Robert V. Stick

Accumulation of arsenic in yelloweye mullet (Aldrichetta forsteri) following oral administration of organoarsenic compounds and arsenate

Groups of yelloweye mullet (Aldrichetta forsteri) were maintained for several weeks on diets containing one of a range of organoarsenic compounds (arsenobetaine, arsenocholine, 2-dimethylarsinylethanol, 2-dimethylarsinylacetic acid, 2-dimethylarsinothioylethanol) or arsenate. Fish fed 2-dimethylarsinylethanol, 2-dimethylarsinylacetic acid or 2-dimethylarsinothioylethanol showed no increase in arsenic concentrations in their muscle tissue, while fish fed arsenate showed small increases. The two groups of fish which received either arsenobetaine or arsenocholine had greatly elevated arsenic concentrations in their muscle tissue resulting from an estimated approximately 40% retention of ingested arsenic. Examination of the form of arsenic accumulated by fish fed arsenocholine showed that most of the arsenic (89%) was accumulated as arsenobetaine.

Structural and biochemical evidence for a boat-like transition state in |[beta]|-mannosidases

Enzyme inhibition through mimicry of the transition state is a major area for the design of new therapeutic agents. Emerging evidence suggests that many retaining glycosidases that are active on alpha- or beta-mannosides harness unusual B2,5 (boat) transition states. Here we present the analysis of 25 putative beta-mannosidase inhibitors, whose Ki values range from nanomolar to millimolar, on the Bacteroides thetaiotaomicron beta-mannosidase BtMan2A. B2,5 or closely related conformations were observed for all tightly binding compounds. Subsequent linear free energy relationships that correlate log Ki with log Km/kcat for a series of active center variants highlight aryl-substituted mannoimidazoles as powerful transition state mimics in which the binding energy of the aryl group enhances both binding and the degree of transition state mimicry. Support for a B2,5 transition state during enzymatic beta-mannosidase hydrolysis should also facilitate the design and exploitation of transition state mimics for the inhibition of retaining alpha-mannosidases--an area that is emerging for anticancer therapeutics.

Effects of triacylglycerol-saturated acyl chains on the clearance of chylomicron-like emulsions from the plasma of the rat

We previously found that a single saturated acyl chain at the glycerol 2-position affected the metabolism of chylomicrons. The explanation for the effect is not clear, but could be reproduced by saturated monoacylglycerols. In the present work we have extended our measurements to several different triacylglycerols containing one or two saturated chains in specific locations in an attempt to define structural features that affect chylomicron clearance. Lipid emulsions containing triacylglycerol, egg yolk phosphatidylcholine, free cholesterol, cholesteryl oleate (CO) and labelled with 3H-CO and [14C]triolein (OOO) were prepared as models of lymph chylomicrons. When injected intravenously into rats, the metabolism of the emulsions was influenced by the acyl chains of the constituent triacylglycerols. Compared with emulsions containing OOO as the only triacylglycerol, plasma clearances of emulsion [3H]CO were extremely slow in emulsions containing either 1,2-dioleoyl-3-stearoylglycerol (OOS) or 1-stearoyl-2,3-dioleoylglycerol (SOO). As little as 10% of SOO in mixture with OOO slowed the clearance, and increasing proportions of SOO in OOO emulsions progressively slowed the removal of OOO and CO labels from plasma. With 50% and 100% SOO in the emulsions clearance was negligible. In emulsions containing the triacyl-sn-glycerols, 1,3-dimyristoyl-2-oleoylglycerol (MOM), 1,3-dipalmitoyl-2-oleoylglycerol (POP), 1-oleoyl-2,3-distearoylglycerol (OSS) or 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS), clearance rates of CO and OOO labels from plasma were significantly decreased compared with control OOO emulsions. With emulsions prepared with the triacylglycerols, 1-oleoyl-2,3-dimyristoylglycerol (OMM) and 1-oleoyl-2,3-dipalmitoylglycerol (OPP), clearances of CO label were significantly slower than with control OOO emulsions, while the removal of OOO label was not significantly affected. The uptake of CO label in the liver was decreased in conjunction with the lower rates of clearance of emulsion CO from the plasma. The clearance from plasma of 1,3-distearoyl-2-oleoylglycerol (SOS) emulsions was similar to the control OOO emulsions, but significantly more emulsion OOO label was taken up by the liver. Emulsions made with the triacylglycerols extracted from natural cocoa butter, which contained a high proportion of saturated acyl chains, were cleared similarly to the control OOO emulsions. Our findings indicate that the plasma clearance of triacylglycerol-rich lipoprotein particles depends upon the specific arrangements of the acyl chains of the constituent triacylglycerols, and not necessarily on the overall saturation of the triacylglycerols.(ABSTRACT TRUNCATED AT 400 WORDS)

Arsenic-containing ribosides from the brown alga Sargassum lacerifolium: X-ray molecular structure of 2-amino-3-[5′-deoxy-5′-(dimethylarsinoyl)ribosyloxy]propane-1-sulphonic acid

