Markus W. Germann

Reactions of salicylaldehydes with alkyl cyanoacetates on the surface of solid catalysts: syntheses of 4H-chromene derivatives

Substituted 4H-chromene derivatives are a new class of compounds that bind Bcl-2 protein and induce apoptosis in tumor cells. Here we report an efficient synthetic method for the preparation of these compounds from salicylaldehyde derivatives and alkyl cyanoacetates under solid-phase catalysis.

Conformational analysis of the hydrophobic peptide αs1-casein(136–196)

Abstract Hydrophobic interactions are important in the self-association of milk proteins, including α s1 -casein. The extent to which casein interaction sites are influenced by local secondary structure is not widely known. Both primary amino acid sequence and local secondary structure are shown to affect the self-association of the hydrophobic peptide α s1 -casein(136–196). The peptide is aggregated at low concentrations (7 μM and above), as determined by 1 H nuclear magnetic resonance (NMR) measurements at pH 6.0 in phosphate buffer. Increase in temperature is shown to induce side chain mobility (melting) as indicated by both 1 H NMR and near-UV circular dichroism (CD) measurements. As determined by far-UV CD, there is also a loss in the global amount of extended structure with increasing temperature, while β-turn structures and some aromatic dichroism are conserved at temperatures as high as 70°C. Similar retention of structure occurs at pH 2 and in 6 M guanidine HCl. The observed stability of β-turns and some side chains in α s1 -casein(136–196) supports previous assumptions that hydrophobic, proline-based turns are important interaction sites in the self-association of α s1 -casein, and possibly in the formation of the calcium transport complexes, the casein micelles. It may be speculated that these areas of the peptide represent a ‘molten globule-like’, heat stable, core structure for α s1 -casein.

RNA facilitates RecA-mediated DNA pairing and strand transfer between molecules bearing limited regions of homology.

The RecA protein ofEscherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule. We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner. A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle. The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation. Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation.

Solution Structures of Casein Peptides: NMR, FTIR, CD, and Molecular Modeling Studies of αs1-Casein, 1–23

To determine its potential for interacting with other components of the casein micelle, the N-terminal section of bovine αs1-casein-B, residues 1-23, was investigated with nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies, and molecular modeling. NMR data were not consistent with conventional α-helical or β-sheet structures, but changes in N-H proton chemical shifts suggested thermostable structures. Both CD and FTIR predicted a range of secondary structures for the peptide (30–40% turns, 25–30% extended) that were highly stable from 5°C to 25°C. Other conformational elements, such as loops and polyproline II helix, were indicated by FTIR only. Molecular dynamics simulation of the peptide predicted 32% turns and 27% extended, in agreement with FTIR and CD predictions and consistent with NMR data. This information is interpreted in accord with recent spectroscopic evidence regarding the nature of unordered conformations, leading to a possible role of αs1-casein (1–23) in facilitating casein-casein interactions.

Conformational dynamics in mixed α/β-oligonucleotides containing polarity reversals: A molecular dynamics study using time-averaged restraints*

Nucleic acid duplexes featuring a single alpha-anomeric thymidine inserted into each DNA strand via 3′-3′ and 5′-5′ phosphodiester linkages exhibit local conformational dynamics that are not adequately depicted by conventional restrained molecular dynamics (rMD) methods. We have used molecular dynamics with time-averaged NMR restraints (MDtar) to explore its applicability to describing the conformational dynamics of two α-containing duplexes – d(GCGAAT-3′-3′-αT-5′-5′-CGC)2 and d(ATGG-3′-3′-αT-5′-5′-GCTC)•r(gagcaccau). In contrast to rMD, enforcing NOE-based distance restraints over a period of time in MDtar rather than instantaneously results in better agreement with the experimental NOE and J-data. This conclusion is based on the dramatic decreases in average distance and coupling constant violations (Δdav, Jrms, and ΔJav) and improvements in sixth-root R-factors (Rx). In both duplexes, the deoxyribose ring puckering behavior predicted independently by pseudorotation analysis is portrayed remarkably well using this approach compared to rMD. This indicates that the local dynamic behavior is encoded within the NOE data, although this is not obvious from the local Rx values. In both systems, the backbone torsion angles comprising the 3′-3′ linkage as well as the (high S-) sugars of the α-nucleotide and preceding residue (α−1) are relatively static, while the conformations of the 5′-5′ linkage and the sugar in the neighboring β-nucleotide (α+1) show enhanced flexibility. To reduce the large ensembles generated by MDtar to more manageable clusters we utilized the PDQPRO program. The resulting PDQPRO clusters (in both cases, 13 structures and associated probabilities extracted from a pool of 300 structures) adequately represent the structural and dynamic characteristics predicted by the experimental data.

