Neil E. Jacobsen

The deazapurine biosynthetic pathway revealed: in vitro enzymatic synthesis of PreQ(0) from guanosine 5'-triphosphate in four steps.

Deazapurine-containing secondary metabolites comprise a broad range of structurally diverse nucleoside analogues found throughout biology, including various antibiotics produced by species of Streptomyces bacteria and the hypermodified tRNA bases queuosine and archaeosine. Despite early interest in deazapurines as antibiotic, antiviral, and antineoplastic agents, the biosynthetic route toward deazapurine production has remained largely elusive for more than 40 years. Here we present the first in vitro preparation of the deazapurine base preQ(0), by the successive action of four enzymes. The pathway includes the conversion of the recently identified biosynthetic intermediate, 6-carboxy-5,6,7,8-tetrahydropterin, to a novel intermediate, 7-carboxy-7-deazaguanine (CDG), by an unusual transformation catalyzed by Bacillus subtilis QueE, a member of the radical SAM enzyme superfamily. The carboxylate moiety on CDG is converted subsequently to a nitrile to yield preQ(0) by either B. subtilis QueC or Streptomyces rimosus ToyM in an ATP-dependent reaction, in which ammonia serves as the nitrogen source. The results presented here are consistent with early radiotracer studies on deazapurine biosynthesis and provide a unified pathway for the production of deazapurines in nature.

N-(furfural) chitosan hydrogels based on Diels-Alder cycloadditions and application as microspheres for controlled drug release.

In this study, chitosan was chemically modified by reductive amination in a two-step process. The synthesis of N-(furfural) chitosan (FC) was confirmed by FT-IR and (1)H NMR analysis, and the degrees of substitution were estimated as 8.3 and 23.8%. The cross-linkable system of bismaleimide (BM) and FC shows that FC shared properties of furan-maleimide chemistry. This system produced non-reversible hydrogel networks by Diels-Alder cycloadditions at 85 °C. The system composed of BM and FC (23.8% substitution) generated stronger hydrogel networks than those of FC with an 8.3% degree of substitution. Moreover, the FC-BM system was able to produce hydrogel microspheres. Environmental scanning electron microscopy revealed the surface of the microspheres to be non-porous with small protuberances. In water, the microspheres swelled, increasing their volume by 30%. Finally, microspheres loaded with methylene blue were able to release the dye gradually, obeying second-order kinetics for times less than 600 min. This behavior suggests that diffusion is governed by the relaxation of polymer chains in the swelled state, thus facilitating drug release outside the microspheres.

Isolation of lignans from Schisandra chinensis with anti-proliferative activity in human colorectal carcinoma: Structure-activity relationships.

Separate benzocyclooctadiene lignans were isolated from the berries of Schisandra chinensis in milligram quantities on analytical reverse phase (RP) HPLC by an automated repeat-injection method and shown to have anti-proliferative activity against human colorectal cancer cells. Structures of the compounds were determined by a combination of NMR and mass spectrometry. Stereospecific NMR assignments for gomisin-N and deoxyschisandrin, gave more complete and accurate data than previously reported, based on 600MHz 2D HSQC, DQF-COSY and HMBC data. Comparison of coupling constants and HMBC crosspeak intensities with calculated and X-ray crystal structures confirmed their stereochemistry and conformation. Analysis of structure-activity relationships revealed the importance of key structural determinants. The S-biphenyl configuration of gomisin N, the most active lignan, correlated with increased anti-proliferative activity, while the presence of a hydroxyl group at the C7 position reduced or abolished this activity. Increased activity was also observed when a methylenedioxy group was present between C12 and C13. The percent yield of the most active compounds relative to the starting plant materials was 0.0156% for deoxyschisandrin and 0.0173% for gomisin N. The results of these studies indicate that automated repeat-injection method of analytical HPLC may provide a superior alternative to the standard semi-preparative HPLC techniques for separation of complex mixtures.

The Importance of Micelle-Bound States for the Bioactivities of Bifunctional Peptide Derivatives for δ/μ Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists

To provide new insight into the determining factors of membrane-bound peptide conformation that might play an important role in peptide-receptor docking and further biological behaviors, the dodecylphosphocholine (DPC) micelle-bound conformations of bifunctional peptide derivatives of delta-preferring opioid agonists and NK1 antagonists (1: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bzl(CF 3) 2; 2: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3,5-Bzl(CF 3) 2; 3: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bzl) were determined based on 2D NMR studies. Although the differences in the primary sequence were limited to the C-terminus, the obtained NMR conformations were unexpectedly different for each compound. Moreover, their biological activities showed different trends in direct relation to the compound-specific conformations in DPC micelles. The important result is that not only were the NK1 antagonist activities different (the pharmacophore located at the C-terminus)but the opioid agonist activities (this pharmacophore was at the structurally preserved N-terminus) also were shifted, suggesting that a general conformational change in the bioactive state was induced due to relatively small and limited structural modifications.

