Optimizing the Readout of Lanthanide-DOTA Complexes for the Detection of Ligand-Bound Copper(I).

The CuAAC 'click' reaction was used to couple alkyne-functionalized lanthanide-DOTA complexes to a range of fluorescent antennae. Screening of the antenna components was aided by comparison of the luminescent output of the resultant sensors using data normalized to account for reaction conversion as assessed by IR. A maximum 82-fold enhanced signal:background luminescence output was achieved using a Eu(III)-DOTA complex coupled to a coumarin-azide, in a reaction which is specific to the presence of copper(I). This optimized complex provides a new lead design for lanthanide-DOTA complexes which can act as irreversible 'turn-on' catalytic sensors for the detection of ligand-bound copper(I).

Exploring the substrate specificity of OxyB, a phenol coupling P450 enzyme involved in vancomycin biosynthesis.

OxyB is a cytochrome P450 enzyme that catalyzes the first oxidative phenol coupling reaction during vancomycin biosynthesis. The preferred substrate is a linear peptide linked as a C-terminal thioester to a peptide carrier protein (PCP) domain of the glycopeptide antibiotic non-ribosomal peptide synthetase. Previous studies have shown that OxyB can efficiently oxidize a model hexapeptide-PCP conjugate (R-Leu(1)-R-Tyr(2)-S-Asn(3)-R-Hpg(4)-R-Hpg(5)-S-Tyr(6)-S-PCP) (Hpg = 4-hydroxyphenylglycine) into a macrocyclic product by phenolic coupling of the aromatic rings in residues-4 and -6. In this work, the substrate specificity of OxyB has been explored using a series of N-terminally truncated peptides related in sequence to this model hexapeptide-PCP conjugate. Deletion of one or three residues from the N-terminus afforded a penta- (Ac-Tyr-Asn-Hpg-Hpg-Tyr-S-PCP) and a tri- (Ac-Hpg-Hpg-Tyr-S-PCP) peptide that were also efficiently transformed into the corresponding macrocyclic cross-linked product by OxyB. The tripeptide, representing the core of the macrocycle in vancomycin created by OxyB, is thus sufficient, as a thioester with the PCP domain, for phenol coupling to occur. The related tetrapeptide-PCP thioester was not cyclized by OxyB, neither was a related model hexapeptide containing tryptophan in place of tyrosine-6, nor were tripeptides (related to the natural product K-13) with the sequence Ac-Tyr-Tyr-Tyr-S-PCP cross-linked by OxyB.

Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of dolichols in plants.

Plant isoprenoids are derived from two biosynthetic pathways, the cytoplasmic mevalonate (MVA) and the plastidial methylerythritol phosphate (MEP) pathway. In this study their respective contributions toward formation of dolichols in Coluria geoides hairy root culture were estimated using in vivo labeling with (13)C-labeled glucose as a general precursor. NMR and mass spectrometry showed that both the MVA and MEP pathways were the sources of isopentenyl diphosphate incorporated into polyisoprenoid chains. The involvement of the MEP pathway was found to be substantial at the initiation stage of dolichol chain synthesis, but it was virtually nil at the terminal steps; statistically, 6-8 isoprene units within the dolichol molecule (i.e. 40-50% of the total) were derived from the MEP pathway. These results were further verified by incorporation of [5-(2)H]mevalonate or [5,5-(2)H(2)]deoxyxylulose into dolichols as well as by the observed decreased accumulation of dolichols upon treatment with mevinolin or fosmidomycin, selective inhibitors of either pathway. The presented data indicate that the synthesis of dolichols in C. geoides roots involves a continuous exchange of intermediates between the MVA and MEP pathways. According to our model, oligoprenyl diphosphate chains of a length not exceeding 13 isoprene units are synthesized in plastids from isopentenyl diphosphate derived from both the MEP and MVA pathways, and then are completed in the cytoplasm with several units derived solely from the MVA pathway. This study also illustrates an innovative application of mass spectrometry for qualitative and quantitative evaluation of the contribution of individual metabolic pathways to the biosynthesis of natural products.

Enabling methodology for the end functionalization of glycosaminoglycan oligosaccharides.

The chemical functionalization of glycosaminoglycans is very challenging due to their structural heterogeneity and polyanionic character; but as an enabling technology it promises rich rewards in terms of the structural and biological data it will afford. This review surveys the known methods for the preparation of glycosaminoglycan oligosaccharides and conditions for the selective functionalization of both the reducing and non-reducing ends. The synthetic merits of each approach are discussed, together with the structural modification of the glycosaminoglycan oligosaccharide which they confer. Recent applications of this methodology are highlighted, including introduction of functional labels for gel mobility shift assays and NMR studies of glycosaminoglycan-protein complexes, and synthesis of immobilised glycosaminoglycan arrays.

