Non-glycosidic analogues of nucleotides: 2′(R),3′(S),5′-trihydroxypentyl derivatives of adenine and cytosine

Chiral 2′,3′,5′-trihydroxypentyl derivatives of adenine and cytosine in which configurations at C-2′ and C-3′ are opposite to those of the natural nucleosides have been synthesized. The nucleoside analogues were converted into 3′-phosphates and dinucleoside phosphate analogues with 3′–5′ phosphodiester linkages. PMR, UV and CD spectra of the compounds are presented.     

Turnover is rate-limited by deglycosylation for Micromonospora viridifaciens sialidase-catalyzed hydrolyses: conformational implications for the Michaelis complex

A panel of seven isotopically substituted sialoside natural substrate analogues based on the core structure 7-(5-acetamido-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonic acid)-(2→6)-β-D-galactopyranosyloxy)-8-fluoro-4-methylcoumarin (1, Neu5Acα2,6GalβFMU) have been synthesized and used to probe the rate-limiting step for turnover by the M. viridifaciens sialidase. The derived kinetic isotope effects (KIEs) on k(cat) for the ring oxygen ((18)V), leaving group oxygen ((18)V), anomeric carbon ((13)V), C3-carbon ((13)V), C3-R deuterium ((D)V(R)), C3-S deuterium ((D)V(S)), and C3-dideuterium ((D)(2)V) are 0.986 ± 0.003, 1.003 ± 0.005, 1.021 ± 0.006, 1.001 ± 0.008, 1.029 ± 0.007, 0.891 ± 0.008, and 0.890 ± 0.006, respectively. The solvent deuterium KIE ((D(2)O)V) for the sialidase-catalyzed hydrolysis of 1 is 1.585 ± 0.004. In addition, a linear proton inventory was measured for the rate of hydrolysis, under saturating condition, as a function of n, the fraction of deuterium in the solvent. These KIEs are compatible with rate-determining cleavage of the enzymatic tyrosinyl β-sialoside intermediate. Moreover, the secondary deuterium KIEs are consistent with the accumulating Michaelis complex in which the sialosyl ring of the carbohydrate substrate is in a (6)S(2) skew boat conformation. These KIE measurements are also consistent with the rate-determining deglycosylation reaction occurring via an exploded transition state in which synchronous charge delocalization is occurring onto the ring oxygen atom. Finally, the proton inventory and the magnitude of the solvent KIE are consistent with deglycosylation involving general acid-catalyzed protonation of the departing tyrosine residue rather than general base-assisted attack of the nucleophilic water.     

Efficient synthesis of MUC4 sialylglycopeptide through the new sialylation using 5-acetamido-neuraminamide donors

Sialylation reactions using a new sialyl donor, diethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-beta-D-glycero-D-galacto-2-nonulopyranosylonamide phosphite (Neu5Ac-1-amide-2-phosphite) derivatives, and the synthesis of the sialyl-T N-MUC4 glycopeptide are described. The sialylation was performed in CH2Cl2 solvent toward the 6-hydroxyl group of several monosugar acceptors and generated alpha-sialoside in good yield under low temperature and TMSOTf activation system. Amide derivatives of sialoside were easily converted into naturally occurring sialoside after hydrolysis of the amide group. Sialyl-alpha(2,6)-GalN3 was also prepared by this new sialylation protocol, and then this sialoside was further converted into a Fmoc-protected sialyl-TN serine derivative for solid-phase glycopeptides synthesis. The solid-phase glycopeptide synthesis using this sialyl-TN serine derivative in which the sugar hydroxyl group was free afforded the target sialyl-TN-MUC4 glycopeptide.     

Investigation of the stability of thiosialosides toward hydrolysis by sialidases using NMR spectroscopy

[formula: see text] 1H NMR spectroscopy has been used to investigate whether the alpha(2-->6)-linked thiosialoside 3 and the alpha(2-->3)-linked thiosialoside 9 are hydrolyzed in the presence of Vibrio cholerae sialidase. Similarly, the hydrolysis of the O-ketosides Neu5Ac-2-O-alpha-(2-->3)-Gal beta Me (4) and the alpha-(2-->6)-sialyllactoside 7, representing natural alpha(2-->3)- and alpha(2-->6)-linked sialosides, respectively, was investigated. The results of the 1H NMR experiments clearly demonstrate that the thiosialosides are not hydrolyzed by Vibrio cholerae sialidase. As expected, the O-sialosides are hydrolyzed to give N-acetyl-alpha-D-neuraminic acid as the first product of substrate cleavage.     

