A cascade reaction for the synthesis of d-fagomine precursor revisited: Kinetic insight and understanding of the system.

The synthesis of aldol adduct (3S,4R)-6-[(benzyloxycarbonyl)amino]-5,6-dideoxyhex-2-ulose, a precursor of the interesting dietary supplement, iminosugar d-fagomine, was studied in a cascade reaction with three enzymes starting from Cbz-N-3-aminopropanol. This system was studied previously using a statistical optimization method which enabled a 79 % yield of the aldol adduct with a 10 % yield of the undesired amino acid by-product. Here, a kinetic model of the cascade, including enzyme operational stability decay rate and the undesired overoxidation of the intermediate product, was developed. The validated model was instrumental in the optimization of the cascade reaction in the batch reactor. Simulations were carried out to determine the variables with the most significant impact on substrate conversion and product yield. As a result, process conditions were found that provided the aldol adduct in 92 % yield with only 0.7 % yield of the amino acid in a one-pot one-step reaction. Additionally, compared to previous work, this improved process outcome was achieved at lower concentrations of two enzymes used in the reaction. With this study the advantages are demonstrated of a modelling approach in developing complex biocatalytical processes. Mathematical models enable better understanding of the interactions of variables in the investigated system, reduce cost, experimental efforts in the lab and time necessary to obtain results since the simulations are carried out in silico.

Metabolism, transformation and dynamic changes of alkaloids in silkworm during feeding mulberry leaves.

Metabolism, transformation and dynamic changes of DNJ, 2-O-α-D-Gal-DNJ, fagomine, isofagomine and 4-O-ß-d-Glc-fagomine from mulberry leaves in silkworms at different instars were observed. UPLC-Q/TOF-MS and UPLC-TQ/MS methods were adopted for qualitative and quantitative analysis respectively. Three species mulberry leaves were used to feed the silkworm as controls. By analyzing and comparing the content changes of DNJ, fagomine and their derivatives in silkworms and silkworm excrement at different instar, we revealed the dynamic changes, confirmed the enrichment effect of the polyhydroxy alkaloids by silkworm, and inferred the conversion process behind this effect. The experimental results indicated that DNJ and its derivatives turned into some intermediate substances in the metabolic process, and finally they converted back and the content increased. Fagomine and its derivatives interconverted into each other in the process, 4-O-ß-d-Glc-fagomine transformed into fagomine, while fagomine transformed into isofagomine.

Stabilization of Glucocerebrosidase by Active Site Occupancy.

Glucocerebrosidase (GBA) is a lysosomal ß-glucosidase that degrades glucosylceramide. Its deficiency results in Gaucher disease (GD). We examined the effects of active site occupancy of GBA on its structural stability. For this, we made use of cyclophellitol-derived activity-based probes (ABPs) that bind irreversibly to the catalytic nucleophile (E340), and for comparison, we used the potent reversible inhibitor isofagomine. We demonstrate that cyclophellitol ABPs improve the stability of GBA in vitro, as revealed by thermodynamic measurements (Tm increase by 21 °C), and introduce resistance to tryptic digestion. The stabilizing effect of cell-permeable cyclophellitol ABPs is also observed in intact cultured cells containing wild-type GBA, N370S GBA (labile in lysosomes), and L444P GBA (exhibits impaired ER folding): all show marked increases in lysosomal forms of GBA molecules upon exposure to ABPs. The same stabilization effect is observed for endogenous GBA in the liver of wild-type mice injected with cyclophellitol ABPs. Stabilization effects similar to those observed with ABPs were also noted at high concentrations of the reversible inhibitor isofagomine. In conclusion, we provide evidence that the increase in cellular levels of GBA by ABPs and by the reversible inhibitor is in part caused by their ability to stabilize GBA folding, which increases the resistance of GBA against breakdown by lysosomal proteases. These effects are more pronounced in the case of the amphiphilic ABPs, presumably due to their high lipophilic potential, which may promote further structural compactness of GBA through hydrophobic interactions. Our study provides further rationale for the design of chaperones for GBA to ameliorate Gaucher disease.

