Purification and characterization of acid phosphatase from Macrotyloma uiflorum seeds.

Acid phosphatases play a crucial role in food processing industries to reduce phosphate content of food. Here in acid phosphatase from the seeds of Macrotyloma uniflorum has been purified to homogeneity using UNOsphere-S cation exchange chromatography followed by gel filtration with 81.85 fold purification. Molecular weight of purified enzyme was 55,000 (± 1040) Daltons under physiological conditions. It was a heterodimer of subunits having molecular weights 27,093 and 28,241 Daltons as determined by MALDI-TOF analysis. The optimum pH and temperature for the purified enzyme was 5.0 and 50 °C respectively. The enzyme was stable in the pH range 3.5-5.5 and showed temperature stability up to 60 °C. Substrate specificity of enzyme was checked with different substrates namely, p-nitrophenyl phosphate (p-NPP), ATP, ADP, glucose 6-phosphate, glucose-1-phosphate, fructose 6-phosphate, phenyl phosphate, α-naphthyl-phosphate, pyridoxyl phosphate and ß-glycerophosphate. Enzyme showed high substrate specificity towards p-NPP, phenyl phosphate, ATP and α-naphthyl phosphate. Km and Vmax of enzyme were found to be 0.934 mM and 1.333 mM/min respectively with respect to p-NPP as a substrate. Chemical modification studies showed that tryptophan was present at the active site of the enzyme.

α-Geminal dihydroxymethyl piperidine and pyrrolidine iminosugars: synthesis, conformational analysis, glycosidase inhibitory activity, and molecular docking studies.

The Jocic-Reeve and Corey-Link type reaction of dichloromethyllithium with suitably protected 5-keto-hexofuranoses followed by treatment with sodium azide and sodium borohydride reduction gave 5-azido-5-hydroxylmethyl substituted hexofuranoses 7a-c with required geminal dihydroxymethyl group. Removal of protecting groups and converting the C-1 anomeric carbon into free hemiacetal followed by intramolecular reductive aminocyclization with in situ generated C5-amino functionality afforded corresponding 5C-dihydroxymethyl piperidine iminosugars 2a-c. Alternatively, removal of protecting groups in 7b and 7c and chopping of C1-anomeric carbon gave C2-aldehyde that on intramolecular reductive aminocyclization with C5-amino gave 4C-dihydroxymethyl pyrrolidine iminosugars 1b and 1c, respectively. On the basis of the (1)H NMR studies, the conformations of 2a/2b were assigned as (4)C(1) and that of 2c as (1)C(4). The glycosidase inhibitory activities of all five iminosugars were studied with various glycosidase enzymes and compared with natural d-gluco-1-deoxynojirimycin (DNJ). All the five compounds were found to be potent inhibitors of rice α-glucosidase with K(i) and IC(50) values in the nanomolar concentration range. Iminosugars 2b and 1b were found to be more potent inhibitors than their parent iminosugar. These results were substantiated by in silico molecular docking studies.

Bicyclic amino acid-carbohydrate-conjugates as conformationally restricted hydroxyethylamine (HEA) transition-state isosteres.

This communication describes a general synthetic route to bicyclic amino acid-carbohydrate-conjugates, which would be useful as conformationally restricted hydroxyethylamine (HEA) transition-state isosteres. The synthesis was achieved in 12 steps starting from D-glucose. The striking features of this system are the bicyclic rigid core displaying an α-amino acid side chain and hydroxyethylamine moiety--both of which would be potentially important for receptor interactions, leading to various biomedical responses, as described in the literature. Crystal structure investigation suggested extensive intermolecular hydrogen-bonding interactions in this system, involving the backbone amide and hydroxyl groups.

Synthesis of new six- and seven-membered 1-N-iminosugars as promising glycosidase inhibitors.

New six- and seven-membered 1-N-iminosugars were prepared from d-glucose by the stereoselective Michael addition of nitromethane to d-glucose derived α,ß-unsaturated ester A followed by one pot reduction of nitro/ester functionality and subsequent amine protection to get N-Cbz protected aminol 6. Hydrolysis of 1,2-acetonide and reductive aminocyclization gave seven membered 1-N-iminosugar 5b. While, hydrolysis of 1,2-acetonide followed by NaIO(4) oxidative cleavage and hydrogenation using 10% Pd(OH)(2)/C, H(2) gave six membered 1-N-iminosugar 4a; the hydrogenation using 10% Pd/C-H(2) however, gave N-methyl substituted 1-N-iminosugar 4b. The hydrochloride salts of 4a/4b and 5b were found to be specific α-galactosidase and moderate α-glucosidae inhibitors, respectively, in micro molar range.

