NMR SOLUTION STRUCTURE AND CONFORMATIONAL ANALYSIS OF THE CALCIUM RELEASE AGENT CYCLIC ADENOSINE 5′-DIPHOSPHATE RIBOSE (cADPR)

A high-resolution NMR study of the solution structure of the calcium release agent cADPR has been performed. Pseudorotationals analysis reveals that in solution both sugar rings in cADPR adopt predominantly (～75%) South conformations, with the A and N rings adopting approximately ²T₃ (C2′-endo(major)-C3′-exo(minor) and ⁴ ₃T (C3′-exo-C4′-endo) conformations, respectively. The backbone torsion angles β and γ have also been determined. While the minor North conformers were not observed in the crystal structure of cADPR, the solution values of the major South conformers compare well to those found in crystal structure.     

SYNTHESIS AND BIOLOGICAL ACTIVITY OF CYCLIC ADP-CARBOCYCLIC-RIBOSE ANALOGS: STRUCTURE-ACTIVITY RELATIONSHIP AND CONFORMATIONAL ANALYSIS OF N-1-CARBOCYCLIC-RIBOSE MOIETY

Several cyclic ADP-carbocyclic-ribose analogs 3–10 modified in the N-1-carbocyclic-ribose moiety were synthesized. Their Ca²⁺-releasing activity was estimated in sea urchin eggs to show that the 3″-deoxy analog 6 shows 5 times more potent activity than cADPcR, but the 2″,3″-didieoxy-2″,3″-unsunsaturated analog 3 has very weak activity. We also calculated their stable conformation and found that 3 and 6 were significantly different in their stable conformation.     

PURIFICATION AND CHARACTERIZATION OF A SULFOTRANSFERASE SPECIFIC TO N-21 OF SAXITOXIN AND GONYAUTOXIN 2+3 FROM THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM (DINOPHYCEAE)

A sulfotransferase (ST) specific to N-21 of saxitoxin (STX) and gonyautoxin 2+3 (GTX2+3) designated as N-ST was purified to homogeneity from the cytosolic fraction of clonal-axenic vegetative cells of the toxic dinoflagellate Gymnodinium catenatum Graham GC21V, which causes paralytic shellfish poisoning. The enzyme transferred a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to N-21 in the carbamoyl group of STX and GTX2+3 to produce GTX5 and C1+2, respectively. The molecular mass of the purified enzyme was determined by SDS-PAGE to be 59 kDa. Gel filtration chromatography showed a native molecular mass of 65 kDa, indicating that the N-ST is a monomeric enzyme. The N-ST was specific to only N-21 of STX and GTX2+3, and O-22 sulfation was not observed. Moreover, the N-ST was not active toward neo STX and GTX1+4, which differed from STX and GTX2+3, respectively, in only N-1 hydroxylation. When various compounds previously reported to be substrates for STs in other organisms and paralytic shellfish poisoning toxins other than STX and GTX2+3 were added to the reaction mixture, N-ST activity was not decreased. The enzyme required PAPS as the sole source of sulfate. The enzyme was optimally active at pH 6.0 and 25° C, and its activity was enhanced by Mg^{2 +} and Co^{2 +} . The K_m values of the N-ST for STX and GTX2+3 were 16.1 μM and 29.8 μM, respectively.     

CYCLIC HETEROPHYLLY IN SYNGONIUM (ARACEAE)

Species in the genus Syngonium germinate on the ground and mature on the trunks of the trees. These vines consist of relatively unbranched shoots which grow through the forest for considerable distances both horizontally and vertically. In the three species of this study, the shape of the segment, measured as internode diam/length, falls into two distinct classes, leafy and elongate, and varies cyclicly along single shoots. Two cycles are recognized. One cycle occurs in small diam terrestrial shoots in which cycling seems to be controlled by endogenous factors, and occurs with a period of a few tens of segments. The other cycle occurs in shoots of larger diam which climb and descend from trees. In this cycle, alternation between the two forms is controlled by gain or loss of contact with trees, and shoots may remain within a phase of the cycle for hundreds of segments, as long as the substrate does not change.     