Ab initio conformational space study of model compounds of O-glycosides of serine diamide

Relative stabilities of rotamers of the N-acetyl-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-L-seryl-N'-methyl amide (1) and eleven analogous molecules containing beta-galactose, alpha- and beta-mannose, alpha- and beta-glucose, and L-threonine were calculated to learn whether they could explain the natural preference for 1 in linkages between the carbohydrate and protein in glycoproteins. The lowest energy rotamers of four O-glycoside models of serine diamide were identified with a Monte Carlo search coupled with molecular mechanics (MM2*). These rotamers were further optimized with an ab initio level of theory (HF/6-31G(d)). Subsequently, B3LYP/6-31 + G(d) single point energies were calculated for the most stable HF structures. The most favorable interactions are present in 1 and its glucose analogue. The monosaccharide for the carbohydrate antenna is anchored to the serine residue with an AcNH...O=C-NHMe hydrogen bond in the most stable rotamers. The mannose analogue and the beta-anomers are considerably less stable according to the MM2* and especially to the ab inito energy values. The three analogues have HF/6-31 G(d) energies which are 4-6 kcal mol-1 higher; the single point B3LYP/6-31 + G(d)//HF/6-31 G(d) calculations yield preferences of 3-5 kcal mol-1 for 1. The most stable L-threonine analogues show a behaviour very similarly to the corresponding serine analogues. The ZPE and thermal correction components of the calculated delta H298 and delta G298 values are relatively small (< 0.4 kcal mol-1). However, the T delta S298 term can be as large as 2.6 kcal mol-1. The entropy terms stabilize the alpha-anomers relative to beta-anomers, and ManNAc relative to GalNAc. The largest stabilization effect is observed for one of the rotamers of the alpha-anomer of ManNAc.     

Structures and cytotoxic properties of sponge-derived bisannulated acridines

A reinvestigation of sponge natural products from additional Indo-Pacific collections of Xestospongiacf. carbonaria and X. cf. exigua has provided further insights on the structures, biological activities, and biosynthetic origin of bisannulated acridines. These alkaloids include one known pyridoacridine, neoamphimedine (2), and three new analogues, 5-methoxyneoamphimedine (4), neoamphimedine Y (5), and neoamphimedine Z (6). A completely new acridine, alpkinidine (7), was also isolated. A disk diffusion soft agar assay, using a panel of five cancer cell lines (solid tumors and leukemias) and two normal cells, was used to evaluate the differential cytotoxicity (solid tumor selectivity) of the sponge semipure extracts and selected compounds including amphimedine (1), 2, 4, and 7. While all four compounds were solid tumor selective, 1 and 2 were the most potent and 4 was the most selective. The rationale used to characterize the new structures is outlined along with the related biosynthetic pathways envisioned to generate 2 and 7.     

Design and efficient synthesis of 2 alpha-(omega-hydroxyalkoxy)-1 alpha,25-dihydroxyvitamin D3 Analogues, including 2-epi-ED-71 and their 20-epimers with HL-60 cell differentiation activity

A concise and efficient synthetic approach to 2 alpha-(omega-hydroxyalkoxy)-1 alpha,25-dihydroxyvitamin D(3) (4a-c), including 2-epi-ED-71, was developed starting from D-glucose as a chiral template for the construction of the 2 alpha-modified A-ring precursors (11a-c). It was found that the best ligand for the bovine thymus vitamin D receptor (VDR) in this series is 4b, which has 1.8 times greater binding affinity for the bovine thymus VDR than that of the natural hormone 1. Interestingly, potency in the induction of HL-60 cell differentiation for 4a-c was almost the same or weaker than that of 1 despite the strong binding affinity for the VDR. Next, we were interested in the "double modification"of 1 based on 4a-c with C20-epimerization, affording 2 alpha-(omega-hydroxyalkoxy)-20-epi-1 alpha,25-dihydroxyvitamin D(3) (20-epi-4a-c). All three 2 alpha-substituted 20-epi analogues of 1 (20-epi-4a-c) exhibited stronger binding affinities for the VDR, and their conformations in the ligand binding domain of VDR were analyzed by molecular modeling. Double-modified analogues of 20-epi-4a-c showed marked HL-60 cell differentiation activity, and 20-epi-4a possesses an activity 58-fold higher than that of the natural hormone 1.     

