Synthesis, structural elucidation, DNA-PK inhibition, homology modelling and anti-platelet activity of morpholino-substituted-1,3-naphth-oxazines

A number of new angular 2-morpholino-(substituted)-naphth-1,3-oxazines (compound 10b), linear 2-morpholino-(substituted)-naphth-1,3-oxazines (compounds 13b-c), linear 6, 7 and 9-O-substituted-2-morpholino-(substituted)-naphth-1,3-oxazines (compounds 17-22, 24, and 25) and angular compounds 14-16 and 23 were synthesised. The O-substituent was pyridin-2yl-methyl (15, 18, and 21) pyridin-3yl-methyl (16, 19, and 22) and 4-methylpipreazin-1-yl-ethoxy (23-25). Twelve compounds were tested for their inhibitory effect on collagen induced platelet aggregation and it was found that the most active compounds were compounds 19 and 22 with IC(50)=55±4 and 85±4 μM, respectively. Furthermore, the compounds were also assayed for their ability to inhibit DNA-dependent protein kinase (DNA-PK) activity. The most active compounds were 18 IC(50)=0.091 μM, 24 IC(50)=0.191 μM, and 22 IC(50)=0.331 μM. Homology modelling was used to build a 3D model of DNA-PK based on the X-ray structure of phosphatidylinositol 3-kinases (PI3Ks). Docking of synthesised compounds within the binding pocket and structure-activity relationships (SAR) analyses of the poses were performed and results agreed well with observed activity.     

Synthesis,spectroscopic and structural elucidation of tyrosinamide hydrogensquarate monohydrate

Synthesis, isolation, spectroscopic and structural elucidation of tyrosinamide hydrogensquarate monohydrate (I) is reported on the basis of quantum chemical DFT calculations, vibrational analysis and experimental linear-polarized IR-spectroscopy in solid state. These data are compared with those obtained using single crystal X-ray diffraction, which show that the molecules of (I) in the unit cell formed 3D network through moderate intermolecular (Tyr)OH...O = C(Sq) (2.727 A), O=C-NH2...OH(Tyr) (2.991 A), O=C-NH2...OH(Sq) (2.988 A), O=C-NH2...O=C-NH2 (3.068 A), N+H3...O=C(Sq) (2.737, 2.953, 2.954 A), OH2...O=C(Sq) (2.839 A) and (Sq)OH...OH2 (2.607 A) hydrogen bonds. The relationship between the structure and spectroscopic properties is studied.     

Synthesis,spectroscopic and structural elucidation of sympathomimetic amine,tyraminium dihydrogenphosphate

The synthesis, isolation, spectroscopic and structural elucidation of sympathomimetic amine, tyramine dihydrogenphosphate are of interest due to its biological activity and the establishing correlation between spectroscopic properties and structure. The complex approach for investigation included single crystal X-ray diffraction, new technique in linear-polarized IR-spectroscopy in solid state and quantum chemical calculations with a view to predict the electronic structure and vibrational data of interacting species in entitled compound, the correlation structure–spectroscopic properties as well as the influence of intermolecular interaction on IR-characteristic bands are carried out.     

A facile enzymatic synthesis of sphingosine-1-phosphate and dihydrosphingosine-1-phosphate

A procedure is described to prepare sphingosine-1-phosphate by treatment of sphingosylphosphocholine with phospholipase D, isolated from Streptomyces chromofuscus. The phosphorylated long chain bases were purified by selective precipitation and differential extraction. Milligram quantities can be obtained in a yield of about 70%. Application of the procedure to dihydrosphingosylphosphocholine results in the synthesis of dihydrosphingosine-1-phosphate.     

Synthesis,structural elucidation and in vitro antiparasitic activity against Trypanosoma cruzi and Leishmania chagasi parasites of novel tetrahydro-1-benzazepine derivatives

Forty six new 1,4-epoxy-2-exo-aryl- and cis-2-aryl-4-hydroxytetrahydro-1-benzazepine derivatives were synthesized and fully characterized. All compounds were tested in vitro against both Trypanosoma cruzi and Leishmania chagasi parasites and also for cytotoxicity using Vero and THP-1 mammalian cell lines. Many of the evaluated compounds showed remarkable activity against the epimastigote and intracellular amastigote forms of T. cruzi, with IC₅₀ values comparable with that of control drug nifurtimox, a nitrofuran derivative currently used in the treatment of Chagas’ disease. Other derivatives were found to have good activity against L. chagasi promastigotes, with low toxicity against the mammalian cells, but neither of them was active on intracellular amastigotes of L. chagasi infecting THP-1 macrophages.     

NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides.

The potential of one- and two-dimensional NMR techniques for the identification of individual sugar residues, their anomeric configuration, interglycosidic linkages, sequencing and the site of any appended group, in establishing the structures of naturally occurring oligosaccharides and glycosides is presented.     

Synthesis of arabinitol 1-phosphate and its use for characterization of arabinitol-phosphate dehydrogenase

D-arabinitol 1-phosphate (Ara-ol1-P), a substrate for D-arabinitol-phosphate dehydrogenase (APDH), was chemically synthesized from D-arabinonic acid in five steps (O-acetylation, chlorination, reduction, phosphorylation, and de-O-acetylation). Ara-ol1-P was used as a substrate for the characterization of APDH from Bacillus halodurans. APDH converts Ara-ol1-P to xylulose 5-phosphate in the oxidative reaction; both NAD(+) and NADP(+) were accepted as co-factors. Kinetic parameters for the oxidative and reductive reactions are consistent with a ternary complex mechanism.     

Synthesis and biological evaluation of prodrugs of 2-fluoro-2-deoxyribose-1-phosphate and 2,2-difluoro-2-deoxyribose-1-phosphate

We report in this Letter the synthesis of prodrugs of 2-fluoro-2-deoxyarabinose-1-phosphate and 2,2-difluoro-2-deoxyribose-1-phosphate. We demonstrate the difficulty of realising a phosphorylation step on the anomeric position of 2-deoxyribose, and we discover that introduction of fluorine atoms on the 2 position of 2-deoxyribose enables the phosphorylation step: in fact, the stability of the prodrugs increases with the degree of 2-fluorination. Stability studies of produgs of 2-fluoro-2-deoxyribose-1-phosphate and 2,2-difluoro-2-deoxyribose-1-phosphate in acidic and neutral conditions were conducted to confirm our observation. Biological evaluation of prodrugs of 2,2-difluoro-2-deoxyribose-1-phosphate for antiviral and cytotoxic activity is reported.     

Isolation, structural elucidation and immunomodulatory activity of fructans from aged garlic extract

Traditionally, garlic (Allium sativum) is known to be a significant immune booster. Aged garlic extract (AGE) possesses superior immunomodulatory effects than raw garlic; these effects are attributed to the transformed organosulfur compounds. AGE is also known to contain fructans; the amount of fructans in AGE represents a small fraction (0.22%) of the total fructans in raw garlic. In order to evaluate the biological activity of fructans present in AGE, both high molecular weight (>3.5 kDa; HF) and low molecular weight (<3 kDa; LF) fructans were isolated. The structures of purified HF and LF from AGE determined by (1)H NMR and (13)C NMR spectroscopy revealed that both have (2→1) β-D-fructofuranosyl bonds linked to a terminal glucose at the non-reducing end and β-D-fructofuranosyl branching on its backbone. Biological activity of fructans was assessed by immunostimulatory activity using murine lymphocytes and peritoneal exudate cells (source of macrophages). Both HF and LF displayed mitogenic activity and activation of macrophages including phagocytosis. These activities were comparable to that of known polysaccharide immunomodulators such as zymosan and mannan. This study clearly demonstrates that garlic fructans also contribute to the immunomodulatory properties of AGE, and is the first such study on the biological effects of garlic fructans.     

Expression of a synthetic gene coding for ostrich egg-white lysozyme in Pichia pastoris and its enzymatic activity