N-(4-hydroxyphenyl)retinamide (Fenretinide) and nephrectomy alter normal plasma retinol-binding protein metabolism

We have reported previously that injecting vitamin A-deficient rats with N-(4-hydroxyphenyl)retinamide causes a significant reduction in the liver retinol-binding protein concentration and a 2 fold rise in the kidney retinol-binding protein concentration. This presumably reflects a rapid translocation of retinol-binding protein from the liver to the kidney through the plasma, although no rise in plasma retinol-binding protein is detected. In the present studies, nephrectomized rats were used to determine if retinol-binding protein accumulating in kidneys passes through the plasma. Bilateral nephrectomy in control rats caused the plasma retinol-binding protein concentration to approximately double by 5 hr postsurgery. However, nephrectomy plus N-(4-hydroxyphenyl)retinamide treatment did not result in an increase in the plasma retinol-binding protein concentration. Therefore, the lowering of liver retinol-binding protein concentration in response to N-(4-hydroxyphenyl)retinamide treatment was not accounted for by an accumulation of retinol-binding protein in the plasma compartment. Interestingly, the muscle retinol-binding protein concentration increased with nephrectomy plus N-(4-hydroxyphenyl)retinamide treatment. The ratio of muscle retinol-binding protein:plasma retinol-binding protein in vitamin A-deficient nephrectomized rats treated with N-(4-hydroxyphenyl)retinamide was significantly higher than in comparable rats treated with either carrier or retinol. We conclude that in vivo N-(4-hydroxyphenyl)retinamide induces the secretion of retinol-binding protein from the liver. Since the N-(4-hydroxyphenyl)retinamide-retinol-binding protein complex does not bind with transthyretin it rapidly leaves the plasma. In non-nephrectomized rats this complex is rapidly filtered by the kidney. Nephrectomizing rats causes the retinol-binding protein secreted in response to N-(4-hydroxyphenyl)retinamide to diffuse into interstitial fluid.     

Determination of the enantiomeric purity of (−−) 2-(N-propyl-n-2-thienylethylamino)-5-hydroxytetralin (n-0923) by chiral stationary phase HPLC

The determination of the enantiomeric impurity, i.e., the percentage of (+) N?0437 (= N?0924) in several batches of (??) N-0437 (= N-0923) by chiral HPLC is described. Enantiomeric impurities were calculated based on the peak areas of the two baseline separated enantiomers in the chromatogram. The enantiomeric impurities found in different batches ranged from 0.02% to 0.11%. Calibration curves of the two isomers of N-0437 (Fig. 1,) were made twice to study the reproducibility and linearity of the method. The absorbance ratio, N-0923/N-0924, was found to be 1.02 with a relative standard deviation (RSD) of 9% over the whole concentration range used for the calibration curves.     

A New Simple Synthesis of N-2-Methylguanosine and Its Analogues Via Derivatives of 4-Desmethylwyosine (Nucleosides and Nucleotides. Part 631)

Abstract

Guanosine and 2′-deoxyguanosine have been converted into the corresponding N-2-methyl and N-2-ethyl derivatives in a simple, three-step procedure by N-5-alkylation of N-4-desmethylwyosines (4,5) and subsequent deprotection with N-bromosuccinimide.     

Synthesis of cationic cyclo-oligo(ethyleneadenine)

A novel cationic and cyclic oligo (ethyleneadenine) whose main chain has adenine ring was synthesized by the thermal polymerization of 9-(2-bromoethyl) adenine, in the solid phase. The chain extends through N-9 - N-1 and N-9 - N-7 of the adenine rings. Its molecular weight was determined to be 3960 by the sedimentation method. The micro-structure was confirmed mainly with NMR measurements. The cationic and cyclic oligo(ethyleneadenine) interacts with nucleotides and polynucleotides in neutral aqueous solution.     

N-(3-(4-Hydroxyphenyl)-propenoyl)-amino acid tryptamides as SIRT2 inhibitors

A series of N-(3-(4-hydroxyphenyl)-propenoyl)-amino acid tryptamides was based on a previously reported new SIRT2 inhibitor from our group, and it was designed to study if the molecular size of the compound could be reduced. The most potent compounds, N-(3-(4-hydroxyphenyl)-propenoyl)-2-aminoisobutyric acid tryptamide and N-(3-(4-hydroxyphenyl)-propenoyl)-L-alanine tryptamide, were equipotent, 30% smaller in molecular weight, and slightly more selective (SIRT2/SIRT1) than the parent compound.     