Synthesis, cytotoxicity and pro-apoptosis of novel benzoisoindolin hydrazones as anticancer agents

A series of benzoisoindolin hydrazones as analogues of natural lignan diphyllin were synthesized and the structures of these compounds were established by (1)H-NMR, (13)C-NMR, Mass and high resolution (HR)-MS. The compounds were evaluated for in vitro cytotoxicity against KB, A549 and HCT-116 cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Compound 4 possessed the highest growth inhibitory effect. Significant apoptosis of HCT-116 cells treated with compound 4 was observed by Hoechst33342-propidium iodide (PI) and acridine orange (AO)-ethidium bromide (EB) staining assay. Western blot analysis disclosed that compound 4 induced apoptosis via the mitochondrial pathway accompanied by an increased expression of Bax and a decreased expression of Bcl-2.     

Biotransformation of perfumery terpenoids, ()- ambrox(R) by a fungal culture Macrophomina phaseolina and a plant cell suspension culture of Peganum harmala

ABSTRACT: BACKGROUND: Biotransformation offers chemo enzymatic system to modify the compounds into their novel analogues which are difficult to synthesize by chemical methods. This paper describes the biotransformational studies of ambrox, one of the most important components of natural Ambergris (wale sperm) with fungal and plant cell culture. RESULTS: Biotransformation of ()-ambrox (1) with a fungal cell culture of Macrophomina phaseolina and a plant cell suspension cultures of Peganum harmala yielded oxygenated products, 3beta- hydroxyambrox (2), 6beta-hydroxyambrox (3), 1alpha-hydroxy-3oxoambrox (4), 1alpha,3beta- dihydroxyambrox (5), 13,14,15,16-tetranorlabdane-3-oxo-8,12-diol (6), 3-oxoambrox (7), 2alpha- hydroxyambrox (8), 3beta-hydroxysclareolide (9), and 2alpha,3beta-dihydroxyambrox (10). Metabolite 4 was found to be new compound. These metabolites were structurally characterized on the basis of spectroscopic studies. CONCLUSION: Nine oxygenated metabolites of ()-ambrox (1) were obtained from Macrophomina phaseolina and Peganum harmala. Enzymatic system of screened organisms introduced hydroxyl and keto functionalities at various positions of compound 1 in a stereo- and regiocontrolled manner.     

Rationalization of the difference in lifetime of two covalent sialosyl-enzyme intermediates of Trypanosoma rangeli sialidase

The difference in lifetime with respect to hydrolysis of two covalent syalosyl-enzyme intermediates of two difluorinated sialic acid analogues ( 1 and 2) bound to Trypanosoma rangeli sialidase is rationalized based on quantum mechanical calculations. The two intermediates differ only in a single functional group, acetamide in the sialidase- 1 complex and hydroxyl in the sialidase- 2 complex. It is shown that the acetamide group, which is also present in the natural substrate, increases the pKa of a catalytic base (Asp60) through electrostatic repulsion with the carbonyl oxygen on the ligand. This oxygen is absent in 2, resulting in a less basic Asp60 residue and, hence, a longer lifetime of the silaidase- 2 complex. Presumably, the lifetime of a sialidase inhibitor complex could be increased further by substituents that stabilize the negative charge on (and lowers the pKa value of) Asp60 in T. rangeli sialidase.     

N-(4-Nitrophenyl)oxamic Acid and Related N-Acylanilines Are Non-competitive Inhibitors of vibrio cholerae sialidase but do not inhibit trypanosoma cruzi or trypanosoma brucei trans-sialidases

N-(4-Nitrophenyl)oxamic acid 1 , N-(2-fluoro-4-nitrophenyl)oxamic acid 7 , N-(4-nitrophenyl)-trifluoroacetamide 3 , and N-(2-methoxy-4-nitrophenyl)trifluoroacetamide 9 are non-competitive inhibitors of Vibrio cholerae sialidase with K_i-values ranging from 2.66 to 5.18 · 10^?4 M . These compounds, and the N-acetylneuraminic-acid analogues 11–13 do not inhibit the sialidase and trans-sialidase activities from Trypanosoma cruzi; nor does N-(4-nitrophenyl)oxamic acid ( 1 ) inhibit the corresponding enzyme activities from T. brucei.     

Synthesis of difluoromethyl substituted lysophosphatidic acid analogues

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) displays an intriguing cell biology that is mediated via interactions both with G-protein coupled seven transmembrane receptors and with nuclear hormone receptor PPARγ. We describe a new and efficient route to enantiomerically homogeneous lysophospholipid analogues from (S)-1,2,4-butanetriol to give two 3-difluoromethyl substituted analogues of 2-acyl-sn-glycerol 3-phosphate. These compounds are migration-blocked analogues of the liable sn-2 LPA species. Preliminary studies were conducted on a nuclear reporter assay in which monocytic cells were transfected with a luciferase construct activated by a PPARγ nuclear receptor response element and have shown that the 3-difluoromethyl substituted analogues are fully active as natural LPA.     

