Equatorial Hydroxyl Research Articles

Study of the Steric Structure of 7-Alkoxyalkyl-3-oxa-7-azabicyclo[3.3.1]nonan-9-ones and Their Derivatives Using1H NMR Spectroscopy

Using the 1H NMR spectroscopic method it has been shown that 7-alkoxyalkyl-3-oxa-7-azabicyclo[3.3.1]nonan-9-ones and 7-alkoxyalkyl-3-oxa-7-azabicyclo[3.3.1]nonanes exist in deuterochloroform solution in a double chair conformation. 7-(3-Butoxypropyl)-3-oxa-7-azabicyclo[3.3.1]nonan-9-ol is a 1:1 mixture of the two stereoisomeric alcohols. One of them exists in a double chair conformation having an equatorial hydroxyl group with relation to the piperidine ring and the other in a chair-boat conformation having an axial hydroxyl group which involves an intramolecular hydrogen bond with the unshared electron pair of the nitrogen atom.

Actinobacillus actinomycetemcomitans serotype b lipopolysaccharide mediates coaggregation with Fusobacterium nucleatum.

Purified Actinobacillus actinomycetemcomitans serotype b lipopolysaccharide (LPS) was found to be able to bind Fusobacterium nucleatum cells and to inhibit binding of F. nucleatum to A. actinomycetemcomitans serotype b. Sugar binding studies showed that the requirements for binding of A. actinomycetemcomitans serotype b LPS to the F. nucleatum lectin are the presence of a metal divalent ion, an axial free hydroxyl group at position 4, and free equatorial hydroxyl groups at positions 3 and 6 of D-galactose, indicating that the beta-N-acetyl-D-galactosamine in the serotype b LPS trisaccharide repeating unit is the monosaccharide residue recognized by the F. nucleatum lectin. These data strongly suggest that A. actinomycetemcomitans serotype b LPS is one of the receptors responsible for the lactose-inhibitable coaggregation of A. actinomycetemcomitans to fusobacteria.

Theoretical pKaestimates for solvated P(OH)5from coordination constrained Car–Parrinello molecular dynamics

Car–Parrinello molecular dynamics simulations have been performed on P(OH)5 and its monanionic and dianionic derivatives in liquid water, representing minimal models of the transition complex occurring in the hydrolysis of RNA. P(OH)5 and PO5H4− have been found to be stable in aqueous solution, while PO5H32− is reduced to PO5H4− within femtoseconds by solvent–solute proton transfer. The deprotonated hydroxyl group of the monoanion exhibits pronounced equatoriphilicity. This observation has been underlined by theoretical pKa determination of axial and equatorial hydroxyl groups of P(OH)5, respectively, by means of coordination constrained ab initio molecular dynamics, which has shown the equatorial site to be more acidic by 4.4 units, the absolute values being 14.2 and 9.8.

The role of amino-acid residues Q39 and E451 in the determination of substrate specificity of the Spodoptera frugiperda beta-glycosidase.

The design of beta-glycosidases with planed substrate specificity for biotechnological application has received little attention. This is mostly a consequence of the lack of data on the molecular basis of the beta-glycosidase specificity, namely data on the energy of the noncovalent interactions in the enzyme-transition state complex. In an attempt to fill this gap, site-directed mutagenesis and enzyme steady-state kinetic experiments with different substrates were conducted, using as model a digestive beta-glycosidase (glycoside hydrolase family 1) from Spodoptera frugiperda (Lepidoptera) (Sfbetagly50). The active site of this enzyme was modeled based on its sequence and on crystallographic data of similar enzymes. Energy of noncovalent interactions in transition state between Sfbetagly50 amino acids and glycone hydroxyls was determined. Sfbetagly50 residue E451 seems to be a key residue in determining beta-glycosidase preference for glucosides vs. galactosides based on the following data: (a) The energy of the noncovalent interaction between glycone equatorial hydroxyl 4 with E451 in the transition state is about 60% higher than its interaction with Q39. (b) The energy of the E451-hydroxyl 4 interaction decreases more than the Q39-hydroxyl 4 interaction when hydroxyl 4 is changed from equatorial to axial position. (c) A Sfbetagly50 mutant, where E451 was substituted by A, hydrolyzes galactosides faster than glucosides. It was also shown that glycone hydroxyl 6 interacts favorably with Q39, but not with E451, probably due to steric hindrance. These interactions result in the beta-glycosidase hydrolyzing fucosides (6-deoxygalactosides) faster than glucosides and galactosides.