Oxidative phenol coupling reactions catalyzed by OxyB: a cytochrome P450 from the vancomycin producing organism. implications for vancomycin biosynthesis.

OxyB is a cytochrome P450 enzyme that catalyzes the first phenol coupling reaction during the biosynthesis of vancomycin-like glycopeptide antibiotics. The phenol coupling reaction occurs on a linear peptide intermediate linked as a C-terminal thioester to a peptide carrier protein (PCP) domain within the multidomain glycopeptide nonribosomal peptide synthetase (NRPS). Using model peptides with the sequence (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP and (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-(S)Dpg-S-PCP (where Hpg = 4-hydroxyphenylglycine, and Dpg = 3,5-dihydroxyphenylglycine), or containing (R)Leu instead of (R)(NMe)Leu, attached to recombinant PCPs derived from modules-6 and -7 in the vancomycin NRPS, we show that cross-linking of Hpg4 and Tyr6 by OxyB can occur in both hexapeptide- and heptapeptide-PCP conjugates. Thus, whereas OxyB may act preferentially on a hexapeptide still linked to the PCP-6 in NRPS subunit-2, it is possible that a linear heptapeptide intermediate linked to PCP-7 in NRPS subunit-3 may also be transformed into monocyclic product. For turnover, OxyB requires electrons, which in vitro can be supplied by spinach ferredoxin and E. coli flavodoxin reductase. Turnover is also dependent upon the presence of molecular oxygen. The model substrate (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP is transformed into cross-linked product by OxyB with a kcat of 0.1 s-1 and Km in the range 4-13 muM. Equilibrium binding of this substrate to OxyB, monitored by UV-vis, is accompanied by a typical low-to-high spin state change in the heme, characterized with a Kd of 17 +/- 5 muM.

Lewis acid mediated regioselective ring opening of benzylglycidol with dibenzyl phosphate: Short and attractive synthesis of dihydroxyacetone phosphate.

A novel, mild, and efficient method was described to introduce a dibenzyl phosphate by ring opening of benzylglycidol mediated by Lewis acids. This methodology was used as a key step for synthesizing the dihydroxyacetone phosphate (DHAP) in only three steps with an overall yield of 74% from the commercially available racemic benzylglycidol.

Isoprenoid biosynthesis via the MEP pathway. Synthesis of (3,4)-3,4-dihydroxy-5-oxohexylphosphonic acid, an isosteric analogue of 1-deoxy-D-xylulose 5-phosphate, the substrate of the 1-deoxy-D-xylulose 5-phosphate reducto-isomerase.

(3,4)-3,4-Dihydroxy-5-oxohexylphosphonic acid, an isosteric analogue of 1-deoxy-D-xylulose 5-phosphate (DXP), was obtained in enantiomerically pure form from (+)-2,3--benzylidene--threitol by a seven-step sequence. This phosphonate did not affect the growth of. It did not inhibit the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), but was converted by this enzyme into (3,4)-3,4,5-trihydroxy-3-methylpentylphosphonic acid, an isosteric analogue of 2-C-methyl-D-erythritol 4-phosphate. The enzyme was, however, less efficient with the methylene phosphonate analogue than with the natural substrate.

Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells.

In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate, the universal precursor for isoprenoid biosynthesis. The key enzyme of the cytoplasmic mevalonic acid (MVA) pathway is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Treatment of Tobacco Bright Yellow-2 (TBY-2) cells by the HMGR-specific inhibitor mevinolin led to growth reduction and induction of apparent HMGR activity, in parallel to an increase in protein representing two HMGR isozymes. Maximum induction was observed at 24 h. 1-Deoxy-d-xylulose (DX), the dephosphorylated first precursor of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, complemented growth inhibition by mevinolin in the low millimolar concentration range. Furthermore, DX partially re-established feedback repression of mevinolin-induced HMGR activity. Incorporation studies with [1,1,1,4-2H4]DX showed that sterols, normally derived from MVA, in the presence of mevinolin are synthesized via the MEP pathway. Fosmidomycin, an inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, the second enzyme of the MEP pathway, was utilized to study the reverse complementation. Growth inhibition by fosmidomycin of TBY-2 cells could be partially overcome by MVA. Chemical complementation was further substantiated by incorporation of [2-13C]MVA into plastoquinone, representative of plastidial isoprenoids. Best rates of incorporation of exogenous stably labeled precursors were observed in the presence of both inhibitors, thereby avoiding internal isotope dilution.