Impact of sulfur substitution on biotin binding affinity to streptavidin.

In this study, we investigated how the replacement of the tetrahydrothiophene ring of biotin with either an oxolane or (methyl)pyrrolidine moiety may affect its molecular interactions, in an effort to identify alternative affinity ligands suitable for in vitro and in vivo applications in synthetic biology. Initial molecular dynamics (MD) simulations suggested the potential formation of a hydrogen bond between either the oxygen or nitrogen atom of the envisaged tetrahydroheteryl analogues and the Thr90 residue of streptavidin, mirroring the sulfur-centered hydrogen bond detected by the crystallographic analysis of the biotin-streptavidin interaction. Therefore, oxy-, aza-, and N-methylazabiotin were readily synthesized starting from chiral five- or six-carbon sugar precursors. Based on fluorescence-based titration experiments using the corresponding fluorescein conjugates, oxybiotin showed a binding behavior similar to biotin with streptavidin, while both amino analogues displayed lower binding capacities. Notably, azabiotin exhibited a pH-dependent interaction profile, demonstrating enhanced binding under acidic conditions but weaker binding under basic pH, which could be exploited for various purposes.

Synthesis of Protected Amino Hexitol Nucleosides as Building Blocks for Oligonucleotide Synthesis.

A new synthesis protocol for the preparation of hitherto unknown 1',5'-anhydro-4'-amino-trityl/MMTr hexitol nucleosides has been developed. Key steps in the synthesis of the pyrimidine analogues (U and C) include the regioselective d- allo-hexitol oxirane and 2',4'-anhydronucleoside ring opening by uracil and azide, respectively. A different strategy using a regioselective epoxide ring opening of d- gulo-oxirane, followed by a SN2 type of azidation reaction, has been adopted for the purine analogues (A and G). These compounds can be easily converted to 6'-phosphoramidites for the solid-phase synthesis of N4' → P6' phosphoramidates of amino hexitol nucleic acids (AHNA).

Methylated Nucleobases: Synthesis and Evaluation for Base Pairing In†Vitro and In†Vivo.

The synthesis, base pairing properties and in vitro (polymerase) and in vivo (E. coli) recognition of 2'-deoxynucleotides with a 2-amino-6-methyl-8-oxo-7,8-dihydro-purine (X), a 2-methyl-6-thiopurine (Y) and a 6-methyl-4-pyrimidone (Z) base moiety are described. As demonstrated by Tm measurements, the X and Y bases fail to form a self-complementary base pair. Despite this failure, enzymatic incorporation experiments show that selected DNA polymerases recognize the X nucleotide and incorporate this modified nucleotide versus X in the template. In vivo, X is mainly recognized as a A/G or C base; Y is recognized as a G or C base and Z is mostly recognized as T or C. Replacing functional groups in nucleobases normally involved in W-C recognition (6-carbonyl and 2-amino group of purine; 6-carbonyl of pyrimidine) readily leads to orthogonality (absence of base pairing with natural bases).

Preparation of a disulfide-linked precipitative soluble support for solution-phase synthesis of trimeric oligodeoxyribonucleotide 3´-(2-chlorophenylphosphate) building blocks.

The preparation of a disulfide-tethered precipitative soluble support and its use for solution-phase synthesis of trimeric oligodeoxyribonucleotide 3´-(2-chlorophenylphosphate) building blocks is described. To obtain the building blocks, N-acyl protected 2´-deoxy-5´-O-(4,4´-dimethoxytrityl)ribonucleosides were phosphorylated with bis(benzotriazol-1-yl) 2-chlorophenyl phosphate. The "outdated" phosphotriester strategy, based on coupling of P(V) building blocks in conjunction with quantitative precipitation of the oligodeoxyribonucleotide with MeOH is applied. Subsequent release of the resulting phosphate and base-protected oligodeoxyribonucleotide trimer 3'-pTpdC(Bz)pdG(ibu)-5' as its 3'-(2-chlorophenyl phosphate) was achieved by reductive cleavage of the disulfide bond.

