Synthesis of the Spirosuccinimide Moiety of Asperparaline A

2,6,6-Trimethyl-2-azaspiro[4.4]nonane-1,3-dione (9), a spirosuccinimide moiety of asperparaline A (1), was synthesized by starting from 2,2-dimethylcyclopentanone (4) via trinitrile 6 in five steps in a moderate yield. This conversion establishes a model study for synthesis of the spirosuccinimide moiety of asperparaline A (1).     

A fungal metabolite asperparaline a strongly and selectively blocks insect nicotinic acetylcholine receptors: the first report on the mode of action

Asperparalines produced by Aspergillus japonicus JV-23 induce paralysis in silkworm (Bombyx mori) larvae, but the target underlying insect toxicity remains unknown. In the present study, we have investigated the actions of asperparaline A on ligand-gated ion channels expressed in cultured larval brain neurons of the silkworm using patch-clamp electrophysiology. Bath-application of asperparaline A (10 μM) had no effect on the membrane current, but when delivered for 1 min prior to co-application with 10 μM acetylcholine (ACh), it blocked completely the ACh-induced current that was sensitive to mecamylamine, a nicotinic acetylcholine receptor (nAChR)-selective antaogonist. In contrast, 10 μM asperparaline A was ineffective on the γ-aminobutyric acid- and L-glutamate-induced responses of the Bombyx larval neurons. The fungal alkaloid showed no-use dependency in blocking the ACh-induced response with distinct affinity for the peak and slowly-desensitizing current amplitudes of the response to 10 μM ACh in terms of IC(50) values of 20.2 and 39.6 nM, respectively. Asperparaline A (100 nM) reduced the maximum neuron response to ACh with a minimal shift in EC(50), suggesting that the alkaloid is non-competitive with ACh. In contrast to showing marked blocking action on the insect nAChRs, it exhibited only a weak blocking action on chicken α3β4, α4β2 and α7 nAChRs expressed in Xenopus laevis oocytes, suggesting a high selectivity for insect over certain vertebrate nAChRs.     

Selective cytotoxicity of recombinant STXA1-GM-CSF protein in hematopoetic cancer cells

Chimeric proteins are composed of a cell-targeting moiety and a cell-killing moiety. In this study, a chimeric protein, STXA1-GM-CSF, composed of catalytic domain of Shiga toxin (A1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed and expressed in E. coli. Cytotoxicity, receptor blocking, and neutralization experiments revealed that the chimeric protein induced cytotoxic effect on different cell lines. This effect was found to be specific, due to the presence of the killing moiety (A1), which exerts its effect through a specific GM-CSF-targeting domain, by binding to its receptor present on those cell lines. These initial investigations indicate that the chimeric protein was functional; further analyses are required for its application.     

Synthesis and in vitro binding studies of substituted piperidine naphthamides. Part I: Influence of the substitution on the basic nitrogen and the position of the amide on the affinity for D2L, D4.2, and 5-HT2A receptors

A series of 1- and 2-naphthamides has been prepared and tested for in vitro binding to D(2L), D(4.2), and 5-HT(2A) receptors. Different compounds display selectivity for D(4.2) and 5-HT(2A) receptors versus D(2L) receptors. N-(1-Arylalkyl-piperidin-4-yl) carboxamides have higher affinities than the corresponding N-(4-arylalkylamino-piperidin-1-yl) carboxamide analogues. A benzyl moiety in position 1 of the piperidine in the 2-naphthamide series (2) appears to be the best choice for a favorable interaction with D(4.2) and 5-HT(2A) receptors. Increasing the linker length between the phenyl ring and the basic nitrogen led to a decreased affinity for these receptors. In the 1-naphthamide series, the most potent D(4.2) ligand (7) possesses a phenylpropyl moiety while its affinity for 5-HT(2A) receptors is strongly reduced. All compounds with significant affinity for D(4.2) and 5-HT(2A) receptors were antagonists.     

