Polysaccharide recognition by surfactant protein D: novel interactions of a C-type lectin with nonterminal glucosyl residues.

Surfactant protein D (SP-D), a C-type lectin, is an important pulmonary host defense molecule. Carbohydrate binding is critical to its host defense properties, but the precise polysaccharide structures recognized by the protein are unknown. SP-D binding to Aspergillus fumigatus is strongly inhibited by a soluble beta-(1-->6)-linked but not by a soluble beta-(1-->3)-linked glucosyl homopolysaccharide (pustulan and laminarin, respectively), suggesting that SP-D recognizes only certain polysaccharide configurations, likely through differential binding to nonterminal glucosyl residues. In this study we have computationally docked alpha/beta-D-glucopyranose and alpha/beta-(1-->2)-, alpha/beta-(1-->3)-, alpha/beta-(1-->4)-, and alpha/beta-(1-->6)-linked glucosyl trisaccharides into the SP-D carbohydrate recognition domain. As with the mannose-binding proteins, we found significant hydrogen bonding between the protein and the vicinal, equatorial OH groups at the 3 and 4 positions on the sugar ring. Our docking studies predict that alpha/beta-(1-->2)-, alpha-(1-->4)-, and alpha/beta-(1-->6)-linked but not alpha/beta-(1-->3)-linked glucosyl trisaccharides can be bound by their internal glucosyl residues and that binding also occurs through interactions of the protein with the 2- and 3-equatorial OH groups on the glucosyl ring. By using various soluble glucosyl homopolysaccharides as inhibitors of SP-D carbohydrate binding, we confirmed the interactions predicted by our modeling studies. Given the sequence and structural similarity between SP-D and other C-type lectins, many of the predicted interactions should be applicable to this protein family.     

Involvement of beta-1,4-galactosyltransferase V in malignant transformation-associated changes in glycosylation

In spite of marked changes in the glycosylation upon malignant transformation of cells, no biological significance of beta-1, 4-galactosyltransferase (beta-1,4-GalT) activities has been elucidated. When beta-1,4-GalT activities toward 1 mM GlcNAcbeta-S-pNP were determined using homogenates of NIH3T3 and its transformant, MTAg, MTAg contained 1.3 times higher activities. Northern blot analysis, however, revealed that the beta-1,4-GalT V gene expression increases by three times with a decrease in that of beta-1,4-GalT II by one-fifth and without significant changes in those of other beta-1,4-GalTs in MTAg. Analysis of beta-1,4-GalT V acceptor-specificity showed that the GlcNAcbeta1-->6Man group of the GlcNAcbeta1-->6(GlcNAbeta1-->2)Manalpha1- branch is galactosylated. These results indicate that changes in beta-1,4-GalT II and V activities are important for the altered glycosylation.     

Cross metathesis for the synthesis of novel C-sialosides

Cross metathesis of both anomers of C-allyl sialoside (3alpha/3beta) with styrene catalyzed by the second generation Grubbs or Hoveyda-Grubbs catalysts gave the corresponding aryl derivatives (4alpha/4beta) in virtually quantitative yields. The products were hydrogenated to model compounds 5alpha/5beta. Similarly, reaction of the alpha-anomer 3alpha with galactose derivative 8 gave the olefin-linked disaccharide mimetic 9. Following hydrogenation and deprotection, the ethylene-bridged Neu5Acalpha(2-->6)Gal analogue 11 could be obtained.     

Occurrence of poly-N-acetyllactosamine synthesis in Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase I, II, III, IV, V and VI cDNAs

Lectin blot analysis of membrane glycoprotein samples from Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase (beta-1,4-GalT) I, II, III, IV, V et VI cDNAs showed that the endogenous N-linked oligosaccharides are galactosylated (Guo et al., Glycobiology (2001), in press). Further analysis revealed that membrane glycoprotein samples from all the gene-transfected cells are also reactive to Lycopersicon esculentum agglutinin (LEA) et Datura stramonium agglutinin (DSA), both of which bind to oligosaccharides with poly-N-acetyllactosamine chains while no lectin reactive protein bands are detected when blots are pretreated with a mixture of diplococcal beta-1,4-galactosidase et jack bean beta-N-acetylhexosaminidase or N-glycanase. Analysis of endo-beta-galactosidase-digestion products revealed the presence of the Gal1-->GlcNAc1-->Gal and/or GlcNAc1-->Gal structures in the gene-transfected cells. When the homogenates of the gene-transfected cells were used as enzyme sources towards oligosaccharides with the GlcNAc beta 1-->(3Gal beta 1-->4GlcNAc)(1-3) structures, human recombinant beta-1,4-GalTs I et II galactosylated these oligosaccharides more effectively than other beta-1,4-GalTs. These results indicate that beta-1,4-GalTs I-VI can synthesize poly-N-acetyllactosamine chains with beta-1,3-N-acetylglucosaminyltransferase.     