Solution phase synthesis of short oligoribonucleotides on a precipitative tetrapodal support.

An effective method for the synthesis of short oligoribonucleotides in solution has been elaborated. Novel 2'-O-(2-cyanoethyl)-5'-O-(1-methoxy-1-methylethyl) protected ribonucleoside 3'-phosphoramidites have been prepared and their usefulness as building blocks in RNA synthesis on a soluble support has been demonstrated. As a proof of concept, a pentameric oligoribonucleotide, 3'-UUGCA-5', has been prepared on a precipitative tetrapodal tetrakis(4-azidomethylphenyl)pentaerythritol support. The 3'-terminal nucleoside was coupled to the support as a 3'-O-(4-pentynoyl) derivative by Cu(I) promoted 1,3-dipolar cycloaddition. Couplings were carried out with 1.5 equiv of the building block. In each coupling cycle, the small molecular reagents and byproducts were removed by two quantitative precipitations from MeOH, one after oxidation and the second after the 5'-deprotection. After completion of the chain assembly, treatment with triethylamine, ammonia and TBAF released the pentamer in high yields.

Phytochemical analysis and free radical scavenging activity of medicinal plants Gnidia glauca and Dioscorea bulbifera.

Gnidia glauca and Dioscorea bulbifera are traditional medicinal plants that can be considered as sources of natural antioxidants. Herein we report the phytochemical analysis and free radical scavenging activity of their sequential extracts. Phenolic and flavonoid content were determined. Scavenging activity was checked against pulse radiolysis generated ABTS(•+) and OH radical, in addition to DPPH, superoxide and hydroxyl radicals by biochemical methods followed by principal component analysis. G. glauca leaf extracts were rich in phenolic and flavonoid content. Ethyl acetate extract of D. bulbifera bulbs and methanol extract of G. glauca stem exhibited excellent scavenging of pulse radiolysis generated ABTS(•+) radical with a second order rate constant of 2.33 × 10(6) and 1.72 × 10(6), respectively. Similarly, methanol extract of G. glauca flower and ethyl acetate extract of D. bulbifera bulb with second order rate constants of 4.48 × 10(6) and 4.46 × 10(6) were found to be potent scavengers of pulse radiolysis generated OH radical. G. glauca leaf and stem showed excellent reducing activity and free radical scavenging activity. HPTLC fingerprinting, carried out in mobile phase, chloroform: toluene: ethanol (4: 4: 1, v/v) showed presence of florescent compound at 366 nm as well as UV active compound at 254 nm. GC-TOF-MS analysis revealed the predominance of diphenyl sulfone as major compound in G. glauca. Significant levels of n-hexadecanoic acid and octadecanoic acid were also present. Diosgenin (C27H42O3) and diosgenin (3á,25R) acetate were present as major phytoconstituents in the extracts of D. bulbifera. G. glauca and D. bulbifera contain significant amounts of phytochemicals with antioxidative properties that can be exploited as a potential source for herbal remedy for oxidative stress induced diseases. These results rationalize further investigation in the potential discovery of new natural bioactive principles from these two important medicinal plants.

Linear and cyclic glycopeptide as HIV protease inhibitors.

Novel linear and cyclic glycotetrapeptides were designed, synthesized and tested for inhibition of the wild type C-SA HIV-1 protease enzyme. The incorporation of ß-amino acid sugar to the linear and cyclic peptides resulted in a series of fifteen novel compounds. Linear glycopeptide 4a and cyclic glycopeptide 6a displayed significant activities against the HIV protease enzyme. The experimental results were compared with a computational approach using molecular docking. The sugar hydroxyl group at the C(3) position in linear (4a) as well as cyclic glycopeptide (6a), shows hydrogen bonding interaction with the enzymatic Asp25/Asp25' residues in docking studies.

Gnidia glauca flower extract mediated synthesis of gold nanoparticles and evaluation of its chemocatalytic potential.

