Iminosugar idoBR1 Isolated from Cucumber Cucumis sativus Reduces Inflammatory Activity.

Cucumbers have been anecdotally claimed to have anti-inflammatory activity for a long time, but the active principle was not identified. idoBR1, (2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid, is an iminosugar amino acid isolated from fruits of certain cucumbers, Cucumis sativus (Cucurbitaceae). It has no chromophore and analytically behaves like an amino acid making detection and identification difficult. It has anti-inflammatory activity reducing lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α) in THP-1 cells and ex vivo human blood. It showed selective inhibition of human α-l-iduronidase and sialidases from both bacteria (Tannerella forsythia) and human THP-1 cells. idoBR1 and cucumber extract reduced the binding of hyaluronic acid (HA) to CD44 in LPS-stimulated THP-1 cells and may function as an anti-inflammatory agent by inhibiting induced sialidase involved in the production of functionally active HA adhesive CD44. Similar to the related iminosugars, idoBR1 is excreted unchanged in urine following consumption. Its importance in the diet should be further evaluated.

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.

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.

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.

Triacetonide of Glucoheptonic Acid in the Scalable Syntheses of d-Gulose, 6-Deoxy-d-gulose, l-Glucose, 6-Deoxy-l-glucose, and Related Sugars.

Ease of separation of petrol-soluble acetonides derived from the triacetonide of methyl glucoheptonate allows scalable syntheses of rare sugars containing the l-gluco or d-gulo structural motif with any oxidation level at the C6 or C1 position of the hexose, usually without chromatography: meso-d-glycero-d-guloheptitol available in two steps is an ideal entry point for the study of the biotechnological production of heptoses.

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.

Synthetic Chemical Inducers and Genetic Decoupling Enable Orthogonal Control of the rhaBAD Promoter.

External control of gene expression is crucial in synthetic biology and biotechnology research and applications, and is commonly achieved using inducible promoter systems. The E. coli rhamnose-inducible rhaBAD promoter has properties superior to more commonly used inducible expression systems, but is marred by transient expression caused by degradation of the native inducer, l-rhamnose. To address this problem, 35 analogues of l-rhamnose were screened for induction of the rhaBAD promoter, but no strong inducers were identified. In the native configuration, an inducer must bind and activate two transcriptional activators, RhaR and RhaS. Therefore, the expression system was reconfigured to decouple the rhaBAD promoter from the native rhaSR regulatory cascade so that candidate inducers need only activate the terminal transcription factor RhaS. Rescreening the 35 compounds using the modified rhaBAD expression system revealed several promising inducers. These were characterized further to determine the strength, kinetics, and concentration-dependence of induction; whether the inducer was used as a carbon source by E. coli; and the modality (distribution) of induction among populations of cells. l-Mannose was found to be the most useful orthogonal inducer, providing an even greater range of induction than the native inducer l-rhamnose, and crucially, allowing sustained induction instead of transient induction. These findings address the key limitation of the rhaBAD expression system and suggest it may now be the most suitable system for many applications.

3-Fluoroazetidinecarboxylic Acids and trans,trans-3,4-Difluoroproline as Peptide Scaffolds: Inhibition of Pancreatic Cancer Cell Growth by a Fluoroazetidine Iminosugar.

Reverse aldol opening renders amides of 3-hydroxyazetidinecarboxylic acids (3-OH-Aze) unstable above pH 8. Aze, found in sugar beet, is mis-incorporated for proline in peptides in humans and is associated with multiple sclerosis and teratogenesis. Aze-containing peptides may be oxygenated by prolyl hydroxylases resulting in potential damage of the protein by a reverse aldol of the hydroxyazetidine; this, rather than changes in conformation, may account for the deleterious effects of Aze. This paper describes the synthesis of 3-fluoro-Aze amino acids as hydroxy-Aze analogues which are not susceptible to aldol cleavage. 4-(Azidomethyl)-3-fluoro-Aze and 3,4-difluoroproline are new peptide building blocks. trans,trans-2,4-Dihydroxy-3-fluoroazetidine, an iminosugar, inhibits the growth of pancreatic cancer cells to a similar degree as gemcitabine.

