Calculations of the conformations of Iturin A in relation with NMR studies

Iturin A is an antifungal antibiotic which was isolated from a strain of Bacillus subtilis, and contains a lipophilic beta amino acid closing an heptapeptide cycle with polar L and D residues. Iturin A belongs to a lipopeptide family of which the LDDLLDL sequence is kept constant. NMR spectroscopy and semi-empirical energy calculations are combined to design the conformations of Iturin A in pyridine solution. J coupling constants and nOes (nuclear Overhauser enhancements) are used as guiding line for energy calculations. This preliminary study shows that Iturin A in pyridine appears as rather rigid, especially in the L Pro 5-D Asn 6 region, probably involved in a beta turn. The polar side chains can form different networks of intramolecular hydrogen bonds. The Tyr side chain, relatively mobile, could be involved in interactions with an hydrophobic environment as the beta amino acid side chain found away from the peptide cycle.     

Bi- and tricyclic nucleoside derivatives restricted in S-type conformations and obtained by RCM-reactions

Ring-closing metathesis (RCM) is applied as a new and powerful technology in the construction of nucleoside analogues that are conformationally restricted in S-type conformations due to additional 3',4'- and/or 3',5'-linkages.     

Calculations of the conformational properties of acyclonucleosides. Part I. Stable conformations of acyclovir

Conformational properties of te antiherpes agent acyclovir (acycloguanosine, ACG) were calculated using molecular mechanics approximation. Eighty two different stable conformations have been determined. The large number of local minima of the total enery, and small differences between them, point to the marked flexibility of the acyclic chain. The barrier to rotation around N9-C1 bond was calculated and found to be asymmetric (the lower equals 17 kJ/mol, the higher 63 kJ/mol). An energetic preference for the compact from of ACG was demonstrated. A comparison of the calculated conformations with the crystallographic structures is presented.     

Acyclonucleosides: acyclobenzimidazole nucleoside and nucleotide analogues and conformations of the acyclic chains by means of NMR spectroscopy

A number of acyclo nucleosides of benzimidazole derivatives has been synthesized, in which the benzimidazole ring includes substituents at C(5), C(6) and C(2). The acyclic chains which replace the sugar moiety are 2',3'-dihydroxypropyl, 2'-hydroxyethoxymethyl and 1',5'-dihydroxy-4'-hydroxymethyl-3'- oxypentyl -2' (R), each of which corresponds to some fragment of the ribose ring. 1H NMR spectroscopy has been employed to determine the conformations of these acyclic chains in solutions of fully deuterated dimethylsulfoxide and methanol, utilizing for this purpose vicinal proton-proton coupling constants, and the new Karplus relation developed by Haasnoot , de Leeuw & Altona ( Tetrahedron , 36, 2783-2792, 1980). The data thus obtained are compared with those available for the solid state from X-ray diffraction data, and should be applicable to other classes of acyclonucleosides . Nucleotides of the three types of acyclo benzimidazole nucleosides have also been prepared, and their susceptibilities to snake venom 5'-nucleotidase examined. In contrast to acycloG , the nucleoside analogues did not exhibit significant in vitro activity against herpes simplex virus type 1 or influenza virus.     

GlcNAc-Thiazoline conformations

The title compound, a powerful inhibitor of retaining N-acetylhexosaminidases, can move freely among three pyranose solution conformations of similar energy—two twist boats and the ⁴C₁ chair—as revealed by NMR, calculational, and crystallographic studies. It binds in the enzyme active site only in the pseudo-⁴C₁ conformation, however, in which it most closely resembles the hypothetical bound substrate transition state, a ⁴E sofa that is approximately trigonal bipyramidal at the anomeric carbon.     