BACKGROUND: Novel approaches for synthesis of gold nanoparticles (AuNPs) are of utmost importance owing to its immense applications in diverse fields including catalysis, optics, medical diagnostics and therapeutics. We report on synthesis of AuNPs using Gnidia glauca flower extract (GGFE), its detailed characterization and evaluation of its chemocatalytic potential. RESULTS: Synthesis of AuNPs using GGFE was monitored by UV-Vis spectroscopy and was found to be rapid that completed within 20 min. The concentration of chloroauric acid and temperature was optimized to be 0.7 mM and 50°C respectively. Bioreduced nanoparticles varied in morphology from nanotriangles to nanohexagons majority being spherical. AuNPs were characterized employing transmission electron microscopy, high resolution transmission electron microscopy. Confirmation of elemental gold was carried out by elemental mapping in scanning transmission electron microscopic mode, energy dispersive spectroscopy and X-ray diffraction studies. Spherical particles of size ~10 nm were found in majority. However, particles of larger dimensions were in range between 50-150 nm. The bioreduced AuNPs exhibited remarkable catalytic properties in a reduction reaction of 4-nitrophenol to 4-aminophenol by NaBH4 in aqueous phase. CONCLUSION: The elaborate experimental evidences support that GGFE can provide an environmentally benign rapid route for synthesis of AuNPs that can be applied for various purposes. Biogenic AuNPs synthesized using GGFE exhibited excellent chemocatalytic potential.

Synthesis and molecular modelling studies of novel carbapeptide analogs for inhibition of HIV-1 protease.

Novel glycopeptides containing amino acids such as valine and alanine were designed, synthesized and tested for inhibition of the wild type C-SA HIV-1 protease enzyme. The incorporation of dipeptide sequences Val-Ala/Ala-Val to the sugar B-amino acid at two side chain positions resulted in a series of nine novel compounds. Compounds 3a, 3b, 3c, 3d, 4a, 4b and 5 displayed significant activities against the HIV protease enzyme. The glycopeptides are orders of magnitude less toxic to human MT-4 cells than lopinavir. Computational results were in good agreement with the experimental HIV-PR activity. The sugar hydroxyl group at the C(3) position interacts with the enzymatic Asp25/Asp25' residues. The docked position of the inhibitor is preserved during MD simulations and at least five hydrogen bond forms between the inhibitor and the enzymatic pocket. The results provide a platform for the progress of more effective carbohydrate supported inhibitors of HIV-1 and other aspartic proteases.

Synthesis of silver nanoparticles using Dioscorea bulbifera tuber extract and evaluation of its synergistic potential in combination with antimicrobial agents.

BACKGROUND: Development of an environmentally benign process for the synthesis of silver nanomaterials is an important aspect of current nanotechnology research. Among the 600 species of the genus Dioscorea, Dioscorea bulbifera has profound therapeutic applications due to its unique phytochemistry. In this paper, we report on the rapid synthesis of silver nanoparticles by reduction of aqueous Ag(+) ions using D. bulbifera tuber extract. METHODS AND RESULTS: Phytochemical analysis revealed that D. bulbifera tuber extract is rich in flavonoid, phenolics, reducing sugars, starch, diosgenin, ascorbic acid, and citric acid. The biosynthesis process was quite fast, and silver nanoparticles were formed within 5 hours. Ultraviolet-visible absorption spectroscopy, transmission electron microscopy, high-resolution transmission electron microscopy, energy dispersive spectroscopy, and x-ray diffraction confirmed reduction of the Ag(+) ions. Varied morphology of the bioreduced silver nanoparticles included spheres, triangles, and hexagons. Optimization studies revealed that the maximum rate of synthesis could be achieved with 0.7 mM AgNO(3) solution at 50°C in 5 hours. The resulting silver nanoparticles were found to possess potent antibacterial activity against both Gram-negative and Gram-positive bacteria. Beta-lactam (piperacillin) and macrolide (eryth-romycin) antibiotics showed a 3.6-fold and 3-fold increase, respectively, in combination with silver nanoparticles selectively against multidrug-resistant Acinetobacter baumannii. Notable synergy was seen between silver nanoparticles and chloramphenicol or vancomycin against Pseudomonas aeruginosa, and was supported by a 4.9-fold and 4.2-fold increase in zone diameter, respectively. Similarly, we found a maximum 11.8-fold increase in zone diameter of streptomycin when combined with silver nanoparticles against E. coli, providing strong evidence for the synergistic action of a combination of antibiotics and silver nanoparticles. CONCLUSION: This is the first report on the synthesis of silver nanoparticles using D. bulbifera tuber extract followed by an estimation of its synergistic potential for enhancement of the antibacterial activity of broad spectrum antimicrobial agents.