Purification and characterization of a novel enoyl coenzyme A reductase from Streptomyces collinus.

A novel NADPH-dependent enoyl reductase, catalyzing the conversion of 1-cyclohexenylcarbonyl coenzyme A (1-cyclohexenylcarbonyl-CoA) to cyclohexylcarbonyl-CoA, was purified to homogeneity from Streptomyces collinus. This enzyme, a dimer with subunits of identical M(r) (36,000), exhibits a Km of 1.5 +/- 0.3 microM for NADPH and 25 +/- 3 microM for 1-cyclohexenylcarbonyl-CoA. It has a pH optimum of 7.5, is most active at 30 degrees C, and is inhibited by both divalent cations and thiol reagents. Two internal peptide sequences were obtained. Ansatrienin A (an antibiotic produced by S. collinus) contains a cyclohexanecarboxylic acid moiety, and it is suggested that the 1-cyclohexenylcarbonyl-CoA reductase described herein catalyzes the final reductive step in the conversion of shikimic acid into this moiety.     

Crystal structure of 3-(adenin-9-yl)propionhistamide hydrochloride monohydrate and the role of protonation in protein-nucleic acid interactions

The three-dimensional structure of 3-(adenin-9-yl)propionhistamide hydrochloride, as a model of interactions between the protonated imidazolyl group and adenine moiety, has been determined by X-ray crystallography. The crystal data are a = 10.314 (1), b = 7.854 (1), c = 20.780 (1) A, beta = 92.765 (4), Z = 4, Dm = 1.32 g X cm-3, space group P2(1)/n. The imidazolium group interacts with adenine moiety by stacking. A comparison with the related neutral derivatives leads to a hypothesis that adenine stacks with the protonated, but not with the neutral, imidazolyl group. Such a characteristic behaviour of the imidazolyl group depending on protonation was shown by ultraviolet and NMR spectroscopy, and by polarographic experiments in solution. We note that a role of the essential histidine in RNAase reaction is a switch between capture and ejection of the substrate, which depends upon proton uptake and its release, respectively.     

Synthesis and structure-activity relationships of a new set of 1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives as adenosine receptor antagonists

In a previous paper (Colotta V. et al., J. Med. Chem. 2000, 43, 1158), we reported the synthesis and the binding activity of some 4-oxo (A) and 4-amino (B) substituted 1,2,4-triazolo[4,3-a]quinoxalin-1-ones, bearing different substituents on the appended 2-phenyl ring (region 1), some of which were potent and selective A(1) or A(3) antagonists. To further investigate the SAR in this class of antagonists, in the present paper some 2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives of both series A and B, bearing simple substituents on the benzofused moiety (region 2), are reported. The binding data at bovine A(1) (bA(1)) and A(2A)(bA(2A)) and at human A(3) (hA(3)) adenosine receptors (ARs) show that in series A (compounds 1, 4-11) the presence of substituents on the benzofused moiety is, in general, not advantageous for anchoring at all three AR subtypes, while within series B (compounds 12-21) it exerts a beneficial effect for both bA(1) and hA(3) AR affinities which span the low nanomolar range. In particular, among the 4-amino derivatives 12-21, the 8-chloro-6-nitro (compound 17) and the 6-nitro (compound 18) substitutions afford, respectively, the highest bA(1) and hA(3) AR affinity. Moreover, compound 18, additionally investigated in binding assays at human A(1) (hA(1)) receptors, shows a 183-fold selectivity for hA(3) versus hA(1) receptors. Finally, the SAR studies provide some new insights about the steric and lipophilic requirements of the hA(3) receptor binding pocket which accommodates the benzofused moiety of our 4-amino-triazoloquinoxalin-1-one derivatives.     

Synthesis of a new N-9 ribityl analogue of cyclic inosine diphosphate ribose (cIDPR) as a mimic of cyclic ADP ribose (cADPR)

A new analogue of cyclic inosine diphosphate ribose (cIDPR), in which the N-1 and N-9 ribosyl moieties were substituted by a carbocyclic moiety and a hydroxyl-alkyl chain, has been synthesized and characterized.     

