Zinc-bound thiolate-disulfide exchange: a strategy for inhibiting metallo-beta-lactamases

The mononuclear zinc thiolate complexes [(Tp(PhMe))Zn(S-R)], where Tp(PhMe) is hydrotris((3-methyl-5-phenyl)pyrazolyl)borate and (S-R) is benzyl thiolate, 4-nitrophenylthiolate, 4-trifluoromethylphenylthiolate, 4-chlorophenylthiolate, phenylthiolate, 2-methylphenylthiolate, 4-methylphenylthiolate, 4-methoxyphenylthiolate, or 4-hydroxyphenylthiolate, were synthesized. Representative members of the class were also characterized structurally. The benzyl thiolate complex undergoes a thiolate-disulfide exchange reaction with a variety of diphenyl and dipyridyl disulfides. Kinetic studies revealed that the reaction shows saturation behavior in both complex and disulfide for most of the disulfides studied. Combined with studies of the lability of the coordinated thiolate, a mechanism is proposed where the reactive species is the zinc-coordinated thiolate. When the free benzyl thiol was allowed to react with the same disulfides, the reaction was slower by a factor of 20-200 than that for the zinc-thiolate complex, depending on the particular disulfide employed. Since most metallo-beta-lactamases contain one or more cysteine residues, the one in the active site being coordinated to zinc, the present study was extended to examine whether disulfides can be used as inhibitors of these enzymes by selective oxidation of the metal-bound cysteine. Several disulfides allowed to react with metallo-beta-lactamase CcrA from Bacteroides fragilis were moderate to potent irreversible inhibitors of the enzyme.     

Current mass spectrometry strategies for selenium speciation in dietary sources of high-selenium

This document reviews the most relevant mass spectrometry approaches to selenium (Se) speciation in high-Se food supplements in terms of qualitative and quantitative Se speciation and Se-containing species identification, with special reference to high-Se yeast, garlic, onions and Brazil nuts. Important topics such as complexity of Se speciation in these materials and the importance of combining Se-specific detection and molecule-specific determination of the particular species of this element in parallel with chromatography, to understand their nutritional role and cancer preventive properties are critically discussed throughout. The versatility and potential of mass spectrometric detection in this field are clearly demonstrated. Although great advances have been achieved, further developments are required, especially if speciatedcertified reference materials (CRMs) are to be produced for validation of measurements of target Se-containing species in Se-food supplements.     

Platinum chromophore-based systems for photoinduced charge separation: a molecular design approach for artificial photosynthesis

Photoinduced charge separation is a fundamental step in photochemical energy conversion. In the design of molecularly based systems for light-to-chemical energy conversion, this step is studied through the construction of two- and three-component systems (dyads and triads) having suitable electron donor and acceptor moieties placed at specific positions on a charge-transfer chromophore. The most extensively studied chromophores in this regard are ruthenium(II) tris(diimine) systems with a common 3MLCT excited state, as well as related ruthenium(II) bis(terpyridyl) systems. This Forum contribution focuses on dyads and triads of an alternative chromophore, namely, platinum(II) di- and triimine systems having acetylide ligands. These d8 chromophores all possess a 3MLCT excited state in which the lowest unoccupied molecular orbital is a pi orbital on the heterocyclic aromatic ligand. The excited-state energies of these Pt(II) chromophores are generally higher than those found for the ruthenium(II) tris(diimine) systems, and the directionality of the charge transfer is more certain. The first platinum diimine bis(arylacetylide) triad, constructed by attaching phenothiazene donors to the arylacetylide ligands and a nitrophenyl acceptor to 5-ethynylphenanthroline of the chromophore, exhibited a charge-separated state of 75-ns duration. The first Pt(tpy)(arylacetylide)+-based triad contains a trimethoxybenzamide donor and a pyridinium acceptor and has been structurally characterized. The triad has an edge-to-edge separation between donor and acceptor fragments of 27.95 Angstroms. However, while quenching of the emission is complete for this system, transient absorption (TA) studies reveal that charge transfer does not move onto the pyridinium acceptor. A new set of triads described in detail here and having the formula [Pt(NO2phtpy)(p-C triple-bond C-C6H4CH2(PTZ-R)](PF6), where NO2phtpy = 4'-{4-[2-(4-nitrophenyl)vinyl]phenyl}-2,2';6',2'-terpyridine and PTZ = phenothiazine with R = H, OMe, possess an unsaturated linkage between the chromophore and a nitrophenyl acceptor. While the parent chromophore [Pt(ttpy)(C triple-bond CC6H5)]PF6 is brightly luminescent in a fluid solution at 298 K, the triads exhibit complete quenching of the emission, as do the related donor-chromophore (D-C) dyads. Electrochemically, the triads and D-C dyads exhibit a quasi-reversible oxidation wave corresponding to the PTZ ligand, while the R = H triad and related C-A dyad display a facile quasi-reversible reduction assignable to the acceptor. TA spectroscopy shows that one of the triads possesses a long-lived charge-separated state of approximately 230 ns.     

