Construction of the isocopalane skeleton: application of a desulfinylative 1,7-hydrogen atom transfer strategy.

Two attractive chirons, aldehyde 6 and chloride 7, exhibiting functionalized ent-spongiane-type tricyclic skeletons (ABC ring system), have been constructed and their absolute configurations have been studied by NMR spectroscopy and confirmed by single-crystal X-ray diffraction. Both of these chirons are derived from commercially available andrographolide in good yield. Aldehyde 6 is obtained through a novel K2 S2 O8 -catalyzed aquatic ring-closing reaction of allylic sodium sulfonate and intramolecular 1,7-hydrogen atom transfer process. Further mechanistic investigations demonstrate that the 1,7-hydrogen atom transfer is a free-radical process, whereby hydrogen migrates from C18 to C17, as evidenced by double-18- deuterium-labeled isotope experiments. Prospective applications of these two chiral sources are also discussed.

A smart library of epoxide hydrolase variants and the top hits for synthesis of (S)-ß-blocker precursors.

Microtuning of the enzyme active pocket has led to a smart library of epoxide hydrolase variants with an expanded substrate spectrum covering a series of typical ß-blocker precursors. Improved activities of 6- to 430-fold were achieved by redesigning the active site at two predicted hot spots. This study represents a breakthrough in protein engineering of epoxide hydrolases and resulted in enhanced activity toward bulky substrates.

Synthesis of the scalarane sesterterpenoid 16-deacetoxy-12-epi-scalarafuranacetate.

The marine natural product 16-deacetoxy-12-epi-scalarafuranacetate, isolated from Spongua officinalis , was synthesized in 18 linear steps, starting from (-)-sclareol, with high stereoselectivity and an overall yield of 6.1%. The intermediate 16-deacetoxy-12-epi-scalarafuran could be easily transformed into a series of natural scalarane sesterterpenoids in a few steps.

Structure-guided engineering of Pseudomonas dacunhael-aspartate ß-decarboxylase for l-homophenylalanine synthesis.

Structure-guided engineering of Pseudomonas dacunhael-aspartate ß-decarboxylase (AspBDC) resulted in a double mutant (R37A/T382G) with remarkable 15 400-fold improvement in specific activity reaching 216 mU mg-1, towards the target substrate 3(R)-benzyl-l-aspartate. A novel strategy for enzymatic synthesis of l-homophenylalanine was developed by using the variant as a biocatalyst affording 75% product yield within 12 h. Our results underscore the potential of engineered AspBDC for the biocatalytic synthesis of pharmaceutically relevant and value added unnatural l-amino acids.

A molecular target for suppression of the evolution of antibiotic resistance: inhibition of the Escherichia coli RecA protein by N(6)-(1-naphthyl)-ADP.

We report that N(6)-(1-naphthyl)-ADP inhibits the Escherichia coli RecA protein in vitro. A novel rapid screen identified it as a potent inhibitor of RecA nucleoprotein filament formation, and further characterization established it as an ATP-competitive inhibitor of RecA-catalyzed ATP hydrolysis. This and other inhibitors of RecA activities represent a new approach for understanding the molecular targets and pathways involved in the evolution of antibiotic resistance in bacteria.

Apoptosis induction of ZBB-006, a novel synthetic diterpenoid, in the human hepatocellular carcinoma cell line HepG2 in vitro and in vivo.

Diterpenes, present in many medicinal plants, have been the focus of continuous studies for the development of new anticancer agents. ZBB-006 is a new synthetic diterpenoid derivative which exhibited significant anti-proliferation activity against various cancer cell lines in our previous study. Here, we investigated the antitumor effect of ZBB-006 and its potential mechanisms in the human hepatocellular carcinoma cell line HepG2, both in vitro and in vivo. We found that oral administration of ZBB-006 effectively suppressed the growth of HepG2 xenograft tumor in nude mice without body weight decline as compared with the control group. Meanwhile, the growth inhibitory effect of ZBB-006 on HepG2 cells was observed with MTT assay. Apoptosis induced by ZBB-006 in HepG2 cells was evidenced by DAPI staining and Annexin V/PI double staining assay. ZBB-006 also dissipated the mitochondrial membrane potential (ΔΨm) apparently as revealed by JC-1 staining. Furthermore, the cleavage of PARP, activation of caspase-3 and caspase-9 but not caspase-8 was demonstrated by western blot assay both in vitro and in vivo. Additionally, the proapoptotic protein Bax was markedly elevated, while the antiapoptotic protein Bcl-2 was downregulated. Collectively, our data indicated that ZBB-006 exerted a strong antitumor effect on HepG2 cells by initiating the mitochondrial-dependent apoptosis, and it has potential to be explored as a new promising therapeutic agent against human hepatoma.