[9] Parallel-stranded duplex DNA: An NMR perspective

Publisher Summary This chapter describes the various forms of parallel-stranded duplex DNA that have been encountered and pays attention to the base pairing that defines the structures. The base pairing schemes that have been reported to date for homo and hetero base pairs are discussed in the chapter. The formation of parallel-stranded structures is not restricted to RNA, but has also been observed for oligodeoxy- and polydeoxyribonucleotides. Based on the spectroscopic and model-building studies, Brown et al. proposed a parallel-stranded double helical model for poly[d(CT)] at low pH 5 . A parallel-stranded double helical structure was found for the duplex formed from [α] anomeric deoxyoligonucleotides and the complementary natural [β] anomeric deoxyoligonucleotides. The structures of related sequences have been investigated by two-dimensional NMR. This study provided evidence for a right-handed double helical structure with parallel strand alignment. The bases of the [β] anomeric strand are in the anti-orientation and those of the [α] strand are in the syn orientation; the base pairing is of the Watson-Crick type. Parallel strand disposition can also be induced by ligands that bind to nucleic acids. The nonself-complementary dinucleoside monophosphate CpA forms a right-handed base-paired parallel-stranded structure, containing one intercalated proflavine.

STRUCTURAL, DYNAMIC, AND ENZYMATIC PROPERTIES OF MIXED α/β-OLIGONUCLEOTIDES CONTAINING POLARITY REVERSALS

We employ NMR structure determination, thermodynamics, and enzymatics to uncover the structural, thermodynamic and enzymatic properties of α/β-ODNs containing 3′-3′ and 5′-5′ linkages. RNase H studies show that α/β-gapmers that are designed to target erbB-2 efficiently elicit RNase H activity. NMR structures of DNA · DNA and DNA · RNA duplexes reveal that single α-anomeric residues fit well into either duplex, but alter the dynamic properties of the backbone and deoxyriboses as well as the topology of the minor groove in the DNA · RNA hybrid.

Calculation of immobilized enzyme reaction progress curves from nested ordered-sequential rate expressions

Abstract A method for estimating immobilized enzyme reaction progress curves, using simultaneous non-linear regression analysis of 2–3 substrate concentrations with time, is presented. These facile procedures involve using nested Gauss–Newton curve fitting algorithms on a Microsoft EXCEL spreadsheet. We refer to our technique as nested because the analysis consists of two or three mutually parameter-dependent sets of computations associated with bi- or termolecular enzyme-catalyzed reactions, respectively. We have applied the method to immobilized glucose oxidase-catalyzed reactions ([ d -glucose] and [O 2 ] with time) and found that the kinetic parameters from initial velocity data were similar to those determined by the nested curve fitting method discussed herein.

Antiparallel DNA duplex formation between alternating α d(GA)n and β d(GA)n sequences

Alternating polypurine d(GA)n sequences exhibit a considerable polymorphism. Here we report that α d(GA)⋅d(GA) sequences form an antiparallel stranded duplex DNA at neutral pH. The spectroscopic, electrophoretic and thermodynamic properties of the α/β chimeric oligodeoxynucleotide, 5′‐d(GA)4(T)4 α d(AG)4T‐3′, support the formation of a hairpin structure with antiparallel strands in the stem. The optical properties of this novel antiparallel structure are different from the parallel stranded homoduplex formed by d(GA)G7. This α/β hairpin has a remarkably high T m of 44.5°C in 0.4 M NaCl with a vant Hoff enthalpy comparable to that of a parallel d(GA)n duplex. Base pairing was confirmed by T4 polynucleotide ligase catalyzed joining of the α/β hairpin to an antiparallel bimolecular duplex and by non‐denaturing gel electrophoresis using duplexes containing sequence constraints. Both support the presence of αG‐G and αA‐A base pairing in the antiparallel 5′‐d(GA)4(T)4 α d(AG)4T‐3′ intramolecular duplex. This study adds to the polymorphic nature of alternating d(GA)n sequences as well as providing novel homopurine base pairing approaches for probing polypurine polypyrimidine sequences.

Homooligomeric dA·dU and dA·dT Sequences in Parallel and Antiparallel Strand Orientation: Consequence of the 5-methyl Groups on Stability, Structure and Interaction with the Minor Groove Binding Drug HOECHST 33258

Oligodeoxyribonucleotides containing dA.dU base combinations were shown to form parallel stranded DNA. CD spectra and hyperchromicity profiles provide evidence that the structure is very similar to that of a related parallel stranded dA.dT oligomer. Thermal denaturation studies show that these parallel dA.dU sequences are significantly less stable than their dA.dT analogues in either antiparallel or parallel stranded orientations. The stabilizing effect of the 5-methyl group is similar for parallel and antiparallel sequences. The minor groove binding drug Hoechst 33258 binds with similar affinity to APS dA.dT and APS dA.dU sequences. However, binding to the PS dA.dT hairpin is significantly impaired as a consequence of the different groove dimensions and the presence of thymine methyl groups at the binding site. This results in an 8.6 kJmol-1 reduced free energy of binding for the PS dA.dT sequence. Replacement of the bulky methyl group with a hydrogen (ie. T-->U) results in significantly stronger Hoechst 33258 binding to the parallel dA.dU sequences with a penalty of only 4.1 kJmol-1. Our data demonstrate that although Hoechst 33258 detects the altered groove, it is still able to bind a PS duplex containing dA.dU base pairs with high affinity, despite the large structural differences from its regular binding site in APS DNA.