Galloyl-Derived Orthoquinones as Reactive Partners in Nucleophilic Additions and Diels-Alder Dimerizations: A Novel Route to the Dehydrodigalloyl Linker Unit of Agrimoniin-Type Ellagitannins

Orthochloranil-mediated oxidation of galloyl monoethers furnishes the derived orthoquinones in excellent yield. These reactive electrophiles participate in a variety of nucleophilic addition reactions with heteroatomic and carbanionic partners. In addition, Lewis acid-mediated dimerization of the orthoquinones provides an efficient route to dehydrodigalloyl-type diaryl ether units characteristic of several ellagitannin natural products. The implications for ellagitannin biosynthesis and gallotannin-protein covalent attachment are discussed.     

Liquid chromatography/tandem mass spectrometry analysis of chloramphenicol in cooked crab meat

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method is described for the extraction, cleanup, determination, and confirmation of chloramphenicol (CAP) in cooked crab meat. The method involves pulverization of cooked crab meat with dry ice; extraction of the CAP into ethyl acetate (EtOAc); evaporation (by N2) of the EtOAc; addition of methanol, aqueous NaCl, and heptane; extraction of the lipids into the heptane, followed by extraction of the aqueous phase with EtOAc; evaporation (by N2) of the EtOAc; dissolution into methanol-water; filtration; and separation/detection/confirmation using LC/MS/MS. Crab meat was fortified at 0.25, 0.50, and 1.0 ng/g (ppb) chloramphenicol. Average absolute recoveries were 67, 84, and 86%, respectively, with relative standard deviation values all less than 1%. Four daughter ions (m/z 152, 176, 194, and 257) were monitored off the m/z 321 precursor ion. Determination was based on a standard curve using the peak areas of the m/z 152 daughter ion (the base peak) for standard solutions equivalent to 0.10, 0.20, 0.50, and 1.0 ppb in tissue (made with control crab extract). A set of 6 matrix controls (unfortified crab meat) was also analyzed, in which no chloramphenicol was detected. For identification purposes, the ion ratios (of each daughter ion versus the base daughter ion) of the fortified crab versus those of the chloramphenicol standards agreed within 10% (relative) at fortified chloramphenicol concentrations of 0.25-1.0 ppb.     

Anti-HIV cyclotides

The cyclotides are a recently discovered, structurally unique family of bioactive plant peptides. Their discovery spawned a series of structural analyses, synthetic efforts, and studies to define the biosynthesis and biological properties of these novel peptide metabolites. Cyclotides have a head-to-tail cyclized amino acid backbone and a conserved cystine knot motif that provides an extremely stable structural framework. They all share a common global fold and are highly resistant to denaturation and to cleavage by proteolytic enzymes. However, these macrocyclic peptides are quite permissive to amino acid substitutions or additions in several peripheral loop regions, since changes in these loops do not alter the core cyclotide structure. These features make cyclotides attractive templates for incorporating desired amino acid sequences and then delivering these peptide sequences in a well defined, highly stable framework. Cyclotides likely function in a defensive role in the source plants since they exhibit a broad spectrum of antimicrobial activity and are detrimental to the growth and survival of herbivorous insects. Cyclotides are gene-encoded polypeptides that are cleaved from larger precursor proteins and then cyclized. This review summarizes research done on a subset of cyclotides that were discovered due to their HIV inhibitory properties. It details the isolation and characterization of these compounds and describes this work in the context of our current state of knowledge of the entire cyclotide family.     