Synthesis and biological evaluation of rebeccamycin analogues modified at the imide moiety

Glycosylated indolocarbazoles related to the antibiotic rebeccamycin represent an important class of antitumour drugs. In the course of our structure-activity relationship studies, new rebeccamycin analogues modified at the imide moiety were synthesised. The antiproliferative activity of the compounds was evaluated on three human cancer cell lines, A2780 (ovarian cancer), H460 (lung cancer), and GLC4 (small-cell lung cancer). The in vitro cytotoxicity of compounds 2 and 4, characterised respectively by a 1,3-dioxolan and (1,3-dioxolan-4-yl)methylene groups linked to the imide moiety, was higher than the reference compound, edotecarin. The effect of compound 2 in inducing tumour regression in the A2780 xenograft model was also investigated.     

The influence of the long chain fatty acid on the antagonistic activities of Rhizobium sin-1 lipid A

The lipid A from nitrogen-fixing bacterial species Rhizobium sin-1 is structurally unusual due to lack of phosphates and the presence of a 2-aminogluconolactone and a very long chain fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0), moiety. This structurally unusual lipid A can antagonize TNF-alpha production by human monocytes induced by Escherichia coli LPS. To establish the relevance of the unusual long chain 27-hydroxyoctacosanoic acid for antagonistic properties, a highly convergent strategy for the synthesis of several derivatives of the lipid A of R. sin-1 has been developed. Compound 1 is a natural R. sin-1 lipid A having a 27-hydroxyoctacosanoic acid at C-2', compound 2 contains an octacosanoic acid moiety at this position, and compound 3 is modified by a short chain tetradecanoic acid. Cellular activation studies with a human monocytic cell line have shown that the octacosanoic acid is important for optimal antagonistic properties. The hydroxyl of the natural 27-hydroxyoctacosanoic moiety does, however, not account for inhibitory activity. The resulting structure-activity relationships are important for the design of compounds for the treatment of septic shock.     

Metabolism of Lignin Model Compounds of the Arylglycerol-beta-Aryl Ether Type by Pseudomonas acidovorans D(3)

A natural bacterial isolate that we have classified as Pseudomonas acidovorans grows on the lignin model compounds 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (compound 1) and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (compound 1'), as well as on the corresponding 1-oxo compounds (2 and 2') as sole sources of carbon and energy. Metabolic intermediates present in cultures growing on compound 1 included compound 2, 2-methoxyphenol (guaiacol [compound 3]), beta-hydroxypro-pioveratrone (compound 4), acetoveratrone (compound 5), and veratric acid (compound 6). Also identified were compounds 1', 2', beta-hydroxypropiovanillone (compound 4'), and acetovanillone (compound 5'), indicating that 4-O demethylation also occurs. The phenolic intermediates were the same as those found in cultures growing on compound 1'. Compounds 2 and 2' were in part also reduced to compounds 1 and 1', respectively. Compound 3 was shown to be derived from the 2-methoxyphenoxy moiety. A suggested degradation scheme is as follows: compound 1-->2-->(3 + 4)-->5-->6 (and similarly for 1'). In this scheme, the key reaction is cleavage of the ether linkage between C-2 (C(beta)) of the phenylpropane moiety and the 2-methoxyphenoxy moiety in compounds 2 and 2' (i.e., beta-aryl ether cleavage). On the basis of compounds identified, viz., 3 and 4 (4'), cleavage appears formally to be reductive. Because this is unlikely, the initial cleavage products probably were not detected. The implications of these results for the enzyme(s) responsible are discussed.     