Antidiabetic potential of α-amylase inhibitor from the seeds of Macrotyloma uniflorum in streptozotocin-nicotinamide-induced diabetic mice.

CONTEXT: Macrotyloma uniflorum (Lam.) Verdc. (Leguminosae) seeds, known as the poor man's pulse crop in India, have been used as a food and also used in the traditional method for treatment of kidney stones, diabetes, obesity, etc. OBJECTIVE: To investigate the antidiabetic effect of α-amylase inhibitor isolated from the seeds of Macrotyloma uniflorum seeds in streptozotocin-nicotinamide induced diabetic mice. MATERIALS AND METHOD: α-Amylase inhibitor was purified using a carboxymethyl cellulose (CMC) column. Kinetic studies were done using mouse pancreatic and human salivary α-amylase. Its antidiabetic effect was studied in streptozotocin-nicotinamide-induced diabetic mice. Biochemical parameters such as serum total cholesterol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined. Histopathological investigation was performed on the pancreas, kidney, and liver tissue samples. RESULTS: Macrotyloma uniflorum α-amylase inhibitor (MUAI) inhibited both the mouse pancreatic and human salivary α-amylase in a non-competitive manner with K(i) values of 11 and 8.8 µM and IC(50) value of 30 and 12.5 µg/mL, respectively. It decreased the serum glucose level in the treated diabetic mice. Histological findings suggested minimum pathological changes in the treated diabetic mice as compared to the diabetic control. DISCUSSION AND CONCLUSION: The results suggest that MUAI has an antihyperglycemic activity and therefore can be used in the dietary treatment of non-insulin dependent diabetes mellitus.

Intramolecular reductive cyclization strategy to the synthesis of (-)-6-methyl-3-hydroxy-piperidine-2-carboxylic acid, (+)-6-methyl-(2-hydroxymethyl)-piperidine-3-ol and their glycosidase inhibitory activity.

The first stereoselective synthesis of (2S,3R,6S)-6-methyl-3-hydroxy-piperidine-2-carboxylic acid (-)-6 and (2R,3R,6S)-6-methyl-(2-hydroxymethyl)-piperidine-3-ol (+)-7 was achieved starting from readily available d-glucose in 14 steps with 17% overall yield for both the compounds. The key feature of the present strategy includes the Wittig-olefination for the preparation of required conjugated keto-azide 9 and construction of 2,3,6-trisubstituted piperidine skeleton 11 by applying intramolecular reductive cyclization of conjugated keto-azide intermediate. The glycosidase inhibitory activity of compounds 6 and 7 towards several glycosidases has been evaluated.

A versatile access to calystegine analogues as potential glycosidases inhibitors.

An efficient metathetic strategy and nitrone chemistry have been suitably tethered to construct 8-azabicyclo[3.2.1]octanes as versatile precursors for the synthesis of several calystegine analogues. This synthetic strategy relies on the ability of mannose-derived nitrone to undergo a highly stereoselective nucleophilic addition of various Grignard reagents to access syn orientation of alkenes, which then smoothly undergo ring-closing metathesis (RCM) to provide this framework. These RCM products 18 and 20 have been successfully used as advance precursors to synthesize many calystegine analogues (27, 36, 38, 40, 43, and 44) either by syn-dihydroxylation or by hydrogenation and followed by global deprotection. Interestingly, both compounds 36 and 40 exhibited significant noncompetitive inhibition against alpha-mannosidase and N-acetyl-beta-D-glucosaminidase.

Simplified purification approach of urinary neutrophil gelatinase-associated lipocalin by tangential flow filtration and ion exchange chromatography.

This investigation reports a simplified approach for the purification of urinary siderocalin known as neutrophil gelatinase-associated lipocalin (NGAL). Urinary NGAL was purified by tangential flow filtration and ion exchange chromatography. Isolated NGAL was analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular mass of NGAL is 23674Da. Peptide mass fingerprinting of the purified NGAL yielded peptides that partially matched with known sequence of P80188 (NGAL_HUMAN). The tryptic digestion profile of isolated NGAL infers that it may be unique and additive molecule in the dictionary of urinary proteins. This is the first report of purification and validation of urinary NGAL from large volume sample by using tangential flow filtration and peptide sequencing respectively. This cost-effective and simplified approach to purification of NGAL, together with the easy availability of urine sample makes the large-scale production of NGAL possible, allowing exploration of various bioclinical as well as biodiagnostic applications.

Halogenated D-xylono-δ-lactams: synthesis and enzyme inhibition study.