Antidiabetic Activity of Gnidia glauca and Dioscorea bulbifera: Potent Amylase and Glucosidase Inhibitors.

Diabetes is a metabolic disorder affecting about 220 million people worldwide. One of the most critical complications of diabetes is post-prandial hyper-glycemia (PPHG). Glucosidase inhibitor and α-amylase inhibitors are class of compounds that help in managing PPHG. Low-cost herbal treatment is recommended due to their lesser side effect for treatment of diabetes. Two plants with significant traditional therapeutic potential, namely, Gnidia glauca and Dioscorea bulbifera, were tested for their efficiency to inhibit α-amylase and α-glucosidase. Stem, leaf, and flower of G. glauca and bulb of D. bulbifera were sequentially extracted with petroleum ether, ethyl acetate, and methanol as well as separately with 70% ethanol. Petroleum ether extract of flower of G. glauca was found to inhibit α-amylase significantly (78.56%). Extracts were further tested against crude murine pancreatic, small intestinal, and liver glucosidase enzyme which revealed excellent inhibitory properties. α-glucosidase inhibition provided a strong in vitro evidence for confirmation of both G. glauca and D. bulbifera as excellent antidiabetic remedy. This is the first report of its kind that provides a strong biochemical basis for management of type II diabetes using G. glauca and D. bulbifera. These results provide intense rationale for further in vivo and clinical study.

Synthesis of new six- and seven-membered 1-N-iminosugars as promising glycosidase inhibitors.

New six- and seven-membered 1-N-iminosugars were prepared from d-glucose by the stereoselective Michael addition of nitromethane to d-glucose derived α,ß-unsaturated ester A followed by one pot reduction of nitro/ester functionality and subsequent amine protection to get N-Cbz protected aminol 6. Hydrolysis of 1,2-acetonide and reductive aminocyclization gave seven membered 1-N-iminosugar 5b. While, hydrolysis of 1,2-acetonide followed by NaIO(4) oxidative cleavage and hydrogenation using 10% Pd(OH)(2)/C, H(2) gave six membered 1-N-iminosugar 4a; the hydrogenation using 10% Pd/C-H(2) however, gave N-methyl substituted 1-N-iminosugar 4b. The hydrochloride salts of 4a/4b and 5b were found to be specific α-galactosidase and moderate α-glucosidae inhibitors, respectively, in micro molar range.

Synthesis, computational study and glycosidase inhibitory activity of polyhydroxylated conidine alkaloids--a bicyclic iminosugar.

New bicyclic conidine iminosugars 1d and 1e were synthesized from D-glucose. Thus, D-glucose was converted to sugar beta-amino acids 3a and 3b in good yields. Individual treatment of 3a/3b with the Mukaiyama reagent afforded sugar beta-lactams 4a/4b that on reduction with LiAlH(4)/AlCl(3) gave azetidines 5a/5b with a sugar appendage. Reductive aminocyclization of sugar azetidines 5a/5b afforded the corresponding conidine iminosugars 1d/1e. Based on the (1)H NMR and DFT calculation studies the conformation of 1d was assigned as half chair A2 and that of 1e as a boat B2. The glycosidase inhibitory activities of 1d and 1e such as alpha-mannosidase, alpha-glucosidase and alpha-galactosidase were studied. The alpha-amylase activity was compared with acarbose. Compound 1d was found to be a moderate inhibitor of glycosidases while 1e was noticed to be a good inhibitor of alpha-mannosidase and a moderate inhibitor of other glycosidases. These results were substantiated by molecular docking studies using WHAT IF software and the AUTODOCK 3.0 program.