Stereoelectronic substituent effects in polyhydroxylated piperidines and hexahydropyridazines.

From the pK(a) values of the conjugate acids of a large series of hydroxylated piperidines and hexahydropyridazines, a consistent difference in basicity was found between stereoisomers having an axial or equatorial hydroxyl (OH) group either beta or gamma to the amine. Compounds with an equatorial OH group in the 3-position were 0.8 pH units more acidic than otherwise identical compounds with an axial OH group, whilst compounds with an equatorial OH group in the 4-position relative to the amine were 0.4 pH units more acidic than the corresponding compound with an axial OH. A similar effect was observed for the COOMe substituent. The difference in electron-withdrawing power of axial and equatorial substituents was explained by a difference in charge-dipole interactions in the two systems. Since this stereoelectronic substituent effect causes differences in basicity in different conformers, certain piperidines and hexahydropyridazines were found to change conformation upon protonation. A method for predicting the pK(a) of piperidines which takes stereochemistry into account is described.

The Presence of a Hydrogen Bond between Asparagine 485 and the π System of FAD Modulates the Redox Potential in the Reaction Catalyzed by Cholesterol Oxidase,

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. An asparagine residue (Asn485) at the active site is believed to play an important role in catalysis. To test the precise role of Asn485, we mutated it to a leucine and carried out kinetic and crystallographic studies. Steady-state kinetic analysis revealed a 1300-fold decrease in the oxidation k(cat)/K(m) for the mutant enzyme whereas the k(cat)/K(m) for isomerization is only 60-fold slower. The primary kinetic isotope effect in the mutant-catalyzed reaction indicates that 3alpha-H transfer remains the rate-determining step. Measurement of the reduction potentials for the wild-type and N485L enzymes reveals a 76 mV decrease in the reduction potential of the FAD for the mutant enzyme relative to wild type. The crystal structure of the mutant, determined to 1.5 A resolution, reveals a repositioning of the side chain of Met122 near Leu485 to form a hydrophobic pocket. Furthermore, the movement of Met122 facilitates the binding of an additional water molecule, possibly mimicking the position of the equatorial hydroxyl group of the steroid substrate. The wild-type enzyme shows a novel N-H...pi interaction between the side chain of Asn485 and the pyrimidine ring of the cofactor. The loss of this interaction in the N485L mutant destabilizes the reduced flavin and accounts for the decreased reduction potential and rate of oxidation. Thus, the observed structural rearrangement of residues at the active site, as well as the kinetic data and thermodynamic data for the mutant, suggests that Asn485 is important for creating an electrostatic potential around the FAD cofactor enhancing the oxidation reaction.

Amino acid residues involved in substrate binding and catalysis in an insect digestive β-glycosidase

A β-glycosidase (Mr 50 000) from Spodoptera frugiperda larval midgut was purified, cloned and sequenced. It is active on aryl and alkyl β-glucosides and cellodextrins that are all hydrolyzed at the same active site, as inferred from experiments of competition between substrates. Enzyme activity is dependent on two ionizable groups (pKa1=4.9 and pKa2=7.5). Effect of pH on carbodiimide inactivation indicates that the pKa 7.5 group is a carboxyl. kcat and Km values were obtained for different p-nitrophenyl β-glycosides and Ki values were determined for a range of alkyl β-glucosides and cellodextrins, revealing that the aglycone site has three subsites. Binding data, sequence alignments and literature β-glycosidase 3D data supported the following conclusions: (1) the groups involved in catalysis were E187 (proton donor) and E399 (nucleophile); (2) the glycone moiety is stabilized in the transition state by a hydrophobic region around the C-6 hydroxyl and by hydrogen bonds with the other equatorial hydroxyls; (3) the aglycone site is a cleft made up of hydrophobic amino acids with a polar amino acid only at its first (+1) subsite.

X-ray photoelectron study of the interaction of the uranyl group UO 2 2+ with hydroxylapatite and fluoroapatite in aqueous solutions

The interaction of the uranyl group UO 2 2+ in aqueous solutions with hydroxylapatite and fluoroapatite was studied by X-ray photoelectron spectroscopy. The apatite samples under study were found to contain CO 2 2+ groups. The reaction of uranyl nitrate with hydroxylapatite in aqueous solutions does not lead to U(IV)-containing compounds but forms U(VI)-containing uranyl compounds with equatorial hydroxyl or carbonate groups partly replaced by fluorine in the uranyl compounds in the case of fluoroapatite. The interaction of the uranyl group with fluoroapatite in aqueous solutions is much more effective than the interaction with hydroxylapatite.

