Design, synthesis and glycosidase inhibition of DAB derivatives with C-4 peptide and dipeptide branches.

A series of DAB-peptide and DAB-dipeptide derivatives were synthesized from D-tartrate-derived nitrone 18. The DAB peptides 16 are derivatives of trans,trans-3,4-dihydroxy-L-proline. Glycosidase inhibition assay found four of them to be weak and selective bovine liver ß-galactosidase inhibitors, and the C-2' methyl substituted compound 23b showed the most potent ß-galactosidase inhibition (IC50 = 0.66 µM). Molecular docking studies revealed different docking modes of compound 23b compared to those of other DAB-peptides, and partial similarity of compound 23b to DGJ.

Design and synthesis of iso-allo-DNJ and L-isoDALDP derivatives: pursuit of potent and selective inhibitors of α-glucosidase.

A series of iso-allo-DNJ and L-isoDALDP derivatives were synthesized from dithioacetal 16 with sequential and highly diastereoselective Ho and Henry reactions, and aziridinium intermediate-mediated ring rearrangement as key steps. Glycosidase inhibition assay found four of them as selective α-glucosidase inhibitors, and the less substituted compound 30 showed more potent α-glucosidase inhibition (IC50 = 9.3 µM) than the others. Molecular docking study revealed different docking modes of the iso-allo-DNJ and L-isoDALDP derivatives from their parent compounds, and also the similarity of compound 30 to isofagomine.

Production of d-aldotetrose from l-erythrulose using various isomerases.

d-Aldotetroses are rare sugars that are obtained via chemical synthesis in low yield. In this study, we demonstrated that d-aldotetroses could be produced using 3 isomerases. First, l-erythrulose was epimerized using d-tagatose 3-epimerase from Pseudomonas cichorii ST-24. The specific optical rotation of the reaction solution gradually decreased to zero, indicating that approximately 50% of the l-erythrulose was converted to d-erythrulose. d, l-Erythrulose mixture was isomerized with d-arabinose isomerase from Klebsiella pneumoniae 40bXX to produce d-threose, resulting in a conversion rate of 9.35%. d-Erythrose production using l-rhamnose isomerase from Pseudomonas stutzeri LL172 resulted in a conversion rate of 12.9%. Because of the low purity of the purchased d-erythrose, the product was reduced by the Raney nickel catalyst compared with authentic erythritol. We confirmed the products using HPLC and 13C-NMR spectra. This is the first report of d-aldotetrose production using an enzymatic reaction.

Diastereoselective Synthesis, Glycosidase Inhibition, and Docking Study of C-7-Fluorinated Casuarine and Australine Derivatives.

C-7-fluorinated derivatives of two important polyhydroxylated pyrrolizidines, casuarine and australine, were synthesized with organocatalytic stereoselective α-fluorination of aldehydes as the key step. The strategy is extensively applicable to some synthetically challenging fluorinated iminosugars and carbohydrates. The docking studies indicated that the potent inhibitions of trehalase and amyloglucosidase by the fluorinated polyhydroxylated pyrrolizidines are due to the interaction modes dominated by fluorine atoms in these iminosugars with the amino acids' residues of the corresponding enzymes. Steady interactions were established between the C-7 fluoride and a hydrophobic pocket in amyloglucosidase by untypical anion-π interactions. These unexpected docking modes and related structure-activity relationship studies emphasize the value of fluorination in the design of polyhydroxylated pyrrolizidine glycosidase inhibitors.

Stable D-xylose ditriflate in divergent syntheses of dihydroxy prolines, pyrrolidines, tetrahydrofuran-2-carboxylic acids, and cyclic ß-amino acids.

Double nucleophilic displacement of D-xylo-ditriflate by amines, water and alkyl cyanoacetates, respectively, gave a series of bicyclic divergent intermediates for the synthesis of a wide range of highly functionalized targets, including hydroxylated prolines, pyrrolidines, furanoic acids, and cyclopentanes.

Introduction of C-alkyl branches to L-iminosugars changes their active site binding orientation.