Isotope-coded, iodoacetamide-based reagent to determine individual cysteine pK(a) values by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Cysteine reactivity in enzymes is imparted to a large extent by the stabilization of the deprotonated form of the reduced cysteine (i.e., the thiolate) within the active site. Although this is likely to be an important chemical attribute of many thiol-based enzymes, including cysteine-dependent peroxidases (peroxiredoxins) and proteases, only relatively few pK(a) values have been determined experimentally. Presented here is a new technique for determining the pK(a) value of cysteine residues through quantitative mass spectrometry following chemical modification with an iodoacetamide-based reagent over a range of pH buffers. This isotope-coded reagent, N-phenyl iodoacetamide (iodoacetanilide), is readily prepared in deuterated (d(5)) and protiated (d(0)) versions and is more reactive toward free cysteine than is iodoacetamide. Using this approach, the pK(a) values for the two cysteine residues in Escherichia coli thioredoxin were determined to be 6.5 and greater than 10.0, in good agreement with previous reports using chemical modification approaches. This technique allows the pK(a) of specific cysteine residues to be determined in a clear, fast, and simple manner and, because cysteine residues on separate tryptic peptides are measured separately, is not complicated by the presence of multiple cysteines within the protein of interest.

Synthesis of chemical probes to map sulfenic acid modifications on proteins.

Cysteine sulfenic acids in proteins can be identified by their ability to form adducts with dimedone, but this reagent imparts no spectral or affinity tag for subsequent analyses of such tagged proteins. Given its similar reactivity toward cysteine sulfenic acids, 1,3-cyclohexadione was synthetically modified to an alcohol derivative and linked to fluorophores based on isatoic acid and 7-methoxycoumarin. The resulting compounds retain full reactivity and specificity toward cysteine sulfenic acids in proteins, allowing for incorporation of the fluorescent label into the protein and "tagging" it based on its sulfenic acid redox state. Control experiments using dimedone further show the specificity of the reaction of 1,3-diones with protein sulfenic acids in aqueous media. These new compounds provide the basis for an improved method for the detection of protein sulfenic acids.

Direct observation of an acyl nitroso species in solution by time-resolved IR spectrocopy.

Benzoyl nitroside (5) was generated in solution by laser photolysis of 3,5-diphenyl-1,2,4-oxadiazole-4-oxide (4) and studied by time-resolved infrared spectroscopy. The second-order rate constants for reaction of 5 with diethylamine and 1,3-cyclohexadiene were determined to be (1.3 +/- 0.5) x 10(5) M(-1) s(-1) and (6.0 +/- 0.5) x 10(3) M(-1) s(-1), respectively. The formation of nitroxyl (HNO), a product of the reaction of 5 with diethylamine, was also observed.

Nitroxyl (HNO) release from new functionalized N-hydroxyurea-derived acyl nitroso-9,10-dimethylanthracene cycloadducts.

A thermal retro-Diels-Alder decomposition of N-hydroxyurea-derived acyl nitroso compounds and 9,10-dimethylanthracene cycloadducts followed by acyl nitroso compound hydrolysis produces nitrous oxide, evidence for the formation of nitroxyl, the one-electron reduced form of nitric oxide that has drawn considerable attention for its potential roles in biological systems. EPR and NMR spectroscopy provide further evidence for nitroxyl formation and kinetic information, respectively. Such compounds may prove to be useful alternative nitroxyl donors.

Straightforward synthesis of two pairs of enantiomeric butenolides 3-tetradecyl- and 3-hexadecyl-5-methyl-2(5H)-furanone.

Based on the synthetic method of optically active 3-alkyl-5-methyl-2(5H)-furanones from lactic acid, two pairs of chiral butenolides 3-tetradecyl- and 3-hexadecyl-5-methyl-2(5H)-furanone have been straightforwardly synthesized from methyl stearate and methyl palmitate respectively by aldol condensation with (R)- or (S)-O-tetrahydropyranyl lactal prepared from corresponding ethyl lactate and beta-elimination.

Mimicry of annonaceous acetogenins: enantioselective synthesis of a (4R)-hydroxy analogue having potent antitumor activity.

The (4R)-hydroxylated analogues of annonaceous acetogenin mimicking compound 2 were designed and synthesized structurally on the basis of the naturally occurring annonaceous acetogenin bullatacin, which was discovered as a typical member of the novel family of polyketides with potent cytotoxicity, antitumoral, and other biological activities. The preliminary screenings show that the IC(50) values of 2 were 1.6 x 10(-3) and 8 x 10(-2) microg/mL against HT-29 and HCT-8, respectively. A remarkable enhancement effect was observed by the activity comparison of 1c and its (4R)-hydroxylated analogue 2.

Studies on mimicry of naturally occurring annonaceous acetogenins: non-THF analogues leading to remarkable selective cytotoxicity against human tumor cells.

A class of structurally simplified analogues of the naturally occurring annonaceous acetogenins were developed, amongst which some non-THF analogues showed remarkable cytotoxicities against tumor cell lines, as well as good selectivity between human tumor cells and normal cells. The synthetic routes were significantly shortened because of the removal of the chiral centers bearing the THF rings on the natural templates. This simplification also provides access to the parallel synthesis of these mimics by a combinatorial strategy. The remaining stereogenic centers at the positions alpha to the ethereal links were introduced by the Chiron approach from the easily accessible chiral building blocks 6a and/or 6b, made in turn from L-ascorbic acid or D-mannitol, while the one in the butenolide segment was taken from L-lactate. All four diastereomeric non-THF analogues 2a-2d showed remarkable activity against the HCT-8 cell line, and better differentiation was found when testing against the HT-29 cell line. It was also discovered that both the butenolide and ethylene glycol subunits play essential roles in the cytotoxicities against tumor cell lines, while the 10-substituted hydroxy group and the absolute configuration of methyl group at the butenolide moiety are less important for their activity.