The synthesis and some biological properties of N-(6-purinyl)peptides

The chemical synthesis and biological properties of N-(6-purinyl)peptides are described. N-(6-Purinyl)amino-acid derivatives were synthesized and condensed with amino acid esters and peptide esters using the dicyclohexylcarbodiimide/N-hydroxysuccinimide method. The products were isolated via gel filtration on Sephadex G-10 in 0.05M NH4HCO3 followed by either ion exchange chromatography on SP-Sephadex or by preparative HPLC. The methyl esters were saponified and the tert-butyl ester group was removed by treatment with trifluoroacetic acid without damaging the purinyl residue. N-(6-Purinyl)peptides were characterised by chromatographic and spectroscopic methods. Acid hydrolysis of N-(6-purinyl)-L-amino acids caused the racemization of the neighbouring L-amino acid. Model studies were performed with N-(6-purinyl)-L-alanine, N-(6-purinyl)-D-alanine, N-(6-purinyl)-L-alanyl-L-leucine and N-(6-purinyl)-D-alanyl-L-leucine. After acid hydrolysis the N-(6-purinyl)amino acids were totally racemized and the N-(6-purinyl)dipeptides formed 14% of the enantiomer of alanine. The N-(6-purinyl)-omega-amino acids and the N-(6-purinyl)peptides were screened in a limited number of tests as immunomodulators (antibody-secretion, phagocytosis, cytostatic activity of macrophages) and as cytotoxic agents.     

N-(1-Methyl)cyclopropylbenzylamine: A novel inactivator of mitochondrial monoamine oxidase

N-(1-Methyl)cyclopropylbenzylamine was synthesized and shown to inactivate pig liver mitochondrial monoamine oxidase. Inactivation is time-dependent, concentration dependent, protected by the substrate and product of the enzyme, and is not reversed by exhaustive dialysis. Unlike N-cyclopropylbenzylamine, the adduct formed between N-(1-methyl)cyclopropylbenzylamine and monoamine oxidase is stable to treatment with benzylamine. A mechanism for the inactivation is proposed.     

Spiramine N-6,一种新的抗血小板和抗血小板-中性粒细胞相互作用的物质

Spiramine N-6属粉花秀线菊植物中提取分离的二十碳二萜生物碱。本实验采用Born,Shen和Hamburger等方法分别观察了spiramine N-6在体外和体内对兔血小板聚集功能的影响。应用荧光分光光度法测定其对血小板5-羟色胺释放反应的作用,同时评价spiramine N-6对激活的血小板与中性粒细胞之间粘附反应的影响。结果表明：spiramine N-6在体外选择性抑制血小板活化因子(PAF)诱导的血小板聚集,并呈量效关系,其IC50=26μmol／L,对花生四烯酸(AA)或腺苷二磷酸(ADP)引起的血小板聚集无明显作用;spiramine N-6静注后明显抑制PAF、AA和ADP诱导的血小板聚集。Spiramine N-6呈浓度依赖性减少AA和PAF引起血小板5-羟色胺的释放,其IC50分别为64．7和33．5μmol／L。Spiramine N-6明显阻抑激活的血小板与中性粒细胞间的粘附率,其IC50为78．6μmol／L。结果提示spiramine N-6作为二十碳二萜生物碱具有较强的抗血小板和阻抑血小板一中性粒细胞相互作用的生物活性。     

Synthesis of N-(beta-D-glucopyranosyl)- and N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl) amides as inhibitors of glycogen phosphorylase

2,3,4,6-Tetra-O-acetyl-beta-D-glucopyranosyl- and 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl azides were transformed into the corresponding per-O-acetylated N-(beta-D-glycopyranosyl) amides via a PMe(3) mediated Staudinger protocol (generation of N-(beta-D-glycopyranosyl)imino-trimethylphosphoranes followed by acylation with carboxylic acids, acid chlorides or anhydrides). The deprotected compounds obtained by Zemplén deacetylation were evaluated as inhibitors of rabbit muscle glycogen phosphorylase b. The best inhibitor of this series has been N-(beta-D-glucopyranosyl) 3-(2-naphthyl)-propenoic amide (K(i)=3.5microM).     