L-ido-Deoxynojirimycin (L-ido-DNJ) itself showed no affinity for human lysosomal acid α-glucosidase (GAA), whereas 5-C-methyl-L-ido-DNJ showed a strong affinity for GAA, comparable to the glucose analog DNJ, with a Ki value of 0.060 µM. This excellent affinity for GAA and enzyme stabilization was observed only when methyl and ethyl groups were introduced. Docking simulation analysis revealed that the alkyl chains of 5-C-alkyl-L-ido-DNJs were stored in three different pockets, depending on their length, thereby the molecular orientation was changed. Comparison of the binding poses of DNJ and 5-C-methyl-L-ido-DNJ showed that they formed a common ionic interaction with Asp404, Asp518, and Asp616, but both the binding orientation and the distance between the ligand and each amino acid residue were different. 5-C-Methyl-L-ido-DNJ dose-dependently increased intracellular GAA activity in Pompe patient fibroblasts with the M519V mutation and also promoted enzyme transport to lysosomes. This study provides the first example of a strategy to design high-affinity ligands by introducing alkyl branches into rare sugars and L-sugar-type iminosugars to change the orientation of binding.

Iminosugar Amino Acid idoBR1 Reduces Inflammatory Responses in Microglia.

(1) Background. Inflammation is reported to be a key factor in neurodegeneration. The microglia are immune cells present in the central nervous system; their activation results in the release of inflammatory cytokines and is thought to be related to aging and neurodegenerative disorders, such as Alzheimer's disease. (2) Methods. A mouse BV-2 microglia cell line was activated using LPS and the anti-inflammatory cucumber-derived iminosugar amino acid idoBR1, (2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid, was used alongside dexamethasone as the control to determine whether it could reduce the inflammatory responses. (3) Results. A dose-dependent reduction in the LPS-induced production of the proinflammatory factors TNFα, IL-6, and nitric oxide and the transcription factor NF-κB was found. (4) Conclusions. Further investigations of the anti-inflammatory effects of idoBR1 in other models of neurodegenerative diseases are warranted.

Stereocomplementary synthesis of casuarine and its 6-epi-, 7-epi-, and 6,7-diepi-stereoisomers.

Four diastereomers belonging to the family of casuarines, including casuarine (1), 6-epi-casuarine (2), 7-epi-casuarine (13) and 6,7-diepi-casuarine (14), have been synthesized from D-arabinose-derived cyclic nitrone 7 and nitrone-derived aldehyde 4 by a stereocomplementary strategy. Glycosidase inhibition comparison showed that 6-epi-casuarine (2) exhibits enhanced inhibition of trehalase (IC50 = 9.7 µM) and 6,7-diepi-casuarine (14) leads to specific inhibition of trehalase.

Synthesis and glycosidase inhibition of N-substituted derivatives of 1,4-dideoxy-1,4-imino-d-mannitol (DIM).

N-Substituted derivatives of 1,4-dideoxy-1,4-imino-d-mannitol (DIM), the pyrrolidine core of swainsonine, have been synthesized efficiently and stereoselectively from d-mannose with 2,3:5,6-di-O-isopropylidene DIM (10) as a key intermediate. These N-substituted derivatives include N-alkylated, N-alkenylated, N-hydroxyalkylated and N-aralkylated DIMs with the carbon number of the alkyl chain ranging from one to nine. The obtained 33 N-substituted DIM derivatives were assayed against various glycosidases, which allowed a systematic evaluation of their glycosidase inhibition profiles. Though N-substitution of DIM decreased their α-mannosidase inhibitory activities, some of the derivatives showed significant inhibition of other glycosidases.

Synthesis of Pyrrolidine Monocyclic Analogues of Pochonicine and Its Stereoisomers: Pursuit ofSimplified Structures and Potent ß-N-Acetylhexosaminidase Inhibition.

Ten pairs of pyrrolidine analogues of pochonicine and its stereoisomers have been synthesized from four enantiomeric pairs of polyhydroxylated cyclic nitrones. Among the ten N-acetylamino pyrrolidine analogues, only compounds with 2,5-dideoxy-2,5-imino-d-mannitol (DMDP) and pochonicine (1) configurations showed potent inhibition of ß-N-acetylhexosaminidases (ß-HexNAcases); while 1-amino analogues lost almost all their inhibitions towards the tested enzymes. The assay results reveal the importance of the N-acetylamino group and the possible right configurations of pyrrolidine ring required for this type of inhibitors.