To investigate the structure-function relationships of goose-type lysozyme, a gene coding for ostrich egg-white lysozyme (OEL) was designed based on the published amino acid sequence and constructed by assembling 32 chemically synthesized oligonucleotides. To obtain the recombinant OEL (rOEL), the synthetic gene was fused to the alpha-factor signal peptide in the expression vector pPIC9K and expressed in the methylotrophic yeast Pichia pastoris. The secreted protein from the transformed yeast was found to be processed at three different sites, including the correct site. The correctly processed rOEL was purified to homogeneity and shown to be indistinguishable from the authentic form in terms of circular dichroism (CD) spectrum and enzyme activity. Furthermore, the time-course of the reaction catalyzed by OEL was studied using (GlcNAc)(n) (n = 5 and 6) as the substrate and compared to that of goose egg-white lysozyme (GEL) [Honda and Fukamizo (1998) BIOCHIM: Biophys. Acta 1388, 53-65]. OEL hydrolyzed (GlcNAc)(6) in an endo-splitting manner producing mainly (GlcNAc)(2), (GlcNAc)(3), and (GlcNAc)(4), and cleavage to (GlcNAc)(3) + (GlcNAc)(3) predominated over that to (GlcNAc)(2) + (GlcNAc)(4). This indicates that OEL hydrolyzes preferentially the third glycosidic linkage from the nonreducing end of (GlcNAc)(6) as in the case of GEL. The cleavage pattern seen for (GlcNAc)(5) was similar to that seen for (GlcNAc)(6). Theoretical analysis of the reaction time-course for OEL revealed that the binding free energy values for subsites B, E, and G were different between OEL and GEL, although these lysozymes were estimated to have the same type of subsite structure.     

Low-resolution structure,oligomerization and its role on the enzymatic activity of a sucrose-6-phosphate hydrolase from Bacillus licheniformis

Amino Acids - Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like β-fructofuranosidase. Gene sacA from...     

NMR elucidation of early folding hierarchy in HIV-1 protease

Folding studies on proteases by the conventional hydrogen exchange experiments are severely hampered because of interference from the autolytic reaction in the interpretation of the exchange data. We report here NMR identification of the hierarchy of early conformational transitions (folding propensities) in HIV-1 protease by systematic monitoring of the changes in the state of the protein as it is subjected to different degrees of denaturation by guanidine hydrochloride. Secondary chemical shifts, HN-Halpha coupling constants, 1H-15N nuclear Overhauser effects, and 15N transverse relaxation parameters have been used to report on the residual structural propensities, motional restrictions, conformational transitions, etc., and the data suggest that even under the strongest denaturing conditions (6 m guanidine) hydrophobic clusters as well as different native and non-native secondary structural elements are transiently formed. These constitute the folding nuclei, which include residues spanning the active site, the hinge region, and the dimerization domain. Interestingly, the proline residues influence the structural propensities, and the small amino acids, Gly and Ala, enhance the flexibility of the protein. On reducing the denaturing conditions, partially folded forms appear. The residues showing high folding propensities are contiguous along the sequence at many locations or are in close proximity on the native protein structure, suggesting a certain degree of local cooperativity in the conformational transitions. The dimerization domain, the flaps, and their hinges seem to exhibit the highest folding propensities. The data suggest that even the early folding events may involve many states near the surface of the folding funnel.     

Functional characterization of human 1-acylglycerol-3-phosphate acyltransferase isoform 8: cloning, tissue distribution, gene structure, and enzymatic activity

Glycerophospholipids and triglycerides are synthesized de novo by cells through an evolutionary conserved process involving serial acylations of phosphorylated glycerol. Various isoforms of the enzyme, 1-acylglycerol-3-phosphate acyltransferase (AGPAT), acylate lysophosphatidic acid at the sn-2 position to produce phosphatidic acid. We cloned a cDNA predicted to be AGPAT isoform and designated it AGPAT8. Human and mouse AGPAT8 proteins are 89% homologous, and their gene structure is also highly conserved. AGPAT8 is most closely related to AGPAT5, and its cDNA is expressed most in the heart, while AGPAT5 is expressed more in the prostate and testis. In cell lysates, AGPAT8 shows moderate acyltransferase activity with [(3)H]oleoyl-CoA but lacks acyl-CoA:lysocardiolipin acyltransferase activity. In whole cells upon incubation with [(14)C]linoleic acid, most of the radioactivity was recovered in phosphatidyl ethanolamine, phosphatidyl choline and phosphatidic acid fraction. Of the two well conserved acyltransferase motifs, NHX(4)D is present in AGPAT8, whereas arginine in the EGTR motif is substituted by aspartate. However, mutation of EGTD to EGTR did not increase enzymatic activity significantly. Based on the X-ray crystallographic structure of a related acyltransferase, squash gpat, a model is proposed in which a hydrophobic pocket in AGPAT8 accommodates fatty acyl chains of both substrates in an orientation where the NHX(4)D motif participates in catalysis.     

