Dehydroalanine-Containing Peptides (pages 1206–1215)

      Hao Zhou, Dawn M. Z. Schmidt, John A. Gerlt and Wilfred A. van der Donk

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300654

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      Bring on the substitute: Fluorinated dehydroalanines are potential substitutes for the dehydroalanine residues that are present in numerous natural products. These electrophiles have enhanced reactivity and their reaction results in irreversible conjugate addition. The target structures were incorporated into peptides either by thermal elimination of α-fluorinated sulfoxides or by enzyme-catalyzed elimination of hydrogen fluoride from difluoroalanine by using the peptide epimerase YcjG (see scheme).

    14. Induction of Cell Death of Gastric Cancer Cells by a Modified Compound of the Annonaceous Acetogenin Family (pages 1216–1221)

      Guo-Rui Huang, Sheng Jiang, Yu-Lin Wu, Yan Jin, Zhu-Jun Yao and Jia-Rui Wu

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300677

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      005, licensed to kill: The effects of the annonaceous acetogenin mimic AA005 (see structure) were investigated. Experiments showed that it can induce the cell death of gastric tumor cells. Further studies indicated that most AA005-induced cell death is due to necrosis but partial and p53-independent apoptosis is also detected. An expanded study indicates that AA005 inhibits NADH:ubiquinone oxidoreductase (complex I, NADH=nicotinamide adenine dinucleotide, reduced form) in the mitochondrial electron transport system.

    15. Synthesis and Evaluation of Three Novel Scyphostatin Analogues as Neutral Sphingomyelinase Inhibitors (pages 1223–1225)

      Emmanuel N. Pitsinos, Veit Wascholowski, Sevasti Karaliota, Chrisoula Rigou, Elias A. Couladouros and Athanassios Giannis

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300667

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      The development of new therapies for various inflammatory and autoimmune diseases could be aided by the efficient synthesis and study of three novel analogues of scyphostatin (1), an inhibitor of sphingomyelinases. Unlike previous analogues, compounds such as 2 lack the epoxy moiety of scyphostatin. This could help resolve the contribution of the various reactive functionalities of the inhibitors to the observed biological activity.

    16. Catalytically Active Tetramodular 6-Deoxyerythonolide B Synthase Fusion Proteins (pages 1225–1228)

      Corinne M. Squire, Rebecca J. M. Goss, Hui Hong, Peter F. Leadlay and James Staunton

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300671

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      Easy as 1, 2, 3? Erythromycin (see scheme) is biosynthesised on a polyketide synthase consisting of three discrete bimodular protein subunits which, in the natural system, must dock together to form the active system. This paper details an experiment in which either two or all three of these proteins are translationally fused through their C and N termini. The tetramodular fusions are shown to be competent in biosynthesis.

    17. Comparison of Fertilinβ-Peptide-Substituted Polymers and Liposomes as Inhibitors of In Vitro Fertilization (pages 1229–1231)

      Kenny S. Roberts, Samidha Konkar and Nicole S. Sampson

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300672

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      Binding an egg can be made more difficult for approaching sperm by fertilinβ-containing inhibitors. A small number of multivalent contacts are required for efficient attachment of fertilinβ-containing inhibitors to receptors on the egg plasma membrane (see figure) but the physical size of the inhibitory complex also contributes to steric blocking of sperm binding to the membrane and can block sperm from binding to a second receptor type.

    18. Peptide Binding by One-Armed Receptors in Water: Screening of a Combinatorial Library for the Binding of Val-Val-Ile-Ala (pages 1232–1238)

      Carsten Schmuck and Martin Heil

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300613

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      A new class of one-armed cationic receptors has been found to bind the tetrapeptide Val-Val-Ile-Ala in water through a combination of electrostatic and hydrophobic interactions, to give a β-sheet-like structure as shown. Even within a small library of only 512 different members, the binding constants for the most efficient receptors of approximately 103–104M−1 are more than a factor of 200 greater than those of the worst.

    19. Synthetic Peptides Derived from the Angiostatin K4 Domain Inhibit Endothelial Cell Migration (pages 1238–1242)

      Monica Dettin, Silvio Bicciato, Claudia Scarinci, Edith Cline, Mark W. Lingen and Carlo Di Bello

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300711

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      Loop the loop: Several linear and cyclic peptides patterned on the angiostatin K4 domain (see picture) have been synthesized and tested in order to identify active sequence candidates for further studies aimed at elucidating the antimigratory activity and functionality of the K4 domain. Migration assays indicate that different K4 regions induce different degrees of inhibition of cell motility. In particular, the linear peptide reproducing the (358–379) fragment of angiostatin exhibits an inhibitory activity comparable to that of the entire K4 domain.

    20. Quantum Dots As A Visual Aid For Screening Bead-Bound Combinatorial Libraries (pages 1242–1245)

      Hernando J. Olivos, Kiran Bachhawat-Sikder and Thomas Kodadek

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300712

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      Seeing red: A new methodology for the on-bead screening of proteins has been developed with the aid of red-emitting quantum dots. Use of this nanomaterial allowed a dual-color fluorescence assay to be developed that overcomes the limitation of the autofluorescence of TentaGel beads. The figure shows a positive control (red fluorescence) and a negative control (background green fluorescence).

    21. Rapid Diversity-Oriented Synthesis in Microtiter Plates for In Situ Screening of HIV Protease Inhibitors (pages 1246–1248)

      Ashraf Brik, John Muldoon, Ying-Chuan Lin, John H. Elder, David S. Goodsell, Arthur J. Olson, Valery V. Fokin, K. Bary Sharpless and Chi-Huey Wong

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200300724

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      Click and go: By using click chemistry based on a new triazole forming reaction condition (see scheme), over 100 triazole compounds generated in microtiter plates from a core structure were screened for HIV protease inhibition in situ without product isolation. Potent inhibitors, active at nanomolar concentrations, against the wild type and drug resistant mutants were identified.

    22. Preview: ChemBioChem 11/2003 (page 1254)

      Version of Record online: 3 NOV 2003 | DOI: 10.1002/cbic.200390119