Synthesis of the aminocyclopentenediol fragment of queuosine by way of the stereoselective addition of an organometallic reagent to a N-t-butanesulfinyl glycosylamine.

An innovative, concise synthesis of the aminocyclopentenediol fragment of queuosine is reported. The synthesis is based on the stereocontrolled addition of a vinylGrignard·LiCl reagent to a t-butanesulfinyl L-ribofuranosylamine, followed by dehydrodeoxygenation to generate a second vinyl group and ring-closing metathesis to form the five-membered ring scaffold of the natural product. This approach has the potential for the development of a larger scale synthesis.

Intramolecular Palladium-Catalyzed Carboamination for the Stereoselective Synthesis of Five-Membered Iminosugar C-Glycosides.

We report here a new method for the stereoselective synthesis of five-membered iminosugar C-glycosides using an intramolecular palladium-catalyzed carboamination. We have prepared efficiently two sugar-derived aminoalkenes, which were submitted to the carboamination conditions in the presence of different aryl bromides. A small library of protected iminosugars carrying a 1-C-arylmethyl substituent was obtained, and some of them were fully deprotected to yield original iminosugar C-glycosides. This methodology provides one of the shortest pathways to this family of molecules.

Tunable Approach to C-Linked Analogs of Glycosamines.

The synthesis of (1R)-2-amino-2-deoxy-ß-l-gulopyranosyl benzene and the α and ß forms of 2-amino-2-deoxy-l-idopyranosyl benzene derivatives was accomplished through stereospecific addition of tributylstannyllithium to readily available (SR)- or (SS)-N-tert-butanesulfinyl-arabinofuranosylamine building blocks, followed by stereoretentive Pd-catalyzed Migita-Kosugi-Stille cross-coupling, stereoselective reduction, and an activation-cyclization strategy. Application of this methodology paves the way to new three-dimensional chemical space and preparation of unknown (non-natural) and complex 2-amino-2-deoxy sugars of biological interest.

Hydrozirconation/bromination, followed by a Michaelis-Arbuzov reaction, as a convenient approach towards polyfunctional glycosylphosphonates.

In this note, an hydrozirconation/bromination/Michaelis-Arbuzov sequence was developped to introduce a trimethylene phosphonate unit on ketopyranosides. Performed on polyfunctional substrates bearing orthogonal protecting groups, this new approach provided a straightforward entry towards a large diversity of glycophosphomimetics having a quaternary anomeric position.

A practical approach to Dideoxy-1,4- and 1,5-iminopentitols from protected sugar hemiacetals.

The convenient and straightforward preparation of dideoxy-1,4- and 1,5-iminopentitol derivatives from protected sugar hemiacetals by way of N-tert-butanesulfinyl glycosylamines and open-chain aminoalditols is reported. The synthetic procedure is a method of choice for the making of these important scaffolds of biological interest.

4-epi-Isofagomine derivatives as pharmacological chaperones for the treatment of lysosomal diseases linked to ß-galactosidase mutations: Improved synthesis and biological investigations.

(5aR)-5a-C-pentyl-4-epi-isofagomine 1 is a powerful inhibitor of lysosomal ß-galactosidase and a remarkable chaperone for mutations associated with GM1-gangliosidosis and Morquio disease type B. We report herein an improved synthesis of this compound and analogs (5a-C-methyl, pentyl, nonyl and phenylethyl derivatives), and a crystal structure of a synthetic intermediate that confirms its configuration resulting from the addition of a Grignard reagent. These compounds were evaluated as glycosidase inhibitors and their potential as chaperones for mutant lysosomal galactosidases determined. Based on these results and on docking studies, the 5-C-pentyl derivative 1 was selected as the optimal structure for further investigations: this compound induces the maturation of mutated ß-galactosidase in fibroblasts of a GM1-gangliosidosis patient and promote the decrease of keratan sulfate and oligosaccharide load in patient cells. Compound 1 is clearly capable of restoring ß-galactosidase activity and of promoting maturation of the protein, which should result in significant clinical benefit. These properties strongly support the development of compound 1 for the treatment of GM1-gangliosidosis and Morquio disease type B patients harboring ß-galactosidase mutations sensitive to pharmacological chaperoning.

Promoting the clearance of neurotoxic proteins in neurodegenerative disorders of ageing.