Asymmetrical ligand binding by abscisic acid 8'-hydroxylase

Abscisic acid (ABA), a plant stress hormone, has a chiral center (C1') in its molecule, yielding the enantiomers (1'S)-(+)-ABA and (1'R)-(-)-ABA during chemical synthesis. ABA 8'-hydroxylase (CYP707A), which is the major and key P450 enzyme in ABA catabolism in plants, catalyzes naturally occurring (1'S)-(+)-enantiomer, whereas it does not recognize naturally not occurring (1'R)-(-)-enantiomer as either a substrate or an inhibitor. Here we report a structural ABA analogue (AHI1), whose both enantiomers bind to recombinant Arabidopsis CYP707A3, in spite of stereo-structural similarity to ABA. The difference of AHI1 from ABA is the absence of the side-chain methyl group (C6) and lack of the alpha,beta-unsaturated carbonyl (C2'C3'-C4'O) in the six-membered ring. To explore which moiety is responsible for asymmetrical binding by CYP707A3, we synthesized and tested ABA analogues that lacked each moiety. Competitive inhibition was observed for the (1'R) enantiomers of these analogues in the potency order of (1'R,2'R)-(-)-2',3'-dihydro-4'-deoxo-ABA (K(I)=0.45 microM)>(1'R)-(-)-4'-oxo-ABA (K(I)=27 microM)>(1'R)-(-)-6-nor-ABA and (1'R,2'R)-(-)-2',3'-dihydro-ABA (no inhibition). In contrast to the (1'R)-enantiomers, the inhibition potency of the (1'S)-analogues declined with the saturation of the C2',C3'-double bond or with the elimination of the C4'-oxo moiety. These findings suggest that the C4'-oxo moiety coupled with the C2',C3'-double bond is the significant key functional group by which ABA 8'-hydroxylase distinguishes (1'S)-(+)-ABA from (1'R)-(-)-ABA.     

Structural snapshots of beta-1,4-galactosyltransferase-I along the kinetic pathway

During the catalytic cycle of beta1,4-galactosyltransferase-1 (Gal-T1), upon the binding of Mn(2+) followed by UDP-Gal, two flexible loops, a long and a short loop, change their conformation from open to closed. We have determined the crystal structures of a human M340H-Gal-T1 mutant in the open conformation (apo-enzyme), its Mn(2+) and Mn(2+)-UDP-Gal-bound complexes, and of a pentenary complex of bovine Gal-T1-Mn(2+)-UDP-GalNAc-Glc-alpha-lactalbumin. These studies show that during the conformational changes in Gal-T1, the coordination of Mn(2+) undergoes significant changes. It loses a coordination bond with a water molecule bound in the open conformation of Gal-T1 while forming a new coordination bond with another water molecule in the closed conformation, creating an active ground-state structure that facilitates enzyme catalysis. In the crystal structure of the pentenary complex, the N-acetylglucosamine (GlcNAc) moiety is found cleaved from UDP-GalNAc and is placed 2.7A away from the O4 oxygen atom of the acceptor Glc molecule, yet to form the product. The anomeric C1 atom of the cleaved GalNAc moiety has only two covalent bonds with its non-hydrogen atoms (O5 and C2 atoms), similar to either an oxocarbenium ion or N-acetylgalactal form, which are crystallographically indistinguishable at the present resolution. The structure also shows that the newly formed, metal-coordinating water molecule forms a hydrogen bond with the beta-phosphate group of the cleaved UDP moiety. This hydrogen bond formation results in the rotation of the beta-phosphate group of UDP away from the cleaved GalNAc moiety, thereby preventing the re-formation of the UDP-sugar during catalysis. Therefore, this water molecule plays an important role during catalysis in ensuring that the catalytic reaction proceeds in a forward direction.     