L-fucose from vitamin C with only acetonide protection.

Addition of human milk oligosaccharides (HMO) to baby foods may protect infants from disease. As many simple HMOs are fucosylated this is likely to increase the demand for L-fucose as a synthetic building block. Any chemical synthesis must be cheap to compete with a biotechnological process. Acetonide is the only protecting group we have used in this new synthesis of L-fucose from vitamin C in 27% overall yield (purification by recrystallization; no chromatography required in the entire sequence).

Nine of 16 stereoisomeric polyhydroxylated proline amides are potent ß-N-acetylhexosaminidase inhibitors.

All 16 stereoisomeric N-methyl 5-(hydroxymethyl)-3,4-dihydroxyproline amides have been synthesized from lactones accessible from the enantiomers of glucuronolactone. Nine stereoisomers, including all eight with a (3R)-hydroxyl configuration, are low to submicromolar inhibitors of ß-N-acetylhexosaminidases. A structural correlation between the proline amides is found with the ADMDP-acetamide analogues bearing an acetamidomethylpyrrolidine motif. The proline amides are generally more potent than their ADMDP-acetamide equivalents. ß-N-Acetylhexosaminidase inhibition by an azetidine ADMDP-acetamide analogue is compared to an azetidine carboxylic acid amide. None of the amides are good α-N-acetylgalactosaminidase inhibitors.

Short and sweet: (D)-glucose to (L)-glucose and (L)-glucuronic acid.

The scarcity and expense of access to L-sugars and other rare sugars have prevented the exploitation of their biological potential; for example D-psicose, only recently available, has been recognized as an important new food. Here we give the definitive and cheap synthesis of 99.4% pure L-glucose from D-glucose which requires purification of neither intermediates nor final product other than extraction into and removal of solvents; a simple crystallization will raise the purity to >99.8%.

An approach to 8 stereoisomers of homonojirimycin from (D)-glucose via kinetic & thermodynamic azido-γ-lactones.

Crystal structures were obtained for the two C2 epimeric azido-γ-lactones 2-azido-2-deoxy-3,5:6,7-di-O-isopropylidene-d-glycero-d-ido-heptono-1,4-lactone and 2-azido-2-deoxy-3,5:6,7-di-O-isopropylidene-d-glycero-d-gulo-heptono-1,4-lactone prepared from kinetic and thermodynamic azide displacements of a triflate derived from d-glucoheptonolactone. Azido-γ-lactones are very useful intermediates in the synthesis of iminosugars and polyhydroxylated amino acids. In this study two epimeric azido-heptitols allow biotechnological transformations via Izumoring techniques to 8 of the 16 possible homonojirimycin analogues, 5 of which were isolated pure because of the lack of stereoselectivity of the final reductive amination. A side-by-side glycosidase inhibition profile of 11 of the possible 16 HNJ stereoisomers derived from d-glucose and d-mannose is presented.

C-branched iminosugars: α-glucosidase inhibition by enantiomers of isoDMDP, isoDGDP, and isoDAB-L-isoDMDP compared to miglitol and miglustat.

The Ho crossed aldol condensation provides access to a series of carbon branched iminosugars as exemplified by the synthesis of enantiomeric pairs of isoDMDP, isoDGDP, and isoDAB, allowing comparison of their biological activities with three linear isomeric natural products DMDP, DGDP, and DAB and their enantiomers. L-IsoDMDP [(2S,3S,4R)-2,4-bis(hydroxymethyl)pyrrolidine-3,4-diol], prepared in 11 steps in an overall yield of 45% from d-lyxonolactone, is a potent specific competitive inhibitor of gut disaccharidases [K(i) 0.081 µM for rat intestinal maltase] and is more effective in the suppression of hyperglycaemia in a maltose loading test than miglitol, a drug presently used in the treatment of late onset diabetes. The partial rescue of the defective F508del-CFTR function in CF-KM4 cells by L-isoDMDP is compared with miglustat and isoLAB in an approach to the treatment of cystic fibrosis.