Comparison of the Antifungal Activity of Cadinane Skeletal Sesquiterpenoids from Taiwania (Taiwania cryptomerioides Hayata) Heartwood

Summary The antifungal activity of cadinane skeletal sesquiterpenoids from Taiwania (Taiwania cryptomerioides Hayata) heartwood is demonstrated. Using spectral analyses, the absolute structures of three main cadinanes, T-cadinol, T-muurolol, and α-cadinol, all isolated from Taiwania with HPLC, were identified. The amount of these cadinanes was also quantified using GC. The results showed that the total amount of cadinanes extracted from heartwood with n-C6H14 was 6.49 mg per kg of wood. This was much more than the essential oils collected by water distillation from leaves (0.04 mg/kg), sapwood (0.36 mg/kg), or heartwood (1.77 mg/kg). Moreover, results obtained from the antifungal assays demonstrated that αcadinol exhibited the highest antifungal index for both Coriolus versicolor and Laetiporus sulphureus, followed by T-cadinol and T-muurolol. As a matter of fact, α-cadinol completely inhibited the growth of C. versicolor and L. sulphureus at the level as low as 100 ppm. Further comparison of the molecular configuration of these cadinanes reveals that cadinane skeletal sesquiterpenoids with an equatorial hydroxyl group at C-9 and a trans configuration at the ring junction, such as the case for α-cadinol, exhibited the strongest antifungal activity.

Structure and binding specificity of the second N-terminal cellulose-binding domain from Cellulomonas fimi endoglucanase C.

The 1,4-beta-glucanase CenC from Cellulomonas fimi contains two cellulose-binding domains, CBD(N1) and CBD(N2), arranged in tandem at its N-terminus. These homologous CBDs are distinct in their selectivity for binding amorphous and not crystalline cellulose. Multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy was used to determine the tertiary structure of CBD(N2) in the presence of saturating amounts of cellopentaose. A total of 1996 experimental restraints were used to calculate an ensemble of 21 final structures for the protein. CBD(Nu2) is composed of 11 beta-strands, folded into two antiparallel beta-sheets, with a topology of a jellyroll beta-sandwich. On the basis of patterns of chemical shift perturbations accompanying the addition of cellooligosaccharides, as well as the observation of intermolecular protein-sugar NOE interactions, the cellulose-binding site of CBD(N2) was identified as a cleft that lies across one face of the beta-sandwich. The thermodynamic basis for the binding of cellooligosaccharides was investigated using isothermal titration calorimetry and NMR spectroscopy. Binding is enthalpically driven and consistent with a structural model involving hydrogen bonding between the equatorial hydroxyls of the glucopyranosyl rings and polar amino acid side chains lining the CBD(N2) cleft. Affinity electrophoresis was used to determine that CBD(N2) also binds soluble beta-1,4-linked polymers of glucose, including hydroxyethylcellulose and beta-1,3-1,4-glucans. This study complements a previous analysis of CBD(N1) [Johnson, P. E., Joshi, M. D., Tomme, P., Kilburn, D. G., and McIntosh, L. P. (1996) Biochemistry 35, 14381-14394] and demonstrates that the homologous CBDs from CenC share very similar structures and sugar binding properties.