A concise synthesis of four C-3 fluoro/chloro-D-xylono-δ-lactams 3/4 has been reported. The methodology involves Corey-Link approach with suitably protected 3-oxo-D-gluco-furanose to introduce F/Cl as well as ester/amide functionalities at C-3 of glucose. In next steps, 5,6-O-isopropylidene group was converted to the 5-azido xylosugars that on opening of 1,2-acetonide group, and intramolecular Schmidt-Boyer reaction with TFA/H2O, in one pot, afforded lactams 3/4. Conformational aspect of δ-lactams was studied by the 1H NMR spectroscopy. The halogenated δ-lactams 3/4 showed no inhibition against different glycosidase enzymes.

Synthesis, conformational study, glycosidase inhibitory activity and molecular docking studies of dihydroxylated 4- and 5-amino-iminosugars.

An efficient methodology for the synthesis of new amino iminosugars 6a, 7a and 8, starting from D-glucose, is reported. The conformational study using (1)H NMR data showed that the amino iminosugar 6a exists in the (2)C5 while; the 7a and 8 exist in the (5)C2 conformation. The inhibition activities with different glycosidases showed that 6a and 7a are poor glycosidase inhibitors. However, amino iminosugar 8 showed selective inhibition against the ß-galactosidase (IC50 = 43 µM, Ki = 153 µM). These results are substantiated by the molecular docking studies.

Polyhydroxylated homoazepanes and 1-deoxy-homonojirimycin analogues: Synthesis and glycosidase inhibition study.

The Johnson-Claisen rearrangement of D-gluco and L-ido-derived allylic orthoesters afforded gamma,delta-unsaturated ester that on ester reduction, epoxidation, regioselective oxirane opening by sodium azide and hydrogenation led to sugar amino alcohols--immediate precursors for 1-deoxy-homonojirimycin 3a,b, and polyhydroxylated homoazepanes 4a,b. Our synthetic approach and glycosidase inhibitory activity is reported.

Synthesis and evaluation of glycosidase inhibitory activity of N-butyl 1-deoxy-D-gluco-homonojirimycin and N-butyl 1-deoxy-L-ido-homonojirimycin.

Conjugate addition of n-butyl amine to d-glucose derived alpha,beta-unsaturated ester 4 afforded beta-amino esters 5a,b that on reduction of ester group, 1,2-acetonide deprotection, and reductive amination led to the formation of corresponding N-butyl 1-deoxy-D-gluco-homonojirimycin 2c and N-butyl 1-deoxy-L-ido-homonojirimycin 2d which were found to be selective beta-glucosidase inhibitors with an IC(50) value in millimolar range.

Synthesis of tetrahydroxy perhydroaza-azulenes: tandem Johnson-Claisen rearrangement of D-glucose-derived allylic alcohols.

The Johnson-Claisen rearrangement of D-glucose-derived allylic alcohols 5a,b, afforded sugar-substituted gamma,delta-unsaturated ester in high yield. Conversion of the ester group to an azidomethyl group, epoxidation of the double bond and hydrogenation gave pyrrolidine ring skeletons 13a and 13b, which were transformed to tetrahydroxy perhydroaza-azulenes 1a and 1b, respectively. Glycosidase inhibitory activity was also evaluated.

Synthesis and evaluation of the glycosidase inhibitory activity of 5-hydroxy substituted isofagomine analogues.

An efficient strategy for the synthesis of 5-hydroxy substituted isofagomine analogues and , having both -CH2OH/CH3 and -OH functionality at the C-5 position, and evaluation of their inhibitory potency is reported. The synthetic methodology involves the aldol-Cannizzaro reaction of easily available alpha-d-xylopentodialdose followed by hydrogenolysis to afford the triol . Selective amidation of the alpha- and beta-hydroxymethyl group at C-4, deprotection of the 1,2-acetonide group and hydrogenation gave the target molecules, which were found to be potent against beta-glycosidases with IC50 values in the micro molar range. Compound showed excellent potency against glycosidases and human salivary amylase.

1,3-Dipolar cycloaddition reaction of D-glucose-derived nitrone with allyl alcohol: synthesis of 2-hydroxy-1-deoxycastanospermine analogues.