Inhibition of growth and cholesterol synthesis in breast cancer cells by oxidation products of β-carotene

Abstract We have isolated and chemically characterized a polar oxidation product of β-carotene and tested the effect of a highly enriched fraction containing this compound on the growth and metabolism of breast cancer (MCF-7) cells. This fraction strongly inhibits cell growth and cholesterol synthesis in MCF-7 cells. Pretreatment of the cells with mevalonate overcomes inhibition of cell growth by the oxidized fraction. Addition of the antioxidant butylated hydroxytoluene protects against inhibition of the growth of MCF-7 cells by β-carotene but not by the oxidized fraction. Pretreatment of cells with mevalonate overcomes inhibition of cell growth by oxidation products of β-carotene but not by retinoic acid. The oxidized fraction neither stimulates activity nor inhibits binding of retinoic acid to its nuclear receptors (RXR-α, RXR-β, RXR-γ, RAR-α, RAR-β, RAR-γ, and peroxisome proliferation receptors) in transfection assays. Mevalonate does not protect retinoic acid-induced growth inhibition of MCF-7 cells. The major compound in the inhibitory fraction was identified by mass spectrometry and nuclear magnetic resonance spectroscopy as 5,8-endoperoxy-2,3-dihydro-β-apocarotene-13-one. Our data suggest that the β-carotene oxidation products we have isolated represent a class of compounds not previously described with potential antineoplastic activity.

Metformin Scavenges Methylglyoxal To Form a Novel Imidazolinone Metabolite in Humans

Methylglyoxal (MG) is a highly reactive dicarbonyl compound involved in the formation of advanced glycation endproducts (AGE). Levels of MG are elevated in patients with type-2 diabetes mellitus (T2DM), and AGE have been implicated in the progression of diabetic complications. The antihyperglycemic drug metformin (MF) has been suggested to be a scavenger of MG. The present work examined and characterized unequivocally the resulting scavenged product from the metformin-MG reaction. The primary product was characterized by (1)H, (13)C, 2D-HSQC, and HMBC NMR and tandem mass spectrometry. X-ray diffraction analysis determined the structure of the metformin and MG-derived imidazolinone compound as (E)-1,1-dimethyl-2-(5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)guanidine (IMZ). A LC-MS/MS multiple reaction monitoring method was developed to detect and quantify the presence of IMZ in metformin-treated T2DM patients. Urine from >90 MF-treated T2DM patients was analyzed, with increased levels of MF directly correlating with elevations in IMZ. Urinary MF was detected in the range of 0.17 μM to 23.0 mM, and simultaneous measurement of IMZ concentrations were in the range of 18.8 nM to 4.3 μM. Since plasma concentrations of MG range from 40 nM to 4.5 μM, the level of IMZ production may be of therapeutic significance. Thus, in addition to lowering hepatic gluconeogenesis, metformin also scavenges the highly reactive MG in vivo, thereby reducing potentially detrimental MG protein adducts, with subsequent reductions in diabetic complications.

Synthesis and structure of m-terphenyl thio-, seleno-, and telluroethers.

Several routes for the synthesis of m-terphenyl thio-, seleno-, and telluroethers were investigated. m-Terphenyl iodides react with diphenyl diselenides or ditellurides (CsOH·H(2)O, DMSO, 110 °C) to give the desired compounds in 19-84% yield which significantly extends the previously reported such reactions because o-benzyne cannot be an intermediate as previously suggested. However, the most general synthetic route was that involving reaction of 2,6-diaryl Grignard reagents with sulfur, selenium, or tellurium electrophiles. The m-terphenyl thio-, seleno-, and telluroethers were characterized spectroscopically and, in one case, by single-crystal X-ray analysis. Certain of these compounds showed atropisomerism and barriers for interconversion of isomers were determined by variable-temperature NMR spectroscopy. The barriers for interconverting the syn and anti atropisomers increase on going from the analogous S to Se to Te compounds. Calculations on this isomerization revealed that the barriers are due to rotation about the aryl-aryl bond and that the barriers for rotation about the aryl-chalcogen bond are much lower.

Insights into the Chemical Biology of Selenium

The long-sought pathway by which selenocysteyl-tRNA[Ser]Sec is synthesized in eukaryotes has been revealed. Seryl-tRNA[Ser]Sec is O-phosphorylated and SecS, a pyridoxal phosphate-dependent protein, catalyzes the reaction of O-phosphoseryl-tRNA[Ser]Sec with monoselenophosphate to give selenocysteyl-tRNA[Ser]Sec . 1 H- 77 Se HMQC-TOCSY NMR spectroscopy has been developed to detect the selenium-containing amino acids present in selenized yeast after protease XIV digestion. An archived selenized yeast sample is found to contain the novel amino acid S-(methylseleno)cysteine in addition to selenomethionine. Arsenite and selenite react with GSH to form (GS) 2 AsSe−. The structure of this compound has been determined by EXAFS, 77 Se NMR and Raman spectroscopic and chromatographic studies. Its formation under biological conditions has been demonstrated.

NMR ASSIGNMENTS AND CONFORMATION OF TARAXERENES

Complete 1H and 13C NMR chemical shift assignments were made for taraxerene from HMQC and HMBC spectra. Methyl proton shifts were assigned for 11 other taraxerenes. Molecular mechanics calculations and NMR evidence supported the p‐bromobenzyl acetylaleuritolate x‐ray conformation for taraxerenes in solution.

Neighboring π-Amide Participation in Thioether Oxidation: Conformational Control

The electrochemical oxidation of thioethers is shown to be facilitated by neighboring amide participation. (1)H NMR spectroscopic analysis in acetonitrile solution of two conformationally constrained compounds with such facilitation shows that two-electron participation by the amide π2 orbital can occur to stabilize the developing sulfur radical cation.