Three novel arsenic-containing ribosides, methyl 5-deoxy-5-(dimethylarsinoyl)-β-D-riboside, 1-O[5′-deoxy-5′-(dimethylarsinoyl)-β-D-ribosyl]mannitol, and a dimethylarsonio-β-D-riboside have been isolated from extracts of the brown alga Sargassum lacerifolium. In addition, five previously reported arsenic-containing ribosides, and some arsenate, were isolated from Sargassum, and dimethylarsinic acid was also shown to be present. The compounds were identified chiefly by NMR spectroscopy, and an X-ray molecular structure is reported for one of them, 2-amino-3-[5′-deoxy-5′-(dimethyl-. arsinoyl)ribosyloxy]propane-1-sulphonic acid. The stereochemistry of the aglycones in these arseniccontaining ribosides is discussed and the configuration of 3-[5′-deoxy-5′-(dimethylarsinoyl)-β-D-ribosyloxy]-2-hydroxypropane-1-sulphonic acid 4 was assigned as 2S on the basis of a comparison of NMR spectra with those of synthetic model compounds.

Plasma clearance of model lipoproteins containing saturated and polyunsaturated monoacylglycerols injected intravenously in the rat

Triacylglycerols, with a saturated long-chain fatty acid at the glycerol-2-position, slow the clearance from plasma of remnants derived from injected chylomicrons and chylomicron-like emulsions. Slowing of remnant clearance also occurs when about 1% of monostearoylglycerol is added to a triolein chylomicron-like emulsion. We have now found that addition of monoacylglycerols, containing a saturated acyl chain from 12 to 20 carbons, slowed the plasma clearance and decreased the liver uptake of the remnants. In contrast, monoacylglycerols with unsaturated acyl chains were inconsistent in their effects on the remnant clearance. Monoarachidonin (M20:4) slowed remnant clearance comparable to that of saturated monoacylglycerols, monolinolenin (M18:3) and monolinolein (M18:2) were less effective, while monoolein had the least effect on remnant clearance. We have confirmed the defective remnant clearance in rats of injected emulsions containing saturated acyl chain by the using the diester-2-ether analogues of triolein and 1,3-dioleoyl-2-stearoylglycerol (OSO). Chylomicron-like lipid emulsions made with the ether analogues had clearance rates similar to their triester counterparts. Preformed remnants derived from emulsions of OSO, its ether analogue, and triolein emulsions or emulsions of triolein with approximately 1% saturated monoacylglycerols were prepared in hepatectomized rats. After intravenous injection into conscious recipient rats, these remnants were cleared from plasma similar to remnants traced in situ by lipolysis of injected chylomicron-like emulsions.

Common Inhibition of Both β-Glucosidases and β-Mannosidases by Isofagomine Lactam Reflects Different Conformational Itineraries for Pyranoside Hydrolysis

The ninety sequence-based families of glycoside hydrolases (GHs) and the correspondingly large diversity of protein topologies are a rich framework for studying variations in the catalytic mechanism of enzymatic glycoside hydrolysis. Such hydrolysis features oxocarbenium-ion-like transition states in which the anomeric centre becomes sp hybridised and partial positive charge accumulates, primarily across the endocyclic O5 C1 bond. For pyranosides, such a species demands planarity of C5, O5, C1 and C2 at or near the transition state; a situation accommodated only by the H3 and H4 (half-chair) conformations (or their closely related envelope forms) and B and B2,5 boats. Initial assumptions that all glycosidases harness H3 conformations are incorrect; indeed, the utilisation of different transition states for the hydrolysis of glycosides is an emerging theme in glycobiology (Scheme 1) and one that suggests a route for specific enzyme inhibition. Isofagomine lactam (1) displays an “in-plane” carbonyl at C2 and is, not surprisingly, a reasonable b-glucosidase inhibitor, with family GH1 b-glucosidases from Thermotoga maritima (TmGH1) and sweet almond inhibited with Ki values of 130 nm (this work, Figure 1) and 29 mm, respectively. Compound 1 has previously been shown to be an equally potent b-mannosidase inhibitor, with the snail b-mannosidase inhibited with a Ki of 9 mm ; this is superficially extremely counter-intuitive. Here, such Ki values for mannosidases are rationalised through structural analysis of 1 in complex with both an exo b-mannanase/b-mannosidase and a b-glucosidase. This work strongly supports previous proposals that b-mannosidases utilise a novel conformational itinerary, featuring a B2,5 transition state. The ground-state axial O2 of mannose is thus pseudo-equatorial at the transition state in a way that should be harnessed in future generations of mannosidase inhibitors. Recently we described the conformational agenda of a retaining GH26 b-mannanase. Trapping of the S5 conformation for the “Michaelis” complex of unhydrolysed substrate, together with the S2 conformation for the covalent intermediate, suggested a novel conformational itinerary for these enzymes through a B2,5 transition state consistent with earlier proposals, notably by Sinnott and Horton. Glycoside hydrolases thus appear to be harnessing the full conformational itinerary in a way that is both enzyme and substrate dependent (this was recently reviewed in the context of inhibition by Vasella and colleagues). Conformational considerations suggest that retaining mannosidase transition-state mimics should thus feature the pseudo-equatorial O2 of the B2,5 conformation of mannose. Isofagomine lactam 1, synthesised by both Stick and Bols, contains an in-plane carbonyl at C2. In elegant work, Bols reports a Ki of 9 mm for the snail b-mannosidase; [4] this inspired us to study the three-dimensional structures of 1 bound to both TmGH1 and a Cellvibrio mixtus exo b-mannanase (CmMan5, which is totally specific for manno-configured substrates). “Dual” inhibition of b-glucosidases and b-mannosidases has also been reported for the hydropyridazone compounds, which show similarity to isofagomine lactam, as discussed below. The three-dimensional structures of TmGH1 and CmMan5 in complex with 1 were determined by X-ray [a] Dr. F. Vincent, T. M. Gloster, Prof. G. J. Davies Structural Biology Laboratory, Department of Chemistry The University of York, Heslington, York, YO10 5YW (UK). Fax: (+44)1904-328266 E-mail : davies@ysbl.york.ac.uk