Biosynthesis of nucleoside analogues via thermostable nucleoside phosphorylase

Biocatalyzed synthesis of nucleoside analogues was carried out using two thermostable nucleoside phosphorylases from the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix K1. The synthesis of the 2,6-diaminopurine nucleoside and 5-methyluridine was used as a reaction model to test the process. Both the purine nucleoside phosphorylase (apPNP) and uridine phosphorylase (apUP) were functionally expressed in Escherichia coli. The recombinant enzymes were characterized after purification, and both enzymes showed high thermostability and broad substrate specificity. Both enzymes retained 100 % of their activity after 60 min at high temperature, and the optimum temperature for the enzymes was 90–100 °C. The nucleoside phosphorylases obtained from A. pernix are valuable industrial biocatalysts for high-temperature reactions that produce nucleoside drugs in high yields.     

Molecular mechanics studies on the conformations of 2',3'-dideoxy-2',3'-didehydroguanine nucleoside, D4G.

Conformational energy calculations have been presented on guanine nucleoside in which the furanose ring is replaced by 2',3'-dideoxy-2',3'-didehydrofuran using molecular mechanics and conformational analysis. Conformational energies have been evaluated using the MM2 and AMBER94 force field parameters at two different dielectric constants. The results are presented in terms of isoenergy contours in the conformational space of the glycosidic (chi) and C4'-C5' (gamma) bonds torsions. In general, the chi-gamma interrelationships differ from the corresponding plots for unmodified nucleosides and nucleotides, reported previously. Consistency of the calculated preferred conformations with the x-ray data is sensitive to the force field employed.     

Studies of nucleoside transporters using novel autofluorescent nucleoside probes

To better understand nucleoside transport processes and intracellular fates of nucleosides, we have developed a pair of fluorescent nucleoside analogues, FuPmR and dFuPmR, that differ only in the sugar moiety (ribofuranosyl versus 2'-deoxy, respectively), for real-time analysis of nucleoside transport into living cells by confocal microscopy. The binding and transportability of the two compounds were assessed for five recombinant human nucleoside transporters (hENT1/2, hCNT1/2/3) produced in Saccharomyces cerevisiae and/or oocytes of Xenopus laevis. The ribosyl derivative (FuPmR) was used to demonstrate proof of principle in live cell imaging studies in 11 cultured human cancer cell lines with different hENT1 activities. The autofluorescence emitted from FuPmR enabled direct visualization of its movement from the extracellular medium into the intracellular compartment of live cells, and this process was blocked by inhibitors of hENT1 (nitrobenzylmercaptopurine ribonucleoside, dipyridamole, and dilazep). Quantitative analysis of fluorescence signals revealed two stages of FuPmR uptake: a fast first stage that represented the initial uptake rate (i.e., transport rate) followed by a slow long-lasting second stage. The accumulation of FuPmR and/or its metabolites in nuclei and mitochondria was also visualized by live cell imaging. Measurements of fluorescence intensity increases in nuclei and mitochondria revealed rate-limited processes of permeant translocation across intracellular membranes, demonstrating for the first time the intracellular distribution of nucleosides and/or nucleoside metabolites in living cells. The use of autofluorescent nucleosides in time-lapse confocal microscopy is a novel strategy to quantitatively study membrane transport of nucleosides and their metabolites that will provide new knowledge of nucleoside biology.     

Phosphorylation of anti-HIV nucleoside analogs by nucleoside diphosphate kinase.

The reaction of NDP kinase with antiviral nucleoside triphosphates used in antiviral therapies was studied at the presteady state by fluorescence stopped-flow and compared with the steady-state parameters. The affinity of the analogs was determined by fluorescence titration of a mutated enzyme with an inserted Trp in the binding site. The lack of the 3' hydroxyl in analogs is shown to decrease the kcat more than the KD.     

Recent studies on nucleoside H-phosphonothioate and nucleoside methylphosphonothioate synthesis

The reaction of suitably protected nucleosides with phosphinic acid in the presence of condensing agents has been investigated and applied for the preparation of nucleoside 3'-H-phosphonothioates. Studies on synthesis and separation of diastereoisomers of nucleoside 3'-methylphosphonothioates are also discussed.     

