Synthesis of the enantiomers of 6-epicastanospermine and 1,6-diepicastanospermine from D- and L-gulonolactone.

The synthesis of the enantiomers of 6-epicastanospermine and of 1,6-diepicastanospermine from the enantiomeric gulonolactones is reported and the structure of the former is established as (1S,6R,7R,8R,8aR)-1,6,7,8-tetrahydroxyoctahydroindolizine. The inhibitory activities of the diastereomers against the amyloglucosidase-catalysed hydrolysis of p-nitrophenyl alpha-D-glucopyranoside were investigated, and the effects of 6-epicastanospermine and of 1,6-diepicastanospermine on 14 human liver glycosidases are reported.

The structural basis of the inhibition of human glycosidases by castanospermine analogues.

A series of epimers and deoxy derivatives of castanospermine has been synthesized to investigate the contribution of the different chiral centres to the specificity and potency of inhibition of human liver glycosidases. Castanospermine inhibits all forms of alpha- and beta-D-glucosidases, but alteration to any of the five chiral centres in castanospermine markedly decreases the inhibition. 6-Epicastanospermine, which is related to D-pyranomannose in the same way as castanospermine is to D-pyranoglucose, does not inhibit lysosomal (acidic) alpha-mannosidase, but is a good inhibitor of the cytosolic or neutral alpha-mannosidase. Conversely, 1-deoxy-6-epicastanospermine inhibits acidic alpha-mannosidase strongly, but not the neutral alpha-mannosidase. An explanation of this different inhibition based on preferential recognition of different configurations of mannose by the different forms of alpha-mannosidase is postulated. All derivatives of 6-epicastanospermine also have the minimum structural feature for the inhibition of alpha-L-fucosidase, but those with a beta-anomeric substituent do not inhibit the enzyme, or do so very weakly. 1-Deoxy-6,8a-diepicastanospermine, which has four chiral centres identical with alpha-L-fucose, is, however, a potent inhibitor of alpha-L-fucosidase (Ki 1.3 microM).

Plagiarizing plants: amino sugars as a class of glycosidase inhibitors.

Many polyhydroxylated alkaloids from plants are specific inhibitors of glycosidases. Information about these has led to the development of a wide range of both naturally occurring and synthetic inhibitors which may be used for mechanistic studies and for the purification of these enzymes. Sugar lactones are starting materials for highly efficient syntheses of deoxymannojirimycin and deoxyfuconojirimycin and of a number of their iminoheptitol analogues. This has allowed an investigation of the relationship between mannosidase and fucosidase inhibition.

Sugar-shaped alkaloids.

A range of simple, natural chemicals almost unknown 20 years ago are now being used to advance research in cancer, AIDS, diabetes, immunology and plant-insect recognition. These alkaloids of the pyrrolidine, piperidine, pyrrolizidine and indolizidine classes resemble sugar molecules in the arrangement of hydroxyl substituents and are often potent and specific glycosidase inhibitors.

Behavioral and electrophysiological study of antifeedant mechanisms associated with polyhydroxy alkaloids.

Eleven polyhydroxy alkaloids of plant origin were tested for anti-feedant effects against larvae of the lepidopteransSpodoptera littoralis, Spodoptera frugiperda, Heliothis virescens, andHelicoverpa armigera. Data from behavioral and electrophysiological investigations were correlated to reveal information on the mode of action of the antifeedants. The pyrrolidine DMDP was an effective antifeedant for all four species, whereas the piperidines fagomine and XZ-1 and the pyrrolizidine alexine were all ineffective as antifeedants. The activity of the pyrrolidines CYB-3 and DAB-1, the piperidines DNJ, DMJ, and BR1, and the bicyclic octahydroindolizine castanospermine varied among species. The investigation focuses on the structural similarities between some of the alkaloids and some common phagostimulatory sugars and illustrates a neural interaction involving the neurons that are differentially responsive to alkaloids and sugars. InS. littoralis, the neurons responding specifically to the alkaloids DMDP, DAB-1, and castanospermine and to the sugars fructose, sucrose, and glucose are more active when the compounds are applied singly than when an alkaloid and a sugar are applied together. The implications for the occurrence and functioning of different sugar receptor sites are discussed.

Loss of cytomegalovirus infectivity after treatment with castanospermine or related plant alkaloids correlates with aberrant glycoprotein synthesis.