Neurodegenerative disorders of ageing (NDAs) such as Alzheimer disease, Parkinson disease, frontotemporal dementia, Huntington disease and amyotrophic lateral sclerosis represent a major socio-economic challenge in view of their high prevalence yet poor treatment. They are often called 'proteinopathies' owing to the presence of misfolded and aggregated proteins that lose their physiological roles and acquire neurotoxic properties. One reason underlying the accumulation and spread of oligomeric forms of neurotoxic proteins is insufficient clearance by the autophagic-lysosomal network. Several other clearance pathways are also compromised in NDAs: chaperone-mediated autophagy, the ubiquitin-proteasome system, extracellular clearance by proteases and extrusion into the circulation via the blood-brain barrier and glymphatic system. This article focuses on emerging mechanisms for promoting the clearance of neurotoxic proteins, a strategy that may curtail the onset and slow the progression of NDAs.

Glycoside Mimics from Glycosylamines: Recent Progress.

Glycosylamines are valuable sugar derivatives that have attracted much attention as synthetic intermediates en route to iminosugar-C-glycosyl compounds. Iminosugars are among the most important glycomimetics reported to date due to their powerful activities as inhibitors of a wide variety of glycosidases and glycosyltransferases, as well as for their use as pharmacological chaperones. As they provide ready access to these important glycoside mimics, we have reviewed the most significant glycosylamine-based methodologies developed to date, with a special emphasis on the literature reported after 2006. The groups of substrates covered include N-alkyl- and N-benzyl-glycosylamines, N-glycosylhydroxylamines, N-(alkoxycarbonyl)-, and N-tert-butanesulfinyl-glycosylamines.

1-C-phosphonomethyl- and 1-C-difluorophosphonomethyl-1,4-imino-l-arabinitols as Galf transferase inhibitors: A comparison.

The convenient preparation of iminopentitol derivatives, based on a 1,4-dideoxy-1,4-imino-l-arabinitol scaffold carrying ß-phosphono(difluoromethyl) or ß-phosphonomethyl appendages, as Galf-1P mimics, is reported. The compounds were tested for their ability to inhibit GlfT2, a vital galactofuranosyltransferase involved in the cell wall biosynthesis of mycobacteria. Interestingly, the Galf-1P mimics lacking a fluorine atom (7 and 8) were very poor inhibitors, showing less than 20% inhibition of GlfT2, whereas compounds 2 and 3, which contains a difluoromethylenephosphonate moiety were more potent inhibitors. Compound 3 that is fully deprotected was the most potent showing a significant IC50 value (0.9 mm), despite the absence of the diphosphate linkage present in the parent sugar nucleotide. This study paves the way to the synthesis of more complex ß-phosphonomethyl-imino-l-arabinitol derivatives as simplified mimics of UDP-α-d-Galf.

Total synthesis of pipecolic acid and 1-C-alkyl 1,5-iminopentitol derivatives by way of stereoselective aldol reactions from (S)-isoserinal.

A short synthesis of iminosugars and pipecolic acid derivatives has been realized through aldol addition of a pyruvate, a range of ketones and (S)-isoserinal, followed by catalytic reductive intramolecular amination. The stereoselective aldol reaction was achieved successfully by using tertiary amines or di-zinc aldol catalysts, thus constituting two parallel routes to optically pure products with good yields and high diastereoselectivities. These carbohydrate analogues may be the inhibitors of potent glycosidases and glycosyltransferases.

Tunable Approach for the Stereoselective Synthesis of 1-C-Diethylphosphono(difluoromethyl) Iminosugars as Glycosyl Phosphate Mimics.

An efficient methodology for the stereoselective and tunable addition of LiCF2P(O)(OEt)2 and BrMgCF2P(O)(OEt)2 reagents to N-t-butanesulfinyl glycosylamines is described with details on the stereochemical effects at play in this process. It provides a practical route to various 1-C-diethylphosphono(difluoromethyl) iminosugars as glycosyl phosphate and sugar nucleotide mimics.

A Specific Activity-Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease.