Farnesyl diphosphate analogues with omega-bioorthogonal azide and alkyne functional groups for protein farnesyl transferase-catalyzed ligation reactions

Eleven farnesyl diphosphate analogues, which contained omega-azide or alkyne substituents suitable for bioorthogonal Staudinger and Huisgen [3 + 2] cycloaddition coupling reactions, were synthesized. The analogues were evaluated as substrates for the alkylation of peptide cosubstrates by yeast protein farnesyl transferase. Five of the diphosphates were good alternative substrates for farnesyl diphosphate (FPP). Steady-state kinetic constants were measured for the active compounds, and the products were characterized by HPLC and LC-MS. Two of the analogues gave steady-state kinetic parameters (kcat and Km) very similar to those of the natural substrate.     

Methyl calcein blue and other analogues of calcein blue

4-Methylumbelliferone-8-methylenesarcosine (Methyl Calcein Blue) and four related metallofluorochromic indicators derived from umbelliferone, 4-methylumbelliferone, and 4-methylesculetin by condensation with formaldehyde and iminodiacetic acid or glycine have been synthesized, the structures established, the absorbance and fluorescence measured as functions of pH, and the reactions with copper(II) and calcium studied with attention to the effects on fluorescence. All of the compounds display a maximum fluorescence at ph about 9. The fluorescence of each is quenched by copper(II). The calcium derivatives of the compounds derived from the umbelliferones and iminodiacetic acid fluoresce at high ph but those from the umbelliferones and glycine or sarcosine do not. At high pH, 4-methylesculetin and the amino-acids derived from it do not fluoresce either alone or in the prescence of calcium.     

Limonoids from Meliaceae with lipid peroxidation inhibitory activity

In this study the antioxidant activity of natural limonoids from Meliaceae swietenolide (1), 3,6-O,O-diacetylswietenolide (2), swietenine (3), swietemahonin G (4) and 2-hydroxyswietenine (5) were investigated along with the semi-synthetic analogues (6-25) of compounds 1, 3-4. Lipid peroxidation (LPO) inhibitory assays revealed 85.6, 13.3, 77.3, 61.2 and 24.6% inhibition for the natural compounds 1-5. Excellent antioxidant activity was seen for the semi-synthetic analogues 10 (98.3%), 16-17, 21-22 and 25 (100%), which were more active than the positive controls BHA (91.3%) and TBHQ (95.7%).     

Evaluation of carborane-containing nematic liquid crystals for electro-optical applications

Three new structurally similar compounds in series 1[2] and 2 containing 12- and 10-vertex p-carborane units (A and B) were prepared. Their thermal properties were investigated in the pure state and compared to those of their carbocyclic analogues. Using several higher homologues in the series 1[n], clearing temperatures for the unknown 1[4] and 1[10] were predicted. Optical, dielectric and rotational viscosity parameters for carborane derivatives 1[2] and 2 and their cyclohexane analogies were obtained by extrapolation of values measured for 10% solutions in a standard nematic host. The observed trends were rationalised using computed molecular parameters.     

Synthesis and Kinetic Evaluation of Inhibitors of the Phosphatidylinositol-Specific Phospholipase C from Bacillus cereus

Substrate analogues of phosphatidylinositol (1) were synthesized and evaluated as potential inhibitors of the bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus. The chiral analogues of the water-soluble phospholipid substrate 5 were designed to probe the effects of varying the inositol C-2 hydroxyl group, which is generally believed to serve as the nucleophile in the first step of the hydrolysis of phosphatidylinositols by PI-PLC. In the analogues 6-9, the C-2 hydroxyl group on the inositol ring of the phosphatidylinositol derivatives was rationally altered in several ways. Inversion of the stereochemistry at C-2 of the inositol ring led to the scyllo derivative 6. The inositol C-2 hydroxy group was replaced with inversion by a fluorine to produce the scyllo-fluoro inositol 7 and with a hydrogen atom to furnish the 2-deoxy compound 8. The C-2 hydroxyl group was O-methylated to prepare the methoxy derivative 9. The natural inositol configuration at C-2 was retained in the nonhydrolyzable phosphorodithioate analogue 10. The inhibition of PI-PLC by each of these analogues was then analyzed in a continuous assay using D-myo-inositol 1-(4-nitrophenyl phosphate) (25) as a chromogenic substrate. The kinetic parameters for each of these phosphatidylinositol derivatives were determined, and each was found to be a competitive inhibitor with K(i)'s as follows: 6, 0.2 mM; 10, 0.6 mM; 8, 2.6 mM; 9, 6.6 mM; and 7, 8.8 mM. This study further establishes that the hydrolysis of phosphatidylinositol analogues by bacterial PI-PLC requires not only the presence of a C-2 hydroxyl group on the inositol ring, but the stereochemistry at this position must also correspond to the natural myo-configuration. For future inhibitor design, it is perhaps noteworthy that the best inhibitors 6 and 10 each possess a hydroxyl group at the C-2 position. Several of the inhibitors identified in this study are now being used to obtain crystallographic information for an enzyme-inhibitor complex to gain further insights regarding the mechanism of hydrolysis of phosphatidylinositides by this PI-PLC.     