Evaluation of fluoropyruvate as nucleophile in reactions catalysed by N-acetyl neuraminic acid lyase variants: scope, limitations and stereoselectivity.

The catalysis of reactions involving fluoropyruvate as donor by N-acetyl neuraminic acid lyase (NAL) variants was investigated. Under kinetic control, the wild-type enzyme catalysed the reaction between fluoropyruvate and N-acetyl mannosamine to give a 90 : 10 ratio of the (3R,4R)- and (3S,4R)-configured products; after extended reaction times, equilibration occurred to give a 30 : 70 mixture of these products. The efficiency and stereoselectivity of reactions of a range of substrates catalysed by the E192N, E192N/T167V/S208V and E192N/T167G NAL variants were also studied. Using fluoropyruvate and (2R,3S)- or (2S,3R)-2,3-dihydroxy-4-oxo-N,N-dipropylbutanamide as substrates, it was possible to obtain three of the four possible diastereomeric products; for each product, the ratio of anomeric and pyranose/furanose forms was determined. The crystal structure of S. aureus NAL in complex with fluoropyruvate was determined, assisting rationalisation of the stereochemical outcome of C-C bond formation.

A fluorescence study of isofagomine protonation in ß-glucosidase.

N-(10-Chloro-9-anthracenemethyl)isofagomine 5 and N-(10-chloro-9-anthracenemethyl)-1-deoxynojirimycin 6 were prepared, and their inhibition of almond ß-glucosidase was measured. The isofagomine derivative 5 was found to be a potent inhibitor, while the 1-deoxynojirimycin derivative 6 displayed no inhibition at the concentrations investigated. Fluorescence spectroscopy of 5 with almond ß-glucosidase at different pH values showed that the inhibitor nitrogen is not protonated when bound to the enzyme. Analysis of pH inhibition data confirmed that 5 binds as the amine to the enzyme's unprotonated dicarboxylate form. This is a radically different binding mode than has been observed with isofagomine and other iminosugars in the literature.

Iminosugar-based galactoside mimics as inhibitors of galactocerebrosidase: SAR studies and comparison with other lysosomal galactosidases.

Several families of iminosugar-based galactoside mimics were designed, synthesized, and evaluated as galactocerebrosidase (GALC) inhibitors. They were also tested as inhibitors of lysosomal ß- and α-galactosidases in order to find new potent and selective pharmacological chaperones for treatment of the lysosomal storage disorder, Krabbe disease. Whereas 1-C-alkyl imino-L-arabinitols are totally inactive toward the three enzymes, 1-C-alkyl imino-D-galactitols were found to be active only toward α-galactosidase A. Finally, 1-N-iminosugars provided the best results, as 4-epi-isofagomine was found to be a good inhibitor of both lysosomal ß-galactosidase and GALC. Further elaboration of this structure is required to achieve selectivity between these two galactosidases.

Glucocerebrosidase enhancers for selected Gaucher disease genotypes by modification of α-1-C-substituted imino-D-xylitols (DIXs) by click chemistry.

A series of hybrid analogues was designed by combination of the iminoxylitol scaffold of parent 1C9-DIX with triazolylalkyl side chains. The resulting compounds were considered potential pharmacological chaperones in Gaucher disease. The DIX analogues reported here were synthesized by CuAAC click chemistry from scaffold 1 (α-1-C-propargyl-1,5-dideoxy-1,5-imino-D-xylitol) and screened as imiglucerase inhibitors. A set of selected compounds were tested as ß-glucocerebrosidase (GBA1) enhancers in fibroblasts from Gaucher patients bearing different genotypes. A number of these DIX compounds were revealed as potent GBA1 enhancers in genotypes containing the G202R mutation, particularly compound DIX-28 (α-1-C-[(1-(3-trimethylsilyl)propyl)-1H-1,2,3-triazol-4-yl)methyl]-1,5-dideoxy-1,5-imino-D-xylitol), bearing the 3-trimethylsilylpropyl group as a new surrogate of a long alkyl chain, with approximately threefold activity enhancement at 10 nM. Despite their structural similarities with isofagomine and with our previously reported aminocyclitols, the present DIX compounds behaved as non-competitive inhibitors, with the exception of the mixed-type inhibitor DIX-28.