Many plants contain polyhydroxyalkaloids which are potent inhibitors of glucosidases, enzymes involved in oligosaccharide trimming. These are important in determining the final configuration of specific glycoproteins. Human cytomegalovirus (CMV) encodes a number of glycoproteins, some of which ultimately reside in the outer envelope of the mature virion and are important for virus infectivity. Treatment with three polyhydroxyalkaloids, castanospermine (CAST), deoxynojirimycin (DNJ) and 2R,5R-dihydroxymethyl-3R,4R-dihydroxypyrrolidine (DMDP) blocked the growth of infectious virus, as determined by yield reduction and plaque reduction assays. However, in the presence of CAST, CMV infected cells continued to shed virions into the extracellular medium, as determined by electron microscopy. Envelope glycoproteins of virions produced after treatment with CAST (2.5 mM) were immunoprecipitated with a monoclonal antibody (F5) specific for the gcI family of glycoproteins. Analysis by PAGE-SDS showed an absence of gcI complex 2 (gp52 disulphide-linked to gp130) with a proportional increase in gcI complex 1 (gp52 disulphide-linked to gp95). The results indicated that gp130 alone, or linked to gp52, was important for CMV infectivity. As well as being potential targets for antiviral agents against CMV, inhibitors of glycoprotein trimming reactions may define components of the virion surface important for infectivity.

Inhibition of HIV replication by amino-sugar derivatives.

The plant alkaloids castanospermine, dihydroxymethyldihydroxypyrrolidine and deoxynojirimycin have recently been shown to have potential anti-HIV activity [(1987) Proc. Natl. Acad. Sci. USA 84, 8120-8124; (1987) Nature 330, 74-77; (1987) Lancet i, 1025-1026]. They are thought to act by inhibiting alpha-glucosidase I, an enzyme involved in the processing of N-linked oligosaccharides on glycoproteins. We report here the relative efficacy of a spectrum of amino-sugar derivatives as inhibition of HIV cytopathicity. Several alpha-glucosidase inhibitors and alpha-fucosidase inhibitors were found to be active at concentrations which were non-cytotoxic.

Inhibition of mammalian digestive disaccharidases by polyhydroxy alkaloids.

Several polyhydroxy alkaloids, including the eight presently known to occur in plants, have been compared as inhibitors of mouse gut digestive disaccharidases. The indolizidine castanospermine inhibited all activities tested, but others showed a selectivity which could be of value in studies of carbohydrate digestion and errors of metabolism.

Molluscicidal activity in the seeds of Millettia thonningii (Leguminosae: Papilionoideae).

Seeds of the leguminous plant Millettia thonningii were shown to possess promising molluscicidal activity against Bulinus trunctatus. The size of the snails was an important determinant of their susceptibility to the molluscicide; specimens with shell lengths of 2 to 3 mm being more susceptible than snails 5 to 6 mm long.

The pyrrolidine alkaloid, 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, inhibits glycoprotein processing.

2,5-Dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP) is a pyrrolidine alkaloid that was isolated from the plant, Lonchocarpus sericeus. In the present study, DMDP was tested as an inhibitor of glycoprotein processing. MDCK cells were infected with influenza virus and the virus was raised in the presence of various amounts of DMDP. The glycoproteins were labeled by the addition of [2-3H]mannose or [1-3H]galactose to the medium. The virus was isolated by differential centrifugation and treated with Pronase to obtain glycopeptides. These glycopeptides were isolated by chromatography on Bio-Gel P-4, then digested with endoglucosaminidase H (Endo H) and rechromatographed on the Bio-Gel P-4 column. In the control virus, more than 70% of the glycopeptides were resistant to Endo H and were previously characterized as complex types of oligosaccharides. The remaining 20-25% are sensitive to Endo H and are of the high-mannose type. However, in the presence of DMDP (250 micrograms/ml), more than 80% of the glycopeptides are susceptible to digestion by Endo H. The oligosaccharide released by this treatment sized like a hexose11-12GlcNAc on a calibrated column of Bio-Gel P-4, and was only slightly susceptible to alpha-mannosidase treatment. This oligosaccharide was also labeled in the glucose moieties by growing the virus in [1-3H]galactose in the presence of DMDP. Following isolation, the oligosaccharide was subjected to complete methylation. Acid hydrolysis of the methylated oligosaccharide gave three methylated glucose derivatives, corresponding to 2,3,4,6-tetramethylglucose, 3,4,6-trimethylglucose, and 2,4,6-trimethylglucose in almost equal amounts. These data indicate that the oligosaccharide is a Glc3Man8-9-GlcNAc and that DMDP inhibits glucosidase I. Similar results were obtained with the cellular glycoproteins. DMDP did not inhibit the incorporation of [3H]leucine into protein in MDCK cells, nor did it inhibit virus production as measured by plaque counts or hemagglutination assays. DMDP did cause some inhibition of mannose incorporation into the lipid-linked monosaccharides, but incorporation into lipid-linked oligosaccharides was not greatly affected, and incorporation into protein was stimulated. These results suggest that the pyrrolidine alkaloids are a new class of processing inhibitors.

Phenylacetic acid in human cerebrospinal fluid and plasma: selected ion monitoring assay.

Phenylacetic acid has been detected in human cerebrospinal fluid and plasma. Free acid and total acid values obtained after hydrolysis have been quantified as the pentafluorobenzyl ester by selected ion monitoring, using [2H7]phenylacetic acid as internal standard.