[reaction: see text] The synthesis and evaluation of glycosidase inhibitory activity of polyhydroxylated indolizidine alkaloids namely 2-hydroxy-1-deoxycastanospermine 3a,b and 2-hydroxy-1-deoxy-8a-epi-castanospermine 3c,d is reported. The key step involves the intermolecular 1,3-dipolar cycloaddition of allyl alcohol to d-glucose-derived nitrone 4, followed by tosylation, that afforded four diastereomeric sugar-substituted isoxazolidines 5a-d with the desired regioselectivity. The one-pot conversion of 5a-d to pyrrolidines 8a-d by hydrogenolysis, removal of 1,2-acetonoide functionality, and hydrogenation afforded corresponding target molecules 3a-d.

Aziridine carboxylate from D-glucose: synthesis of polyhydroxylated piperidine, pyrrolidine alkaloids and study of their glycosidase inhibition.

The D-glucose derived aziridine carboxylate 5 was obtained from (E)-ethyl-6-bromo-1,2-O-isopropylidene-3-O-benzyl-5-deoxy-alpha-D-xylo-5-eno-heptofuranuronate 4 through conjugate addition of benzylamine and in situ intramolecular nucleophilic expulsion of bromine. The regioselective aziridine ring-opening, using water as a nucleophile, resulted in the alpha-hydroxy-beta-aminoester 6, which was exploited in the synthesis of six and five membered azasugars 1b/1c and 2b/2c, respectively. The glycosidase inhibitory activity of the title compounds was evaluated.

N-hydroxyethyl-piperidine and -pyrrolidine homoazasugars: preparation and evaluation of glycosidase inhibitory activity.

An efficient and practical strategy for the synthesis of N-hydroxyethyl-1-deoxy-homonojirimycins 4 and 5 and N-hydroxyethyl-pyrrolidine homoazasugars 6 and 7 with full stereocontrol is being reported. The key step involved is the intermolecular Michael addition of benzylamine to D-glucose derived alpha,beta-unsaturated ester 8 followed by N-alkylation with ethyl bromoacetate. Reduction with LAH, acetylation, hydrogenation and protection with -Cbz group afforded compounds 14a and 14b. Removal of 1,2-acetonide functionality, hydrogenation and deacetylation afforded N-hydroxyethyl-D-gluco-1-deoxyhomonojirimycin (4) and N-hydroxyethyl-L-ido-1-deoxyhomonojirimycin (5), respectively. Compounds 14a and 14b on acetylation followed by removal of 1,2-acetonide functionality, sodium metaperiodate oxidation, hydrogenation and deacetylation gave 1,4,5-trideoxy-1,4-imino-N-hydroxyethyl-D-arabino-hexitol (6) and 1,4,5-trideoxy-1,4-imino-N-hydroxyethyl-L-xylo-hexitol (7), respectively. The glycosidase inhibition activity of compounds 4, 5, 6, 7, 16a and 16b was evaluated using sweet almond seed as a rich source of different glycosidases.

1-Aza-sugars from D-glucose. Preparation of 1-deoxy-5-dehydroxymethyl-nojirimycin, its analogues and evaluation of glycosidase inhibitory activity.

D-glucose derived pentodialdoses 11a-c on reduction followed by tosylation, azide displacement, hydrogenation and protection with -Cbz group gave N-Cbz protected compounds 14a-c, respectively, which on removal of 1,2-acetonide functionality and hydrogenation afforded corresponding 1-aza-sugars 3, 9 and 10 in good overall yields. The glycosidase inhibition activity of these 1-aza-sugars was tested with sweet almond as a rich source of different glycosidases.

Synthesis and evaluation of glycosidase inhibitory activity of octahydro-2H-pyrido[1,2-a]pyrimidine and octahydro-imidazo[1,2-a]pyridine bicyclic diazasugars.

An efficient chiron approach for the synthesis of bicyclic diazasugars 4a and 4b having both -CH(2)OH and -OH functionality at the same carbon atom (C-6) is reported. Thus, easily available alpha-D-xylo-pentodialdo-1,4-furanose 5, obtained from D-glucose, on aldol-crossed Cannizzaro reaction followed by hydrogenolysis afforded 7. The regio-selective beta- and alpha-sulfonylation of hydroxymethyl groups in 7 afforded 8a (beta-sulfonylation) and 11 (alpha-sulfonylation) in good yields. The cleavage of the 1,2-acetonide functionality, individually in 8a and 11, followed by reaction with ethylenediamine gave in situ formation of sugar aminals that undergo concomitant nucleophilic displacement of the sulfonyloxy group, by amino functionality, to give hitherto unknown bicyclic diazasugars 4a and 4b, respectively. The inhibitory potency of the earlier reported bicyclic diazasugars 3a,b and 4a,b was evaluated against alpha- and beta-glycosidases and they were found to be potent and specific against the beta-glycosidases with IC(50) and K(1) values in the micro molar range.