Strategy for Designing Selective Lysosomal Acid α-Glucosidase Inhibitors: Binding Orientation and Influence on Selectivity.

Deoxynojirimycin (DNJ) is the archetypal iminosugar, in which the configuration of the hydroxyl groups in the piperidine ring truly mimic those of d-glucopyranose; DNJ and derivatives have beneficial effects as therapeutic agents, such as anti-diabetic and antiviral agents, and pharmacological chaperones for genetic disorders, because they have been shown to inhibit α-glucosidases from various sources. However, attempts to design a better molecule based solely on structural similarity cannot produce selectivity between α-glucosidases that are localized in multiple organs and tissues, because the differences of each sugar-recognition site are very subtle. In this study, we provide the first example of a design strategy for selective lysosomal acid α-glucosidase (GAA) inhibitors focusing on the alkyl chain storage site. Our design of α-1-C-heptyl-1,4-dideoxy-1,4-imino-l-arabinitol (LAB) produced a potent inhibitor of the GAA, with an IC50 value of 0.44 µM. It displayed a remarkable selectivity toward GAA (selectivity index value of 168.2). A molecular dynamic simulation study revealed that the ligand-binding conformation stability gradually improved with increasing length of the α-1-C-alkyl chain. It is noteworthy that α-1-C-heptyl-LAB formed clearly different interactions from DNJ and had favored hydrophobic interactions with Trp481, Phe525, and Met519 at the alkyl chain storage pocket of GAA. Moreover, a molecular docking study revealed that endoplasmic reticulum (ER) α-glucosidase II does not have enough space to accommodate these alkyl chains. Therefore, the design strategy focusing on the shape and acceptability of long alkyl chain at each α-glucosidase may lead to the creation of more selective and practically useful inhibitors.

Isolation from Stevia rebaudiana of DMDP acetic acid, a novel iminosugar amino acid: synthesis and glycosidase inhibition profile of glycine and ß-alanine pyrrolidine amino acids.

DMDP acetic acid [N-carboxymethyl-2,5-dideoxy-2,5-imino-D-mannitol] 5 from Stevia rebaudiana is the first isolated natural amino acid derived from iminosugars bearing an N-alkyl acid side chain; it is clear from GCMS studies that such derivatives with acetic and propionic acids are common in a broad range of plants including mulberry, Baphia, and English bluebells, but that they are very difficult to purify. Reaction of unprotected pyrrolidine iminosugars with aqueous glyoxal gives the corresponding N-acetic acids in very high yield; Michael addition of both pyrrolidine and piperidine iminosugars and that of polyhydroxylated prolines to tert-butyl acrylate give the corresponding N-propionic acids in which the amino group of ß-alanine is incorporated into the heterocyclic ring. These easy syntheses allow the identification of this new class of amino acid in plant extracts and provide pure samples for biological evaluation. DMDP N-acetic and propionic acids are potent α-galactosidase inhibitors in contrast to potent ß-galactosidase inhibition by DMDP.

Growth inhibition by 1-deoxy-d-allulose, a novel bioactive deoxy sugar, screened using Caenorhabditis elegans assay.

The biological activities of deoxy sugars (deoxy monosaccharides) have remained largely unstudied until recently. We compared the growth inhibition by all 1-deoxyketohexoses using the animal model Caenorhabditis elegans. Among the eight stereoisomers, 1-deoxy-d-allulose (1d-d-Alu) showed particularly strong growth inhibition. The 50% inhibition of growth (GI50) concentration by 1d-d-Alu was estimated to be 5.4 mM, which is approximately 10 times lower than that of d-allulose (52.7 mM), and even lower than that of the potent glycolytic inhibitor, 2-deoxy-d-glucose (19.5 mM), implying that 1d-d-Alu has a strong growth inhibition. In contrast, 5-deoxy- and 6-deoxy-d-allulose showed no growth inhibition of C. elegans. The inhibition by 1d-d-Alu was alleviated by the addition of d-ribose or d-fructose. Our findings suggest that 1d-d-Alu-mediated growth inhibition could be induced by the imbalance in d-ribose metabolism. To our knowledge, this is the first report of biological activity of 1d-d-Alu which may be considered as an antimetabolite drug candidate.