FATE OF THE TOXIC CYCLIC HEPTAPEPTIDES,THE MICROCYSTINS,FROM BLOOMS OF MICROCYSTIS (CYANOBACTERIA) IN A HYPERTROPHIC LAKE1

The in situ fate of the toxic cyclic heptapeptides, the microcystins, produced by blooms of Microcystis was examined at two stations in a hypertrophic Japanese lake. Microcystins were detected in all samples of Microcystis with quantities varying seasonally and spatially (230–950 μg · g dry wt^?1 at St. 1 and 160–746 μg · g dry wt^?1 at St. 2) and composed of microcystin-LR, -RR, and-YR. Microcystin-RR was the dominant toxin in most samples. A large amount of microcystin (1.1 μg · L^?1) was detected in only one sample of filtered lake water. Accumulation of microcystin in zooplankton was indirectly estimated from a newly developed equation model. Large amounts of microcystin (75–1387 μg · g dry wt^?1) were accumulated in the zooplankton community, which consisted of two cladocerans, Bosmina fatalis Burckhardt and Diaphanosoma brachyurum Lieve, and a copepod, Cyclops vicinus Uljanin, that co-occurred with the toxic Microcystis blooms. The maximum percent of microcystin content in zooplankton to that in Microcystis was 202%. Among the three species of zooplankton, only B. fatalis seemed to be responsible for accumulation of the microcystins because C. vicinus appeared to avoid contact with Microcystis cells and D. brachyurum did not consume colonies of Microcystis. Microcystins may be transferred to higher trophic levels through B. fatalis.     

Reaction of (bromoacetamido)nucleoside affinity labels with ribonuclease A: evidence for steric control of reaction specificity and alkylation rate

Four new bromoacetamido pyrimidine nucleosides have been synthesized and are affinity labels for the active site of bovine pancreatic ribonuclease A (RNase A). All bind reversibly to the enzyme and react covalently with it, resulting in inactivation. The binding constants Kb and the first-order decomposition rate constants k3 have been determined for each derivative. They are the following: 3'-(bromoacetamido)-3'-deoxyuridine, Kb = 0.062 M, k3 = 3.3 X 10(-4) s-1; 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil, Kb = 0.18 M, k3 = 1700 X 10(-4) s-1; 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil, Kb = 0.038 M, k3 = 6.6 X 10(-4) s-1; and 3'-(bromoacetamido)-3'-deoxythymidine, Kb = 0.094 M, k3 = 2.7 X 10(-4) s-1. 3'-(Bromoacetamido)-3'-deoxyuridine reacts exclusively with the histidine-119 residue, giving 70% of a monoalkylated product substituted at N-1, 14% of a monoalkylated derivative substituted at N-3, and 16% of a dialkylated species substituted at both N-1 and N-3. Both 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil and 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil react with absolute specificity at N-3 of the histidine-12 residue. 3'-(Bromoacetamido)-3'-deoxythymidine alkylates histidines-12 and -119. The major product formed in 57% yield is substituted at N-3 of histidine-12. A monoalkylated derivative, 8% yield, is substituted at N-1 of histidine-119. A disubstituted species is formed in 14% yield and is alkylated at both N-3 of histidine-12 and N-1 of histidine-119. A specific interaction of the "down" 2'-OH group, unique to 3'-(bromoacetamido)-3'-deoxyuridine, serves to orient the 3'-bromoacetamido residue close to the imidazole ring of histidine-119. The 2'-OH group of 3',5'-dinucleoside phosphate substrates may serve a similar role in the catalytic mechanism, allowing histidine-119 to protonate the leaving group in the transphosphorylation step. (Bromoacetamido)nucleosides are bound in the active site of RNase A in a variety of distinct conformations which are responsible for the different specificities and alkylation rates.     