Chemical synthetic biology.

Although both the most popular form of synthetic biology (SB) and chemical synthetic biology (CSB) share the biotechnologically useful aim of making new forms of life, SB does so by using genetic manipulation of extant microorganism, while CSB utilises classic chemical procedures in order to obtain biological structures which are non-existent in nature. The main query concerning CSB is the philosophical question: why did nature do this, and not that? The idea then is to synthesise alternative structures in order to understand why nature operated in such a particular way. We briefly present here some various examples of CSB, including those cases of nucleic acids synthesised with pyranose instead of ribose, and proteins with a reduced alphabet of amino acids; also we report the developing research on the "never born proteins" (NBP) and "never born RNA" (NBRNA), up to the minimal cell project, where the issue is the preparation of semi-synthetic cells that can perform the basic functions of biological cells.

Docking and SAR studies of calystegines: binding orientation and influence on pharmacological chaperone effects for Gaucher's disease.

We report on the identification of the required configuration and binding orientation of nor-tropane alkaloid calystegines against ß-glucocerebrosidase. Calystegine B2 is a potent competitive inhibitor of human lysosomal ß-glucocerebrosidase with Ki value of 3.3 µM. A molecular docking study revealed that calystegine B2 had a favorable van der Waals interactions (Phe128, Trp179, and Phe246) and the hydrogen bonding (Glu235, Glu340, Asp127, Trp179, Asn234, Trp381 and Asn396) was similar to that of isofagomine. All calystegine isomers bound into the same active site as calystegine B2 and the essential hydrogen bonds formed to Asp127, Glu235 and Glu340 were maintained. However, their binding orientations were obviously different. Calystegine A3 bound to ß-glucocerebrosidase with the same orientations as calystegine B2 (Type 1), while calystegine B3 and B4 had different binding orientations (Type 2). It is noteworthy that Type 1 orientated calystegines B2 and A3 effectively stabilized ß-glucocerebrosidase, and consequently increased intracellular ß-glucocerebrosidase activities in N370S fibroblasts, while Type 2 orientated calystegines B3 and B4 could not keep the enzyme activity. These results clearly indicate that the binding orientations of calystegines are changed by the configuration of the hydroxyl groups on the nor-tropane ring and the suitable binding orientation is a requirement for achieving a strong affinity to ß-glucocerebrosidase.

The presence of D-fagomine in the human diet from buckwheat-based foodstuffs.

Buckwheat (Fagopyrum esculentum Moench) groats contain the iminosugar D-fagomine as a minor component that might contribute to the alleged health benefits of this pseudo-cereal. This study presents analysis of D-fagomine in buckwheat-based foodstuffs by liquid chromatography coupled to mass spectrometry and an estimation of its presence in the human diet based on a published population-based cross-sectional nutrition survey. D-fagomine is present in common buckwheat-based foodstuffs in amounts ranging from 1 to 25 mg/kg or mg/L, it is stable during boiling, baking, frying and fermentation, and it is biosynthesised upon sprouting. The estimated total intake of D-fagomine resulting from a diet that includes such foodstuffs would be between 3 and 17 mg per day (mean for both genders; range from P5 to P95). A diet rich in buckwheat products would provide a daily amount of D-fagomine that may in part explain the beneficial properties traditionally attributed to buckwheat consumption.

Effects of pH and iminosugar pharmacological chaperones on lysosomal glycosidase structure and stability.

Human lysosomal enzymes acid-beta-glucosidase (GCase) and acid-alpha-galactosidase (alpha-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and alpha-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using alpha-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of alpha-Gal A with DGJ. Both GCase and alpha-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase-IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in alpha-Gal A are not seen. Thermodynamic parameters obtained from alpha-Gal A unfolding indicate two-state, van't Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and alpha-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological chaperones for lysosomal storage disorders.

Isofagomine induced stabilization of glucocerebrosidase.