Novel 4-azasteroidal N-glycoside analogues bearing sugar-like D ring: synthesis and anticancer activities

A series of novel N-glycoside analogues with 4-azasteroid moiety bearing sugar-like D ring were conveniently synthesized by constructing the core dihydropyran ring embedded in 4-azasteroidal skeleton which was prepared from 4-aza-5α-androst-3,17-dione 1 in four steps. The structure of 6b were unambiguously proved by the appropriate X-ray structural analysis. Anticancer activity was found for all of the analogues with purinyl moiety against breast cancer (MCF-7), human neuroblastoma (SK-N-SH), cervical cancer cell (HeLa) and prostatic cancer (PC-3), while the analogue 7 containing 1,2,4-triazole heterocycle as the nucleobase was inactive against all of the tested cancer cell lines. The biology results showed the purinyl moiety attached to the pyran ring of 6a-d, substituent at 6'-position of purine base and introduction of a halogen atom at 2'-position of 6'-chloropurine had obviously effect on the evaluated anticancer activity.     

A survey of cyclic replacements for the central diamide moiety of inhibitors of inosine monophosphate dehydrogenase

A series of heterocyclic replacements for the central diamide moiety of 1, a potent small molecule inhibitor of inosine monophosphate dehydrogenase (IMPDH) were explored The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for these new series of inhibitors is given.     

Packing and electrostatic behavior of sn-2-docosahexaenoyl and -arachidonoyl phosphoglycerides

Mammalian synaptic membranes appear to contain high proportions of specific, sn-1-stearoyl-2-docosahexaenoyl- and sn-1-stearoyl-2-arachidonoyl phosphoglycerides, but the structural significance of this is unclear. Here we used a standardized approach to compare the properties of homogeneous monolayers of the corresponding phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and phosphatidic acids with those of control monolayers of sn-1-stearoyl-2-oleoyl- and sn-1-palmitoyl-2-oleoyl phosphoglycerides. Major findings were: 1), that the presence of an sn-2-docosahexaenoyl group or an sn-2-arachidonoyl group increases the molecular areas of phosphoglycerides by 3.8 A(2) (7%) relative to the presence of an sn-2-oleoyl group; 2), that the phosphorylcholine headgroup independently increases molecular areas by a larger amount, 7.1 A(2) (13%); and 3), that the dipole moments of species having an arachidonoyl moiety or an oleoyl moiety are 83 mD (19%) higher than those of comparable docosahexaenoic acid-containing phosphoglycerides. These and other results provide new information about the molecular packing properties of polyenoic phosphoglycerides and raise important questions about the role of these phosphoglycerides in synapses.     

The design,synthesis, and evaluation of organic dithienopyrrole-based D-π-A dyes for use as sensitizers in photodynamic therapy

Dithienopyrrole-based organic dyes that combine an electron-donating moiety (D), a π-conjugated bridge moiety (π), and an electron-accepting moiety (A) were designed and synthesized in short steps by previously developed one-pot Suzuki-Miyaura coupling approach. Absorption wavelengths of the dyes were readily tuned by altering the D and A moieties. The use of a strongly electron-withdrawing cyanopyridone acceptor enabled NIR absorption. A synthesized sensitizer, 2j, exerted potent phototoxicity mainly via a Type I mechanism in cells. A nitrogen atom in the dithienopyrrole ring serves as a connecting point for the introduction of functional building blocks that can improve the properties of sensitizers, which makes this D-π-A sensitizer a valuable template for the further development of sensitizers.     

SYNTHESIS OF A NEW N-9 RIBITYL ANALOGUE OF CYCLIC INOSINE DIPHOSPHATE RIBOSE (cIDPR) AS A MIMIC OF CYCLIC ADP RIBOSE (cADPR)

A new analogue of cyclic inosine diphosphate ribose (cIDPR), in which the N-1 and N-9 ribosyl moieties were substituted by a carbocyclic moiety and a hydroxyl-alkyl chain, has been synthesized and characterized.     

Synthesis of a new ribose modified analogue of cyclic inosine diphosphate ribose

A new analogue of cyclic inosine diphosphate ribose (cIDPR), in which the N-1 and N-9 ribosyl moieties were substituted by an alkyl moiety and an hydroxy-alkyl chain, has been synthesized and characterized.