糖質関連酵素を用いた有用オリゴ糖の開発に関する研究

This paper is composed of the following four research topics, with research carried out at the Osaka Municipal Technical Research Institute. Cyclodextrin glycosyltransferases (CGTases) from Bacillus megaterium, B. circulans, B. macerans and B. stearothermophilus were purified and their catalytic properties were studied. CGTase catalyzed the conversion of α-1, 4-glucans such as starch and glycogen to cyclodextrin (CD) by intramolecular transglycosylation. In the presence of a suitable acceptor such as glucose, CGTase catalyzed the intermolecular transglycosylation, in which the non-reducing end glycosyl residues produced by splitting an α-1, 4-glucan were transferred to the acceptor. In the intramolecular transglycosylation, the enzymes from B. megaterium, B. circulans, B. macerans and B. stearothermophilus produced α-, β- and γ-CDs in ratios of 1.0: 6.3: 1.3, 1.0: 6.4: 1.4, 5.7: 1.0: 0.4 and 1.7: 1.0: 0.3, respectively, on 1 % soluble starch at the initial reaction. B. stearothermophilus CGTase showed the strongest activity in the intermolecular transglycosylation. The effective acceptors of CGTases in the intermolecular transglycosylation were D-glucose, D-xylose, 6-deoxy-D-glucose and L-sorbose, which had a pyranose structure with free equatorial hydroxyl groups at C2, C3 and C4. CGTases transferred glycosyl residues preferentially to the C4-hydroxyl group of D-glucose, D-xylose, and 6-deoxy-D-glucose with the exception of L-sorbose, where the preferred group was the C3-hydroxyl group. The enzyme also catalyzed the hydrolysis of α-1, 4-glucans and CDs. The ratios of hydrolysis to total catalysis were 1.9, 2.0, 2.0 and 8.3 for the CGTases from B. megaterium, B. circulans, B. macerans and B. stearothermophilus, respectively. Using the intermolecular transglycosylation of CGTase, maltooligosyl-sucrose (“coupling sugar, ” commercial name) is produced from the mixture of starch hydrolyzates and sucrose. The cariogenicity of the coupling sugar was studied by a group of the Department of Dental Research, Japanese National Institute of Health, and other universities, and the coupling sugar was proved to be an anticariogenic sweetener. It was the first example of a so-called “functional oligosaccharide” which had a physiological property apart from the conventional functions of sweeteners. Arthrobacter sp. K-1 β-fructofuranosidase (β-FFase), isolated from soil, had very strong transfer activity and broad acceptor specificity. When the β-FFase was incubated with sucrose in the presence of xylose, isomaltose and lactose, the enzyme transferred the fructosyl residue only to the C 1 hydroxyl group of the acceptors and efficiently produced fructosylxyloside (XF), isomaltosylfructoside (IMF) and lactosylfructoside (LacF), respectively. XF competitively inhibited the degradation activity of sucrose by glucosyltransferase (GTase) from Streptococcus mutans as an analogue to sucrose, and IMF acted as an alternative acceptor for the glucosyl transfer reaction of GTase to lessen the formation of insoluble glucan. These saccharides had anticariogenic properties. LacF was nondigestive, but selectively utilized by bifidobacteria in the human intestinal bacteria flora, followed by the improvement of constipation and blood lipid levels of hyperlipemia patients and suppression of putrefactive metabolites such as ammonia, phenol and indole. Stevioside, a sweet steviol glycoside isolated from the leaves of Stevia rebaudiana Bertoni, is about 140-fold as sweet as sucrose, but has a slightly bitter taste and aftertaste. To improve the quality of taste, various stevioside derivatives such as glycosyl-stevioside (G-Ste), fructosyl-stevioside (F-Ste) and galactosyl-stevioside were synthesized with the transfer reaction of CGTase, β-FFase and β-galactosidase.

NMR investigation of structure and conformation of aminodeoxyhexonolactams

The crucial requirement of equatorial hydroxyl group at the carbon next to the carbonyl in the seven-membered lactam ring leading to one preferred conformation was confirmed studying 6-amino-2,6-dideoxy-, 6-amino-3,6-dideoxy and 6-amino-2,3,6-trideoxyhexonolactams. The absence of the hydroxyl group at C-2 in 2-deoxy compounds results in the formation of the mixture of 1,NC4 and 4C1,N conformers. The equilibrium was studied by means of NOE and low temperature NMR experiments.

Purification and characterization of Dolichos lablab lectin

The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has been purified from seeds of Dolichos lablab (hyacinth bean) to electrophoretic homogeneity by affinity chromatography on an ovalbumin-Sepharose 4B column. The purified lectin gave a single symmetric protein peak with an apparent molecular mass of 67 kDa on gel filtration chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE. N-Terminal sequence analysis of these bands revealed subunit heterogeneity due to posttranslational proteolytic truncation at different sites mostly at the carboxyl terminus. The carbohydrate binding properties of the purified lectin were investigated by three different approaches: hemagglutination inhibition assay, quantitative precipitation inhibition assay, and ELISA. On the basis of these studies, it is concluded that the Dolichos lablab lectin has neither an extended carbohydrate combining site, nor a hydrophobic binding site adjacent to it. The carbohydrate combining site of DLL appears to most effectively accommodate a nonreducing terminal alpha-d-mannosyl unit, and to be complementary to the C-3, C-4, and C-6 equatorial hydroxyl groups of alpha-d-mannopyranosyl and alpha-d-glucopyranosyl residues. DLL strongly precipitates murine IgM but not IgG, and the recent finding that this lectin interacts specifically with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor and preserves human cord blood stem cells and progenitors in a quiescent state for prolonged periods in culture, make this lectin a valuable tool in biomedical research.