Structurally destabilizing mutations in acid beta-glucosidase (GCase) can result in Gaucher disease (GD). The iminosugar isofagomine (IFG), a competitive inhibitor and a potential pharmacological chaperone of GCase, is currently undergoing clinical evaluation for the treatment of GD. An X-ray crystallographic study of the GCase-IFG complex revealed a hydrogen bonding network between IFG and certain active site residues. It was suggested that this network may translate into greater global stability. Here it is demonstrated that IFG does increase the global stability of wild-type GCase, shifting its melting curve by approximately 15 degrees C and that it enhances mutant GCase activity in pre-treated N370S/N370S and F213I/L444P patient fibroblasts. Additionally, amide hydrogen/deuterium exchange mass spectroscopy (H/D-Ex) was employed to identify regions within GCase that undergo stabilization upon IFG-binding. H/D-Ex data indicate that the binding of IFG not only restricts the local protein dynamics of the active site, but also propagates this effect into surrounding regions.

Compartmentation of lactate originating from glycogen and glucose in cultured astrocytes.

Brain glycogen metabolism was investigated by employing isofagomine, an inhibitor of glycogen phosphorylase. Cultured cerebellar and neocortical astrocytes were incubated in medium containing [U-(13C)]glucose in the absence or presence of isofagomine and the amounts and percent labeling of intra- and extracellular metabolites were determined by mass spectrometry (MS). The percent labeling in glycogen was markedly decreased in the presence of isofagomine. Surprisingly, the percent labeling of intracellular lactate was also decreased demonstrating the importance of glycogen turnover. The decrease was limited to the percent labeling in the intracellular pool of lactate, which was considerably lower compared to that observed in the medium in which it was close to 100%. These findings indicate compartmentation of lactate derived from glycogenolysis and that derived from glycolysis. Inhibiting glycogen degradation had no effect on the percent labeling in citrate. However, the percent labeling of extracellular glutamine was slightly decreased in neocortical astrocytes exposed to isofagomine, indicating an importance of glycogen turnover in the synthesis of releasable glutamine. In conclusion, the results demonstrate that glycogen in cultured astrocytes is continuously synthesized and degraded. Moreover, it was found that lactate originating from glycogen is compartmentalized from that derived from glucose, which lends further support to a compartmentalized metabolism in astrocytes.

Expression of nitric oxide synthase isoforms and nitrotyrosine formation after hypoxia-ischemia in the neonatal rat brain.

BACKGROUND AND PURPOSE: Production of nitric oxide is thought to play an important role in neuroinflammation. Previously, we have shown that combined inhibition of neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) can reduce hypoxia-ischemia-induced brain injury in 12-day-old rats. The aim of this study was to analyze changes in expression of nNOS, iNOS and endothelial NOS (eNOS), and nitrotyrosine (NT) formation in proteins in neonatal rats up to 48 h after cerebral hypoxia-ischemia. METHODS: Twelve-day-old rats were subjected to unilateral carotid artery occlusion and hypoxia, resulting in unilateral cerebral damage. NOS and nitrotyrosine expression were determined by immunohistochemistry and Western blot analysis at 30 min-48 h after hypoxia-ischemia. RESULTS: nNOS was increased in both hemispheres from 30 min to 3 h after hypoxia-ischemia. In the contralateral hemisphere, eNOS was decreased 1-3 h after hypoxia-ischemia. In the ipsilateral hemisphere, eNOS was decreased at 0.5 h after hypoxia-ischemia, normalized at 1-3 h and was increased 6-12 h after hypoxia-ischemia. At 24 and 48 h after hypoxia-ischemia, eNOS levels normalized. Surprisingly, iNOS expression did not change from 30 min up to 48 h after hypoxia-ischemia in the ipsi- or contralateral hemisphere. In addition, the regional expression of iNOS in the brain as determined by immunohistochemistry did not change after hypoxia-ischemia. Expression of nitrotyrosine was slightly increased in both hemispheres only at 30 min after hypoxia-ischemia. CONCLUSION: In 12-day-old rat pups, cerebral hypoxia-ischemia induced a transient increase in nNOS, eNOS, and nitrotyrosine in proteins, but no change in iNOS expression up to 48 h after the insult.