NMR and molecular modeling reveal key structural features of synthetic nodulation factors

Nod factors are lipochitoligosaccharides originally produced by the soil bacteria Rhizobia that are involved in the symbiotic process with leguminous plants. Some synthetic analogs of the Nod factors present a strong biological activity, and the conformational behavior of these molecules is of interest for structure/function studies. Nod factor analogs containing an insertion of a phenyl group in the acyl chain at the oligosaccharidic non-reducing end were previously synthesized (Grenouillat N, Vauzeilles B, Bono J-J, Samain E, Beau J-M. 2004. Simple synthesis of nodulation-factor analogues exhibiting high affinity towards a specific binding protein. Angew Chem Int Ed Engl. 43:4644). Conformational studies of natural compounds and synthetic analogs have been performed combining molecular dynamics simulations in explicit water and NMR. Data revealed that the glycosidic head group can adopt only restricted conformations, whereas chemical modifications of the lipid chains, highly flexible in a water environment, influence the global shape of the molecules. Collected structural data could be used in the future to rationalize and understand their biological activity and affinity toward a putative receptor.     

Synthesis of fluorescence-labeled sphingosine and sphingosine 1-phosphate; effective tools for sphingosine and sphingosine 1-phosphate behavior

A fluorescence-labeled sphingosine and sphingosine 1-phosphate have been successfully synthesized from the oxazolidinone methyl ester derived from glycidol via monoalkylation and the stereoselective reduction of the resulting ketone. The labeled sphingosine was converted into its phosphate by treatment with sphingosine kinase 1 (SPHK1) from mouse, and in platelets, and it was incorporated into the Chinese Hamster Ovarian (CHO) cells. In addition, MAPK was activated by NBD-Sph-1-P through Edg-1, Sph-1-P receptor.     

Mononuclear Au(III)-complexes with tryptophan-containing dipeptides: Synthesis, spectroscopic and structural elucidation

The coordination ability of dipeptides l-tryptophyl-l-phenylalanine (H-Trp-Phe-OH) and l-tyrosyl-l-tryptophan (H-Tyr-Trp-OH) with Au(III) have been elucidated both in solid state and in solution by means of series of methods as UV, ¹H and ¹³C NMR, conventional and linear-polarized IR-spectroscopic tool in solid-state, based on orientation technique as suspension in nematic liquid crystal, FAB-MS, TGV, DSC methods and elemental analysis. The structures of the Au(III)-complexes have been predicted theoretically by DFT calculations at B3LYP level of theory and Lanl2DZ (Au)/6-31+G(3df) (Cl, C, H) basis set. The last data are compared with IR-LD spectroscopic ones giving the experimental evidence of the structures of the complexes studied. The dipeptides interact as tridentate ligands in obtained mononuclear complexes via their -NH₂, deprotonated N⁻-amide and -groups at molar ratio metal to ligand 1:1. One Cl⁻ ion is joined to the Au(III) as terminal ligand, forming [Au^III(LH₋₁)Cl] species. A near to square-planar flat geometry of the chromophores AuN₂OCl is yielded with maximal deviation of total planarity less than 0.9°.     

Antenna domain mobility and enzymatic reaction of L-rhamnulose-1-phosphate aldolase

The enzyme l-rhamnulose-1-phosphate aldolase from Escherichia coli participates in the degradation pathway of l-rhamnose, a ubiquitous deoxy-hexose. It is a homotetramer of the rare C4-symmetric type with N-terminal domains protruding like antennas from the main body. A mobility analysis of the enzyme gave rise to the hypothesis that an anisotropic thermal antenna motion may support the catalysis (Kroemer et al., Biochemistry 42, 10560, 2003). We checked this hypothesis by generating four single mutants and one disulfide bridge that were designed to reduce the mobility of the antenna domain without disturbing the chain-fold or the active center. The catalytic rates of the mutants revealed activity reductions that correlated well with the expected antenna fixation. Among these mutants, K15W was crystallized, structurally elucidated, and used as a guide for modeling the others. The structure confirmed the design because the mutation introduced a tight nonpolar contact to a neighboring subunit that fixed the antenna but did not affect the main chain. The fixation was confirmed by a comparison of the anisotropic B-factors describing the mobility of the domains. It turned out that the distinctly anisotropic mobility of the wild-type antenna domain has become isotropic in K15W, in agreement with the design. We suggest that, like K15W, the other mutations also followed the design, validating the correlation between antenna mobility and activity. This correlation suggests that the domain mobility facilitates the reaction.