KM+, a mannose-binding lectin from Artocarpus integrifolia: amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the beta-prism fold.

The complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl-blocked N-terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine-rich linker that unites the regions homologous to the alpha- and beta-chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (alpha-D-mannose, alpha-D-glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the beta-prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the beta-prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a beta-hairpin containing two classic-type beta-bulges. We suggest the term beta-prism motif to describe this feature.

High-resolution native and complex structures of thermostable beta-mannanase from Thermomonospora fusca - substrate specificity in glycosyl hydrolase family 5.

. beta-Mannanases hydrolyse the O-glycosidic bonds in mannan, a hemicellulose constituent of plants. These enzymes have potential use in pulp and paper production and are of significant biotechnological interest. Thermostable beta-mannanases would be particularly useful due to their high temperature optimum and broad pH tolerance. The thermophilic actinomycete Thermomonospora fusca secretes at least one beta-mannanase (molecular mass 38 kDa) with a temperature optimum of 80 degreesC. No three-dimensional structure of a mannan-degrading enzyme has been reported until now. . The crystal structure of the thermostable beta-mannanase from T. fusca has been determined by the multiple isomorphous replacement method and refined to 1.5 A resolution. In addition to the native enzyme, the structures of the mannotriose- and mannohexaose-bound forms of the enzyme have been determined to resolutions of 1.9 A and 1.6 A, respectively. . Analysis of the -1 subsite of T. fusca mannanase reveals neither a favourable interaction towards the axial HO-C(2) nor a discrimination against the equatorial hydroxyl group of gluco-configurated substrates. We propose that selectivity arises from two possible mechanisms: a hydrophobic interaction of the substrate with Val263, conserved in family 5 bacterial mannanases, which discriminates between the different conformations of the hydroxymethyl group in native mannan and cellulose; and/or a specific interaction between Asp259 and the axial hydroxyl group at the C(2) of the substrate in the -2 subsite. Compared with the catalytic clefts of family 5 cellulases, the groove of T. fusca mannanase has a strongly reduced number of aromatic residues providing platforms for stacking with the substrate. This deletion of every second platform is in good agreement with the orientation of the axial hydroxyl groups in mannan.

The Synthesis, Structure, and Anticancer Activity of cis- and trans-4‘,7-Dihydroxyisoflavan-4-ols

cis-4‘,7-Dihydroxyisoflavan-4-ol (4) and trans-4‘,7-dihydroxyisoflavan-4-ol (5), two proposed metabolites of daidzein (4‘,7-dihydroxyisoflavone), have been synthesized and fully characterized for the first time. The vicinal coupling constants of the pyran ring protons are compatible with a half-chair conformation. The cis isomer is anancomeric while the trans isomer consists of a 68:32 mixture of two ring inversion conformers. Molecular mechanical calculations are in agreement with the half-chair conformation of the pyran ring and suggest that the cis isomer is biased because of an unfavorable gauche interaction of the equatorial hydroxyl and the axial phenyl group. The isoflavanols 4 and 5 are comparable to genistein (4‘,5,7-trihydroxyisoflavone) in antitumor activity against human prostate cancer cells.

Mitsunobu reactions with methanesulfonic acid; The replacement of equatorial hydroxyl groups by azide with net retention of configuration

Treatment of equatorial cyclohexanols with Ph 3P/DEAD/MsOH gives clean conversion to the axial mesylates. Subsequent reaction with NaN 3 gives the equatorial azides in overall yields of 74–87%. Axial hydroxyl groups are not affected, allowing the regioselective conversion of methyl cholate into a 3α-azidodiol intermediate for steroid-based synthetic receptors.

Purification in high yield and characterisation of the galactose-specific lectin from the seeds of snake gourd (Trichosanthes anguina).

The galactose-specific lectin present in the seeds of snake gourd (Trichosanthes anguina) was purified in high yield by affinity chromatography on cross-linked guar gum. The purified snake gourd seed lectin (SGSL) yielded a single symmetrical peak on gel filtration with an M(r) of 62 kDa and gave a single band in PAGE under non-denaturing conditions. In SDS-PAGE, SGSL gave a single band of M(r) 53 kDa in the absence of beta-mercaptoethanol, and two bands of M(r) 32 and 23 kDa in its presence, indicating that the lectin is a heterodimer in which the subunits are linked by a disulphide bridge. The lectin gave a single precipitin line in immunodiffusion experiments with antiserum raised against the purified SGSL. No cross-reactivity was found between SGSL and antiserum raised against the Momordica charantia lectin and vice versa, suggesting that the two lectins are antigenically dissimilar. Haemagglutination-inhibition data show that Me beta D-Gal is the best monosaccharide inhibitor of SGSL and indicate that an equatorial hydroxyl at C-2 and axial hydroxyl at C-4 in the pyranose form are important binding loci for the lectin.