Galactosylceramidase (GALC) is the lysosomal ß-galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity-based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species. The probe consists of a ß-galactopyranose-configured cyclophellitol-epoxide core, conferring specificity for GALC, equipped with a BODIPY fluorophore at C6 that allows visualization of active enzyme in cells and tissues. Detection of residual GALC in patient fibroblasts holds great promise for laboratory diagnosis of Krabbe disease. We further describe a procedure for in situ imaging of active GALC in murine brain by intra-cerebroventricular infusion of the ABP. In conclusion, this GALC-specific ABP should find broad applications in diagnosis, drug development, and evaluation of therapy for Krabbe disease.

(5aR)-5a-C-Pentyl-4-epi-isofagomine: A powerful inhibitor of lysosomal ß-galactosidase and a remarkable chaperone for mutations associated with GM1-gangliosidosis and Morquio disease type B.

This report is about the identification, synthesis and initial biological characterization of derivatives of 4-epi-isofagomine as pharmacological chaperones (PC) for human lysosomal ß-galactosidase. The two epimers of 4-epi-isofagomine carrying a pentyl group at C-5a, namely (5aR)- and (5aS)-5a-C-pentyl-4-epi-isofagomine, were prepared by an innovative procedure involving in the key step the addition of nitrohexane to a keto-pentopyranoside. Both epimers were evaluated as inhibitors of the human ß-galactosidase: the (5aR)-stereoisomer (compound 1) was found to be a very potent inhibitor of the enzyme (IC50 = 8 nM, 30× more potent than 4-epi-isofagomine at pH 7.3) with a high selectivity for this glycosidase whereas the (5aS) epimer was a much weaker inhibitor. In addition, compound 1 showed a remarkable activity as a PC. It significantly enhanced the residual activity of mutant ß-galactosidase in 15 patient cell lines out of 23, with enhancement factors greater than 3.5 in 10 cell lines and activity restoration up to 91% of normal. Altogether, these results indicated that (5aR)-5a-C-pentyl-4-epi-isofagomine constitutes a promising PC-based drug candidate for the treatment of GM1-gangliosidosis and Morquio disease type B.

Effects of the Selected Iminosugar Derivatives on Pseudomonas aeruginosa Biofilm Formation.

A lack of an effective way to eliminate pathogenic bacteria hidden in the biofilm is a major problem in the treatment of chronic bacterial infections. Iminosugar derivatives are potential candidates for inhibitors of enzymes taking part in the biosynthesis of exopolysaccharides, which are forming bacterial biofilm. Investigated iminosugars were studied either at an early stage of biofilm formation or later on when the mature biofilm of Pseudomonas aeruginosa was already formed. A series of diverse iminosugar structures significantly inhibited biofilm formation, whereas they showed no influence on already formed biofilm. This indicates a possible mechanism of their action based on inhibition of exopolysaccharide backbone synthesis in the early stages of biofilm formation. Moreover, iminosugar derivatives did not show significant effect on the viable bacterial numbers in both early and mature biofilm forms. Importantly, they were not cytotoxic against human Caco-2 cells in vitro, which may be to their advantage in case of their medical application in preventing P. aeruginosa biofilm formation.

N-Alkyl-, 1-C-Alkyl-, and 5-C-Alkyl-1,5-dideoxy-1,5-imino-(L)-ribitols as Galactosidase Inhibitors.

A series of 1,5-dideoxy-1,5-imino-(l)-ribitol (DIR) derivatives carrying alkyl or functionalized alkyl groups were prepared and investigated as glycosidase inhibitors. These compounds were designed as simplified 4-epi-isofagomine (4-epi-IFG) mimics and were expected to behave as selective inhibitors of ß-galactosidases. All compounds were indeed found to be highly selective for ß-galactosidases versus α-glycosidases, as they generally did not inhibit coffee bean α-galactosidase or other α-glycosidases. Some compounds were also found to be inhibitors of almond ß-glucosidase. The N-alkyl DIR derivatives were only modest inhibitors of bovine ß-galactosidase, with IC50 values in the 30-700 µM range. Likewise, imino-L-ribitol substituted at the C1 position was found to be a weak inhibitor of this enzyme. In contrast, alkyl substitution at C5 resulted in enhanced ß-galactosidase inhibitory activity by a factor of up to 1000, with at least six carbon atoms in the alkyl substituent. Remarkably, the 'pseudo-anomeric' configuration in this series does not appear to play a role. Human lysosomal ß-galactosidase from leukocyte lysate was, however, poorly inhibited by all iminoribitol derivatives tested (IC50 values in the 100 µM range), while 4-epi-IFG was a good inhibitor of this enzyme. Two compounds were evaluated as pharmacological chaperones for a GM1-gangliosidosis cell line (R301Q mutation) and were found to enhance the mutant enzyme activity by factors up to 2.7-fold.