(3R,4S,5R,6R,7S)-3,4,5,7-Tetrahydroxyconidine, an azetidine analogue of 6,7-diepicastanospermine and a conformationally constrained d-deoxyaltronojirimycin, from l-arabinose.

The synthesis from l-arabinose of an azetidine analogue of 6,7-diepicastanospermine and its glycosidase inhibition profile are described.

3-Hydroxyazetidine carboxylic acids: non-proteinogenic amino acids for medicinal chemists.

The formation from D-glucose of both enantiomers of 2,4-dideoxy-2,4-iminoribonic acid is the first chemical synthesis of unprotected 3-hydroxyazetidine carboxylic acids. The long-term stability of 3-hydroxyazetidine amides is established at acidic and neutral pH and implies their value as non-proteinogenic amino acid components of peptides, providing medicinal chemists with a new class of peptide isosteres. The structure of N,3-O-dibenzyl-2,4-dideoxy-2,4-imino-D-ribonic acid was established by X-ray crystallographic analysis. An N-methylazetidine amide derivative is a specific inhibitor of ß-hexosaminidases at the micromolar level, and is only the second example of potent inhibition of any glycosidase by an amide of a sugar amino acid related to an iminosugar.

2-N-Benzyl-2,6-dide-oxy-2,6-imino-3,4-O-iso-propyl-idene-d-allono-nitrile.

X-ray crystallography firmly established the relative stereochemistry of the title compound, C16H20N2O3. The acetonide ring adopts an envelope conformation with one of the O atoms as the flap and the piperidine ring adopts a slightly twisted boat conformation. The absolute configuration was determined by use of d-ribose as the starting material. The compound exists as O-H⋯O hydrogen-bonded chains of mol-ecules running parallel to the b axis.

N-(2-Carb-oxy-eth-yl)-2,5-dide-oxy-2,5-imino-d-mannonic acid [(3R,4R,5R)-1-(2-carb-oxy-eth-yl)-3,4-dihy-droxy-5-hy-droxy-methyl-l-proline].

The absolute stereochemistry of the title compound, C(9)H(15)NO(7), was determined from the use of d-glucuronolactone as the starting material. The compound crystallizes as the zwitterion. The five-membered ring adopts an envelope conformation with the -CH(2)OH-substituted C atom forming the flap. An intramolecular N-H⋯O hydrogen-bond occurs. In the crystal, the compound exists as a three-dimensional O-H⋯O intermolecular hydrogen-bonded network with each mol-ecule acting as a donor and acceptor for four hydrogen bonds.

Glycosidase inhibition by all 10 stereoisomeric 2,5-dideoxy-2,5-iminohexitols prepared from the enantiomers of glucuronolactone.

The enantiomers of glucuronolactone are excellent chirons for the synthesis of the 10 stereoisomeric 2,5-dideoxy-2,5-iminohexitols by formation of the pyrrolidine ring by nitrogen substitution at C2 and C5, with either retention or inversion of configuration; the stereochemistry at C3 may be adjusted during the synthesis to give seven stereoisomers from each enantiomer. A definitive side-by-side comparison of the glycosidase inhibition of a panel of 13 glycosidases showed that 8 of the 10 stereoisomers showed significant inhibition of at least one glycosidase.

N-Benzyl-3,5-dide-oxy-3,5-imino-1,2-O-isopropyl-idene-ß-l-lyxofuran-ose.

X-ray crystallography confirmed the formation, structure and relative stereochemistry of the title compound, C(15)H(19)NO(3), which contains a sterically congested four-membered azetidine ring system. The absolute configuration was determined by the use of l-arabinose as the starting material.

Synthesis from D-altrose of (5R,6R,7R,8S)-5,7-dihydroxy-8-hydroxymethylconidine and 2,4-dideoxy-2,4-imino-D-glucitol, azetidine analogues of swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol.

Ring closure of a 3,5-di-O-triflate derived from D-altrose with benzylamine allowed the formation of both monocyclic and bicyclic azetidine analogues of swainsonine.