The synthesis of some epoxyalkyl Beta-C-Glycosides as potential inhibitors of Beta-Glucan hydrolases

The treatment of tetra-O-benzyl-D-glucono-1,5-lactone with various alkenylmagnesium halides gave the intermediate lactols which, upon reduction (Et3SiH/BF3) and protecting group manipulation, yielded alkenyl tetra-O-acetyl-beta-D-C-glucopyranosides in good yield. These beta-D-C-glucosides were precursors of the epoxyalkyl beta-D-G-glucopyranosides, themselves putative inhibitors of beta-glucan hydrolases. Similar additions of Grignard reagents to per-benzylated cellobionolactone were not as successful in yielding epoxyalkyl beta-C-cellobiosides. The addition of Grignard reagents to 1,2-anhydro-3,4,6-tri-O-benzyl-alpha-D-glucose offers a viable alternative route to the prop-2-enyl beta-D-C-glucoside, but not to the but-3-enyl and pent-4-enyl counterparts. Likewise, the addition of Grignard reagents to a 1,2-anhydro cellobiose gave disappointing results. Preliminary results are reported for a novel approach to alkenyl beta-D-C-glucosides by the alkylation of nitromethyl beta-D-C-glucosides.

The effect of monostearoylglycerol on the metabolism of chylomicron-like lipid emulsions injected intravenously in rats

In rats, remnant particles derived from chylomicron-like emulsions containing 1,3-dioleoyl-2-stearoylglycerol (OSO) are removed from plasma more slowly than remnants derived from triolein emulsions. The effect associated with a saturated acyl chain at the glycerol 2-position could be reproduced by incorporating 2-stearoylglycerol (MS) in a triolein emulsion. When MS solubilized with rat albumin or in plasma was injected before the injection of a triolein emulsion, clearance of the triolein emulsion was unchanged. The metabolic fate of MS, monitored with 14C-labelled MS, was similar whether incorporated in triacylglycerol emulsion or injected independently. More than 95% of MS had disappeared from the circulation by 5 min after the injection and the radioactivity was found in liver, spleen, muscle and adipose tissue. Some MS label appeared in plasma triacylglycerol. Remnants made in vitro by incubating triolein or OSO emulsions with post-heparin plasma showed no differences in their disappearance from plasma. With OSO emulsion, the in vitro remnants were found to contain more MS than remnants made in vivo in hepatectomized rats. Simultaneous injections of mixtures containing OSO and triolein emulsions, or triolein emulsions with and without MS, each labelled with either [3H]cholesteryl oleate or [14C]cholesteryl oleate showed consistently slower remnant removal and decreased liver uptake of the emulsions containing OSO or MS. Affinity columns and immunodiffusion all indicated that there was no difference in the amounts of apolipoprotein E associated with OSO or triolein particles. The protein spectra of in vivo remnants derived from OSO and triolein emulsion were also similar when examined by SDS-PAGE and isoelectric focusing gels. Our results show that the effects due to OSO or MS are mediated by the presence of MS in the emulsion particle surface, while indirect effects expressed in plasma or liver are excluded. The precise mechanism of the effect remains to be established, but it does not correlate with measurable changes in the spectra of apolipoproteins associated with the emulsion remnants.

An Improvement in the Preparation of Some Carbohydrate Benzylidene Acetals

Carbohydrate benzylidene and p- methoxybenzylidene acetals are easily prepared by treatment of various sugar derivatives with either benzaldehyde diethyl acetal or p- methoxybenzaldehyde diethyl acetal , respectively, in refluxing chloroform containing camphorsulfonic acid.

Glycerylphosphorylarsenocholine and phosphatidylarsenocholine in yelloweye mullet (Aldrichetta forsteri) following oral administration of arsenocholine

The novel arsenical glycerylphosphorylarsenocholine has been isolated from yelloweye mullet (Aldrichetta forsteri) following oral administration of arsenocholine. Ether-soluble arsenic was shown to be present as phosphatidylarsenocholine.