Iminosugars as a new class of cholinesterase inhibitors.

To further extend the scope of iminosugar biological activity, a systematic structure-activity relationship investigation has been performed by synthesizing and evaluating as cholinesterase inhibitors a library of twenty-three iminoalditols with different substitutions and stereochemistry patterns. These compounds have been evaluated in vitro for the inhibition of cholinesterases (different sources of acetylcholinesterase and butyrylcholinesterase). Some compounds have IC50 values in the micromolar range and display significant inhibition selectivity for butyrylcholinesterase over acetylcholinesterase. These are the first examples of iminosugar-based inhibitors of cholinesterases.

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.

Glucosylceramide mimics: highly potent GCase inhibitors and selective pharmacological chaperones for mutations associated with types†1 and 2 Gaucher disease.

A series of iminoxylitol derivatives carrying a C-linked di-O-acyl or di-O-alkyl glyceryl substituent were prepared and characterized. All of these compounds, which were designed as glucosylceramide (GlcCer) mimics, were nanomolar inhibitors of lysosomal ß-glucosidase (glucocerebrosidase, GCase). Two of these pseudoglycolipids were further evaluated for their ability to enhance the activity of mutant GCase in human Gaucher cells. Although the di-O-hexyl ether was surprisingly devoid of chaperoning activity on both N370S and L444P GCases, the di-O-decanoyl ester was a potent chaperone of the L444P hydrolase, capable of increasing the residual activity of the enzyme by a factor of two at a very low concentration (50 nM); such a significant effect on the L444P mutation in human fibroblasts has not yet been observed. In heat-stress studies, the diether was found to be much more effective in stabilizing the wild-type enzyme than the diester. Four representative pseudoglycolipids were also assayed as inhibitors of GlcCer synthase, because such compounds could find use in the substrate reduction therapy approach to treat lysosomal storage diseases, but these compounds revealed only moderate activity. As efficient pharmacological chaperones, new structures such as the di-C10 -ester constitute leads for the development of therapeutic agents for types 2 and 3 Gaucher disease, the most severe neuronopathic forms of this lysosomal disease.

A reinvestigation of the synthesis and revision of spectral data of 1,2-O-isopropylidene-α-l-sorbofuranose, 1,2:4,6-di-O-isopropylidene-α-L-sorbofuranose and derivatives.

Mono- and di-O-isopropylidene-l-sorbofuranose derivatives are important starting materials for the synthesis of modified sugars and useful chiral compounds. However, several inconsistencies in the spectral data of these compounds and erroneous structural assignments have been noted in the literature. The unambiguous synthesis of 1,2:4,6-di-O-isopropylidene-α-L-sorbofuranose and derivatives of 1,2- and 2,3-O-isopropylidene-α-L-sorbofuranoses has been achieved and definitive spectral data on these compounds are provided.

Synthesis and biological investigation of PIM mimics carrying biotin or a fluorescent label for cellular imaging.

Phosphatidyl inositol mannosides (PIMs) are constituents of the mycobacterial cell wall; these glycolipids are known to exhibit potent inhibitory activity toward the LPS-induced production of cytokines by macrophages, and therefore have potential as anti-inflammatory agents. Recently, heterocyclic analogues of PIMs in which the inositol is replaced by a piperidine (aza-PIM mimics) or a tetrahydropyran moiety (oxa-PIM mimics) have been prepared by short synthetic sequences and shown to retain the biological activity of the parent PIM structures. In this investigation, the aza-PIM analogue was used as a convenient scaffold to link biotin or a fluorescent label (tetramethyl-rhodamine) by way of an aminocaproyl spacer, with the goal of using these conjugates for intracellular localization and for the study of the mechanism of their antiinflammatory action. The synthesis of these compounds is reported, as well as the evaluation of their activities as inhibitors of LPS-induced cytokine production by macrophages (TNFα, IL12p40); preliminary investigations by FACS and confocal microscopy indicated that PIM-biotin conjugate binds to macrophage membranes with rapid kinetics.