Ginnalin B induces differentiation markers and modulates the proliferation/differentiation balance via the upregulation of NOTCH1 in human epidermal keratinocytes.

The red maple and sugar maple (Acer rubrum and A. saccharum, respectively) contain acertannins (ginnalins and maplexins), galloylated derivatives of 1,5-anhydro-d-glucitol (1,5-AG, 1). These compounds have a variety of potential medicinal properties and we have shown that some of them promote the expression of ceramide synthase 3. We now report on the beneficial effects of ginnalin B, (6-O-galloyl-1,5-AG, 5), leading to acceleration of skin metabolism and reduction of the turnover time. Ginnalin B dose-dependently increased the relative amount of keratin 10, keratin 1, and filaggrin gene, with maximal increase of 1.7-, 2.9, and 5.2-fold at 100 µM, respectively. The validation study showed that it had superior capacity to induce multiple stages of keratinocyte differentiation and significantly elevated the immunostaining site of keratin 10 and filaggrin in a 3-dimensional cultured human skin model, by 1.2 and 2.8-fold, respectively. Furthermore, ginnalin B caused the arrest of proliferation at the G0/G1 phase but it did not induce apoptotic cell death in normal human keratinocytes. Molecular studies revealed that ginnalin B up-regulated the levels of NOTCH1 and a concomitant increase p21 expression. Ginnalin B, therefore, represents a new class of promising functional and medical cosmetic compound and it could contribute to the maintenance of homeostasis of the epidermis.

d-Idose, d-Iduronic Acid, and d-Idonic Acid from d-Glucose via Seven-Carbon Sugars.

A practical synthesis of the very rare sugar d-idose and the stable building blocks for d-idose, d-iduronic, and d-idonic acids from ido-heptonic acid requires only isopropylidene protection, Shing silica gel-supported periodate cleavage of the C6-C7 bond of the heptonic acid, and selective reduction of C1 and/or C6. d-Idose is the most unstable of all the aldohexoses and a stable precursor which be stored and then converted under very mild conditions into d-idose is easily prepared.

Synthesis and Glycosidase Inhibition of Broussonetine M and Its Analogues.

Cross-metathesis (CM) and Keck asymmetric allylation, which allows access to defined stereochemistry of a remote side chain hydroxyl group, are the key steps in a versatile synthesis of broussonetine M (3) from the d-arabinose-derived cyclic nitrone 14. By a similar strategy, ent-broussonetine M (ent-3) and six other stereoisomers have been synthesized, respectively, starting from l-arabino-nitrone (ent-14), l-lyxo-nitrone (ent-3-epi-14), and l-xylo-nitrone (2-epi-14) in five steps, in 26%-31% overall yield. The natural product broussonetine M (3) and 10'-epi-3 were potent inhibitors of ß-glucosidase (IC50 = 6.3 µM and 0.8 µM, respectively) and ß-galactosidase (IC50 = 2.3 µM and 0.2 µM, respectively); while their enantiomers, ent-3 and ent-10'-epi-3, were selective and potent inhibitors of rice α-glucosidase (IC50 = 1.2 µM and 1.3 µM, respectively) and rat intestinal maltase (IC50 = 0.29 µM and 18 µM, respectively). Both the configuration of the polyhydroxylated pyrrolidine ring and C-10' hydroxyl on the alkyl side chain affect the specificity and potency of glycosidase inhibition.

In silico analyses of essential interactions of iminosugars with the Hex A active site and evaluation of their pharmacological chaperone effects for Tay-Sachs disease.