Site-specific carbon isotope analysis of aromatic carboxylic acids by elemental analysis/pyrolysis/isotope ratio mass spectrometry

Site-specific carbon isotope composition of organic compounds can provide useful information on their origin and history in natural environments. Site-specific isotope analyses of small amounts of organic compounds (sub-nanomolar level), such as short-chain carboxylic acids and amino acid analogues, have been performed using gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/pyrolysis/IRMS). These analyses were previously limited to volatile compounds. In this study, site-specific carbon isotope analysis has been developed for non-volatile aromatic carboxylic acids at sub-micromolar level by decarboxylation using a continuous flow elemental analysis (EA)/pyrolysis/IRMS technique. Benzoic acid, 2-naphthylacetic acid and 1-pyrenecarboxylic acid were pyrolyzed at 500-1000 degrees C by EA/pyrolysis/IRMS to produce CO2 for delta13C measurement of the carboxyl group. These three aromatic acids were most efficiently pyrolyzed at 750 degrees C. Conventional sealed-tube pyrolysis was also conducted for comparison. The delta13C values of CO2 generated by the continuous flow technique were within 1.0 per thousand of those performed by the conventional technique, indicating that the new continuous flow technique can accurately analyze the carbon isotopic composition of the carboxyl group in aromatic carboxylic acids. The new continuous flow technique is simple, rapid and uses small sample sizes, so this technique will be useful for characterizing the isotopic signature of carboxyl groups in organic compounds.     

Synthesis,structure, and biological evaluation of novel N- and O-linked diinositols

Several O-and N-linked inositols and/or aminoinositols have been prepared by iterative opening of epoxides and aziridines derived from homochiral cyclohexadiene cis-diols. The three inositols and their intermediate conduritols (conduramines) were tested against several glycosidases (alpha- and beta-glucosidase, alpha- and beta-galactosidase, alpha- and beta-mannosidase) in an assay that measured the rate of hydrolysis of p-nitrophenolglycosides rather than the concentration of p-nitrophenolate. Somewhat surprisingly, the best inhibition was seen against beta-galactosidase with several of the compounds. The inositols linked through oxygen or nitrogen were subjected to calcium binding studies performed in NMR experiments. Detailed analysis of the title compounds by NMR spectroscopy has been performed, and full assignments were made. One of the attendant benefits of the preparation of these compounds has been expressed in the design and synthesis of new salen catalysts whose effectiveness has been compared with Jacobsen's catalyst in the epoxidation of styrene.     

Synthesis and anti-influenza evaluation of polyvalent sialidase inhibitors bearing 4-guanidino-Neu5Ac2en derivatives

Polyvalent sialidase inhibitors bearing 4-guanidino-Neu5Ac2en derivatives on a poly-L-glutamine backbone are described. Aiming for a longer retention time of 4-guanidino-Neu5Ac2en (zanamivir) in bronchi and lungs, we focused on supermolecules bearing 4-guanidino-Neu5Ac2en derivatives bound at their C-7 position through noncleavable alkyl ether linkages. We first found that alkylation of the 7-hydroxyl group of sialic acid derivative 8 proceeded smoothly, and produced 7-O-alkyl-4-guanidino-Neu5Ac2en derivatives 13, which exhibited equipotent inhibitory activity against not only influenza A virus sialidase but also influenza A virus in the cell culture. Next, we synthesized poly-L-glutamine bearing 7-O-alkyl-4-guanidino-Neu5Ac2en derivatives linked by amide bonds, 26, which showed enhanced antiviral activity against influenza A virus and more potent efficacy in vivo relative to a monomeric sialidase inhibitor.