Synthesis of D- and L-myo-Inositol 1,4,6-Trisphosphate, Regioisomers of a Ubiquitous Second Messenger.

A regioisomer of the second messenger D-myo-inositol 1,4,5-trisphosphate [D-Ins(1,4,5)P(3), 1], DL-myo-inositol 1,4,6-trisphosphate [DL-Ins(1,4,6)P(3), 4ab], together with the chiral antipodes D-Ins(1,4,6)P(3)(4a) and L-Ins(1,4,6)P(3)(4b), was synthesized from myo-inositol. The racemic diol 6, after removal of the trans-ketal of fully protected 5 was p-methoxybenzylated to give the 6-O-alkylated derivative 9, as the major product in 52% yield. Gentle acidic hydrolysis of 9, followed by benzylation of the resulting triol, gave the fully protected compound 11ab. Isomerization of the two allyl groups followed by acidic hydrolysis of the resulting cis-prop-1-enyl moieties and the p-methoxybenzyl group gave the triol 13ab. Phosphorylation of 13ab followed by deprotection of the resulting compound, 14ab, with sodium in liquid ammonia and purification by ion exchange chromatography provided 4ab in 60% yield. The intermediate 9 was converted into the cis-diol 16ab in two steps. Selective acylation at the equatorial hydroxyl group using (S)-(+)-O-acetylmandelic acid in the presence of DCC and DMAP provided two diastereoisomers, 18 and 19, which were separated by flash chromatography. Further transformations provided the corresponding D- and L-1,4,6 triols, 13a and 13b, respectively, and phosphorylation, followed by deprotection of the fully blocked products as for the racemic 4ab, gave 4a and 4b, respectively. The absolute configuration of fully protected 11a was determined by transformation to the known compound L-1,2,4,5-tetra-O-benzyl-myo-inositol (22). Compound 4a was a full agonist at the platelet Ins(1,4,5)P(3) receptor for Ca(2+) release, but 4b was devoid of activity.

Interaction of soluble cellooligosaccharides with the N-terminal cellulose-binding domain of Cellulomonas fimi CenC 2. NMR and ultraviolet absorption spectroscopy.

The N-terminal cellulose-binding domain (CBDN1) from Cellulomonas fimi beta-1,4-glucanase CenC binds amorphous but not crystalline cellulose. To investigate the structural and thermodynamic bases of cellulose binding, NMR and difference ultraviolet absorbance spectroscopy were used in parallel with calorimetry (Tomme, P., Creagh, A. L., Kilburn, D. G., & Haynes, C. A., (1996) Biochemistry 35, 13885-13894) to characterize the interaction of soluble cellooligosaccharides with CBDN1. Association constants, determined from the dependence of the amide 1H and 15N chemical shifts of CBDN1 upon added sugar, increase from 180 +/- 60 M-1 for cellotriose to 4,200 +/- 720 M-1 for cellotetraose, 34,000 +/- 7,600 M-1 for cellopentaose, and an estimate of 50,000 M-1 for cellohexaose. This implies that the CBDN1 cellulose-binding site spans approximately five glucosyl units. On the basis of the observed patterns of amide chemical shift changes, the cellooligosaccharides bind along a five-stranded beta-sheet that forms a concave face of the jelly-roll beta-sandwich structure of CBDN1. This beta-sheet contains a strip of hydrophobic side chains flanked on both sides by polar residues. NMR and difference ultraviolet absorbance measurements also demonstrate that tyrosine, but not tryptophan, side chains may be involved in oligosaccharide binding. These results lead to a model in which CBDN1 interacts with soluble cellooligosaccharides and, by inference, with single polysaccharide chains in regions of amorphous cellulose, primarily through hydrogen bonding to the equatorial hydroxyl groups of the pyranose rings. Van der Waals stacking of the sugar rings against the apolar side chains may augment binding. CBDN1 stands in marked contrast to previously characterized CBDs that absorb to crystalline cellulose via a flat binding surface dominated by exposed aromatic rings.