The affinity of a series of iminosugar-based inhibitors exhibiting various ring sizes toward Hex A and their essential interactions with the enzyme active site were investigated. All the Hex A-inhibiting iminosugars tested formed hydrogen bonds with Arg178, Asp322, Tyr421 and Glu462 and had the favorable cation-π interaction with Trp460. Among them, DMDP amide (6) proved to be the most potent competitive inhibitor with a Ki value of 0.041 µM. We analyzed the dynamic properties of both DMDP amide (6) and DNJNAc (1) in aqueous solution using molecular dynamics (MD) calculations; the distance of the interaction between Asp322 and 3-OH and Glu323 and 6-OH was important for stable interactions with Hex A, reducing fluctuations in the plasticity of the active site. DMDP amide (6) dose-dependently increased intracellular Hex A activity in the G269S mutant cells and restored Hex A activity up to approximately 43% of the wild type level; this effect clearly exceeded the border line treatment for Tay-Sachs disease, which is regarded as 10-15% of the wild type level. This is a significantly greater effect than that of pyrimethamine, which is currently in Phase 2 clinical trials. DMDP amide (6), therefore, represents a new promising pharmacological chaperone candidate for the treatment of Tay-Sachs disease.

Strategy for designing selective α-l-rhamnosidase inhibitors: Synthesis and biological evaluation of l-DMDP cyclic isothioureas.

This study shows that the cyclization of l-DMDP thioureas to bicyclic l-DMDP isothioureas improved α-l-rhamnosidase inhibition which was further enhanced by increasing the length of the alkyl chain. The addition of a long alkyl chain, such as decyl or dodecyl, to the nitrogen led to the production of highly potent inhibitors of α-l-rhamnosidase; it also caused broad inhibition spectrum against ß-glucosidase and ß-galactosidase. In contrast, the corresponding N-benzyl-l-DMDP cyclic isothioureas display selective inhibition of α-l-rhamnosidase; 3',4'-dichlorobenzyl-l-DMDP cyclic isothiourea (3r) was found to display the most potent and selective inhibition of α-l-rhamnosidase, with IC50 value of 0.22µM, about 46-fold better than the positive control 5-epi-deoxyrhamnojirimycin (5-epi-DRJ; IC50=10µM) and occupied the active-site of this enzyme (Ki=0.11µM). Bicyclic isothioureas of ido-l-DMDP did not inhibit α-l-rhamnosidase. These new mimics of l-rhamnose may affect other enzymes associated with the biochemistry of rhamnose including enzymes involved in progression of tuberculosis.

6-Deoxyhexoses from l-Rhamnose in the Search for Inducers of the Rhamnose Operon: Synergy of Chemistry and Biotechnology.

In the search for alternative non-metabolizable inducers in the l-rhamnose promoter system, the synthesis of fifteen 6-deoxyhexoses from l-rhamnose demonstrates the value of synergy between biotechnology and chemistry. The readily available 2,3-acetonide of rhamnonolactone allows inversion of configuration at C4 and/or C5 of rhamnose to give 6-deoxy-d-allose, 6-deoxy-d-gulose and 6-deoxy-l-talose. Highly crystalline 3,5-benzylidene rhamnonolactone gives easy access to l-quinovose (6-deoxy-l-glucose), l-olivose and rhamnose analogue with C2 azido, amino and acetamido substituents. Electrophilic fluorination of rhamnal gives a mixture of 2-deoxy-2-fluoro-l-rhamnose and 2-deoxy-2-fluoro-l-quinovose. Biotechnology provides access to 6-deoxy-l-altrose and 1-deoxy-l-fructose.

Concise synthesis of calystegines B2 and B3via intramolecular Nozaki-Hiyama-Kishi reaction.

The key step in the concise syntheses of calystegine B2 and its C-2 epimer calystegine B3 was the construction of cycloheptanone 8via an intramolecular Nozaki-Hiyama-Kishi (NHK) reaction of 9, an aldehyde containing a Z-vinyl iodide. Vinyl iodide 9 was obtained by the Stork olefination of aldehyde 10, derived from carbohydrate starting materials. Calystegines B2 (3) and B3 (4) were synthesized from d-xylose and l-arabinose derivatives respectively in 11 steps in excellent overall yields (27% and 19%).