Synthesis and characterization of nucleoside derivatives,n-(benzoyl)-n-(deoxyguanosin-8-yl)4-aminobiphenyl and n-(2'-deoxyguanosin-8-yl)4-aminobiphenyl via alpha-phenyl-n-(4-biphenyl)nitrone

Lead tetraacetate (LTA) oxidation of alpha-Phenyl-N-(4-bipheny])nitrone (8) to give a new ultimate carcinogen, N-acetoxy-N-benzoyl-4-aminobiphenyl (9) which was reacted with deoxyguanosine (dG) at pH 6.9 to give nucleoside derivative, N-(benzoyl)-N-(deoxyguanosin-8-yl)-4-aminobiphenyl (10). Following debenzoylation with sodium carbonate-methanol leads to N-(2'-deoxyguanosin-8-yl)-4-aminobiphenyl (11).     

N-(phosphonomethyl)glycine (glyphosate) tolerance in Euglena gracilis acquired by either overproduced or resistant 5-enolpyruvylshikimate-3-phosphate synthase.

Photoautotrophic cells of Euglena gracilis can be adapted to N-(phosphonomethyl)glycine (glyphosate) by cultivation in media with progressively higher concentrations of the herbicide. Two different mechanisms of tolerance to the herbicide were observed. One is characterized by the overproduction and 40-fold accumulation of the target enzyme. 5-enolpyruvylshikimate-3-phosphate synthase, in cells adapted to 6 mM N-(phosphonomethyl)glycine. The other is connected with a herbicide-insensitive enzyme. No evidence was obtained for the involvement of the putative multifunctional arom protein previously reported to be involved in the biosynthesis of aromatic amino acids in Euglena. Cells adapted to N-(phosphonomethyl)glycine excreted shikimate and shikimate 3-phosphate into the medium: the amounts depended on the actual concentration of the herbicide. Two-dimensional gel electrophoresis and determination of 5-enolpyruvylshikimate-3-phosphate synthase activity in crude extracts, as well as after separation by non-denaturing gel electrophoresis, revealed that the overproduction of the enzyme in adapted cells correlates with the accumulation of a 59-kDa protein. Overproduction of this 59-kDa protein resulted from a selectively increased level of a mRNA coding for a 64.5-kDa polypeptide which appeared in adapted cells, as shown by cell-free translation in the wheat germ system. In contrast to this quantitative, adaptive type of tolerance, the second mechanism causing tolerance to N-(phosphonomethyl)glycine in the Euglena cell line NR 6/50 was probably related to a qualitatively altered 5-enolpyruvylshikimate-3-phosphate synthase, which could not be inhibited by even 2 mM N-(phosphonomethyl)glycine in vitro. In agreement with this observation, the putatively mutated cell line excreted neither shikimate nor shikimate 3-phosphate into the growth medium containing N-(phosphonomethyl)glycine, even if cultivated in the presence of 20 mM or 50 mM N-(phosphonomethyl)glycine.     

Antagonists of the human CCR5 receptor as anti-HIV-1 agents. Part 4: synthesis and structure-activity relationships for 1-[N-(methyl)-N-(phenylsulfonyl)amino]-2-(phenyl)-4-(4-(N-(alkyl)-N-(benzyloxycarbonyl)amino)piperidin-1-yl)butanes

(2S)-2-(3-Chlorophenyl)-1-[N-(methyl)-N-(phenylsulfonyl)amino]-4-[spiro(2,3-dihydrobenzthiophene-3,4'-piperidin-1'-yl)]butane S-oxide (1b) has been identified as a potent CCR5 antagonist having an IC50=10 nM. Herein, structure-activity relationship studies of non-spiro piperidines are described, which led to the discovery of 4-(N-(alkyl)-N-(benzyloxycarbonyl)amino)piperidine derivatives (3-5) as potent CCR5 antagonists.     

Synthesis, resolution and anticonvulsant activity of chiral N-1'-ethyl,N-3'-(1-phenylethyl)-(R,S)-2'H,3H,5'H-spiro-(2-benzofuran-1,4'-imidazolidine)-2',3,5'-trione diastereomers

Four new N-1',N-3'-disubstituted-2'H,3H,5'H-spiro-(2-benzofuran-1,4'-imidazolidine)-2',3,5'-triones bearing a chiral N-3' substituent were synthesized, resolved and their anticonvulsant activity was obtained and determined that the activity was not stereoselective.