Periodic conformations of deoxyribonucleic acids

The conformation of a deoxyribonucleotide unit in a deoxyribonucleic acid molecule can be defined by six angles, each of which specifies the relative orientations between two groups of atoms adjacent to a covalent bond. With the assumption that these atoms are hard spheres with fixed van der Waal radii, conformations are sought to minimize their overlapping. The additional requirement of the polymer having a periodic structure further reduces the allowable conformations.     

Physical characterization of serpin conformations

The native serpin fold is characterized by being metastable. This thermodynamic characteristic is manifested in the conversion of the native state to other more stable conformations. Whilst this structural transition is required for proteinase inhibition and regulation of a range of biological phenomena, inappropriate structural changes can result in a number of disease states. Identification of these alternative conformations has been essential in our understanding of serpin structure and function. However, identifying these alternative forms is also important if we are not to misinterpret data due to the formation of these states during in vitro studies. The different physical properties of these alternative serpin conformational states make it possible to use a range of standard laboratory techniques to identify these structures. In this chapter, we will outline these general approaches that can be used routinely to identify the alternative serpin conformational states.     

Clustering analysis of Enkephalin conformations

A combination of a Monte-Carlo sampling, a clustering method, and a minimization algorithm is proposed for the calculation of conformational properties of molecules in solutions. This approach is applied to the determination of stable conformations of the peptide hormone Enkephalin in solution and it gives good agreement with experimental data.     

Computation of DNA backbone conformations

We present an algorithm for the computation of 2'-deoxyribose-phosphodiester backbone conformations that are stereochemically compatible with a given arrangement of nucleic acid bases in a DNA structure. The algorithm involves the sequential computation of 2'-deoxyribose and phosphodiester conformers (collectively referred to as a backbone 'segment'), beginning at the 5'-end of a DNA strand. Computation of the possible segment conformations is achieved by the initial creation of a fragment library, with each fragment representing a set of bond lengths, bond angles and torsion angles. Following exhaustive searching of sugar conformations, each segment conformation is reduced to a single vector, defined by a specific distance, angle and torsion angle, that allows calculation of the O(1)' position. A given 'allowed' conformation of a backbone segment is determined based on its compatibility with the base positions and with the position of the preceding backbone segment. Initial computation of allowable segment conformations of a strand is followed by the determination of continuous backbone solutions for the strand, beginning at the 3'-end. The algorithm is also able to detect repeating segment conformations that arise in structures containing geometrically repeating dinucleotide steps. To illustrate the utility and properties of the algorithm, we have applied it to a series of experimental DNA structures. Regardless of the conformational complexity of these structures, we are able to compute backbone conformations for each structure. Hence, the algorithm, which is currently implemented within a new computer program NASDAC (Nucleic Acids: Structure, Dynamics and Conformation), should have generally applicability to the computation of DNA structures.     

Intramolecularly hydrogen-bonded peptide conformations

Over the past few years the possible occurrence of intramolecularly hydrogen-bonded structures in linear and cyclic peptides has attracted increasing attention. In this review emphasis is given to solid-state studies, particularly by X-ray diffraction and infrared absorption techniques. Conformational energy calculations are also considered. The discussion is focused both on model peptides and biological activity polypeptide molecules. The tetrapeptide system (Formula: see text), examined allows one to discuss the extended C5 structure and the various folded conformations, namely the C7 (gamma-turn), C8, C10 (beta-turn), C11, and C13 conformations. The four latter forms may include cis peptide configurations. The oxy-analogs to the C7, C10, and C13 conformations and structures containing bifurcated hydrogen bonds are also discussed. The last sections describe intramolecularly hydrogen-bonded peptide structures involving: (1) a side-chain group, (2) the N-protecting group (in synthetic model compounds), and (3) a beta-amino acid.