Regiospecific synthesis of mono-N-substituted indolopyrrolocarbazoles

[reactions: see text] Two complementary and efficient strategies have been developed for the regiospecific synthesis of unsymmetrical indolopyrrolocarbazoles (IPCs) mono-N-substituted with a pentacycle. A halogen in position 2 of the intermediate bisindolylmaleimides 3a-e allows a selective Mitsunobu coupling by exploiting the increased acidity of the 2-chloro-substituted indole nitrogen. It also promotes an easier cyclization of bisindolylmaleimides 4a-e and 7b-e to IPCs. Alkylation of the 2-unsubstituted indole-3-carboxamides 2a,b and further processing to the corresponding IPCs gives access to the opposite regioisomers.     

Expanding the Scope of the Cleavable N-(Methoxy)oxazolidine Linker for the Synthesis of Oligonucleotide Conjugates

Oligonucleotides modified by a 2′-deoxy-2′-(N-methoxyamino) ribonucleotide react readily with aldehydes in slightly acidic conditions to yield the corresponding N-(methoxy)oxazolidine-linked oligonucleotide-conjugates. The reaction is reversible and dynamic in slightly acidic conditions, while the products are virtually stable above pH 7, where the reaction is in a ‘‘switched off-state’’. Small molecular examinations have demonstrated that aldehyde constituents affect the cleavage rate of the N-(methoxy)oxazolidine-linkage. This can be utilized to adjust the stability of this pH-responsive cleavable linker for drug delivery applications. In the present study, Fmoc-β-Ala-H was immobilized to a serine-modified ChemMatrix resin and used for the automated assembly of two peptidealdehydes and one aldehyde-modified peptide nucleic acid (PNA). In addition, a triantennary N-acetyl-d-galactosamine-cluster with a β-Ala-H unit has been synthesized. These aldehydes were conjugated via N-(methoxy)oxazolidine-linkage to therapeutically relevant oligonucleotide phosphorothioates and one DNA-aptamer in 19–47% isolated yields. The cleavage rates of the conjugates were studied in slightly acidic conditions. In addition to the diverse set of conjugates synthesized, these experiments and a comparison to published data demonstrate that the simple conversion of Gly-H to β-Ala-H residue resulted in a faster cleavage of the N-(methoxy)oxazolidine-linker at pH 5, being comparable (T_0.5 ca 7 h) to hydrazone-based structures.     

Improved procedure for the synthesis of thiazolium-type peptide coupling reagents: BMTB as a new efficient reagent

An efficient scalable synthesis of 2-halothiazolium-type peptide coupling reagents has been developed. The key step is the formation of the 2-bromothiazole scaffold through cyclization of α-thiocyanato ketones with hydrogen bromide. Using this method, the new coupling reagent 2-bromo-N-methylthiazolium bromide (BMTB) was synthesized. BMTB was tested in a difficult model coupling reaction of two sterically hindered N-methylated amino acids and showed higher activity than the well-established peptide coupling reagent HATU.     

Determination of putrescine and cadaverine in seafood (finfish and shellfish) by liquid chromatography using pyrene excimer fluorescence

A liquid chromatography (LC) method is described for the easy determination of the biogenic diamines putrescine (PUT) and cadaverine (CAD) in canned tuna, frozen tuna loin, fresh mahimahi fillet, frozen raw shrimp, cooked lump crabmeat, and fresh and cold-smoked salmon. The method is also a useful screen for histamine (HTA). The method involves homogenization of fish tissue, extraction of biogenic amines into borate-trichloroacetic acid solution, centrifugation, and derivatization of supernatant with 1-pyrenebutanoic acid succinimidyl ester. The derivatized diamine species allow for the intramolecular excimer fluorescence of the pyrene moiety at a higher emission wavelength than is possible for the endogenous tissue monoamines, thus providing visual specificity of detection. All seafood species were fortified with 0.5, 1.0, 5.0, 10.0, and 15.0 microg/g (ppm) of PUT and CAD. Determination was based on standard graphs for PUT and CAD using peak areas with standard solutions equivalent to 0.375, 1.0, 5.0, 10.0, and 20.0 ppm in tissue. A set of five matrix controls (unfortified seafood tissue) were also analyzed; endogenous PUT was found in all samples except the canned tuna, and CAD found only in the shrimp, crab, and cold-smoked salmon. The background amines were thus subtracted prior to determining spike recovery. The intra-assay average recoveries ranged from 71 to 94% across species and spike levels.