Systematic synthesis and MAG-binding activity of novel sulfated GM1b analogues as mimics of Chol-1 (alpha-series) gangliosides: highly active ligands for neural siglecs

Systematic synthesis and myelin-associated glycoprotein (MAG)-binding activity of novel sulfated GM1b analogues structurally related to Chol-1 (alpha-series) gangliosides, high-affinity ligands for neural siglecs, are described. The suitably protected gangliotriose derivatives, 2-(trimethylsilyl)ethyl 2-acetamido-2-deoxy-6-O-levulinoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside and 2-(trimethylsilyl)ethyl 2-acetamido-2-deoxy-6-O-levulinoyl-beta-D-galactopyranosyl-(1-->4)-2,6-di-O-benzyl-3-O-levulinoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside were each glycosylated with alpha-NeuAc-(2-->3)-galactose donor to give the corresponding pentasaccharides in 94% (beta1,3 glycoside only) and 90% (beta1,3:beta1,4 = 2:1), respectively. After proper manipulation of the protecting groups, the pentasaccharides were converted into three novel sulfated GM1b gangliosides by the successive introduction of the ceramide and sulfo groups, followed by complete deprotection. Among the synthetic gangliosides, GSC-338 (II3III6-disulfate of iso-GM1b) was surprisingly found to be the most potent MAG binding structure tested to date.     

Large-scale production of N-acetyllactosamine through bacterial coupling.

A large-scale production system of N-acetyllactosamine, a core structure of various oligosaccharides, was established by a whole-cell reaction through the combination of recombinant Escherichia coli strains and Corynebacterium ammoniagenes. Two recombinant E. coli strains over-expressed the UDP-Gal biosynthetic genes and the beta-(1-->4)-galactosyltransferase gene of Neisseria gonorrhoeae, respectively. C. ammoniagenes contributed the production of UTP from orotic acid. N-Acetyllactosamine was accumulated at 279 mM (107 g L-1) after a 38 h reaction (2.5 L in volume) starting from orotic acid, D-galactose, and 2-acetamido-2-deoxy-D-glucose.     

Enzymatic supported synthesis of lacto-N-neotetraose using dendrimeric polyethylene glycol

The lacto-N-neotetraose tetrasaccharide was synthesized on a new dendrimeric support, based on polyethylene glycol. Starting from 1-thio-beta-D-lactose, the trisaccharide (2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1-->3)-O-beta-D-galactopyranosyl-(1-->4)-1-thio-beta-D-glucopyranose was obtained using Neisseria meningitidis beta-(1-->3)-N-acetylglucosaminyltransferase according to a soluble synthesis approach, bound on the support and galactosylated using the milk beta-(1-->4)-galactosyl transferase to give after cleavage the tetrasaccharide lacto-N-neotetraose.     

Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system.

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.     

Chemo-enzymatic synthesis of a tetra- and octasaccharide fragment of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of tetrasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (1) and octasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (2), representing one and two tetrasaccharide repeating units of Streptococcus pneumoniae serotype 14 capsular polysaccharide. In a chemical approach, the intermediate linear trisaccharide 3 and hexasaccharide 4 were synthesized. Galactose residues were beta-(1-->4)-connected to the internal N-acetyl-beta-D-glucosamine residues by using bovine milk beta-1,4-galactosyltransferase. Both title oligosaccharides will be conjugated to carrier proteins to be tested as potential vaccines in animal models.     

Synthesis and biological activity of 4'-thio-2'-deoxy purine nucleosides.

Coupling of 1-O-acetyl-2-deoxy-3,5-di-O-toluoyl-4-thio-D-ribofuranose with 6-chloropurine and 2,6-dichloropurine gave a mixture of 9 alpha and 9 beta anomers as major products. These anomers were separated and converted to 2'-deoxy-4'-thio analogues of adenosine, inosine, guanosine, 2-amino-adenosine, and 2-chloro adenosine as well as their alpha-anomers.     

Synthesis and photolytic activation of 6'-O-2-nitrobenzyl uridine-5'-diphosphogalactose: a 'caged' UDP-Gal derivative

Placing an 2-nitrobenzyl group on O-6 of the galactosyl residue in uridine-5'-diphosphogalactose (UDP-Gal) gives 6'-O-2-nitrobenzyl-UDP-Gal that is shown to be inactive as a donor substrate for beta-(1-->4)-galactosyltransferase (GalT). On irradiation at 365 nm, the nitrobenzyl group is completely removed yielding native UDP-Gal that then transfers normally to produce the expected betaGal-(1-->4)-betaGlcNAc disaccharidic linkage. 6'-O-2-Nitrobenzyl-UDP-Gal thus fulfils the minimum requirements of a 'caged' UDP-Gal for application in time-resolved crystallographic studies of beta-(1-->4)-GalT.     

Steroidal glycosides from the rhizomes of Ruscus hypophyllum

Seven steroidal glycosides, along with one known glycoside, were isolated from the rhizomes of Ruscus hypophyllum (Liliaceae). Comprehensive spectroscopic analysis, including 2D NMR spectroscopy, and the results of acid hydrolysis allowed the chemical structures of the compounds to be assigned as (23S,25R)-23-hydroxyspirost-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (1), 1beta-hydroxyspirosta-5,25(27)-dien-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (2), (22S)-16beta,22-dihydroxycholest-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (3), (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (4), (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-3beta-yl beta-d-glucopyranoside (5), (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-3beta,22-dihydroxycholest-5-en-1beta-yl O-alpha-l-rhamnopyranosyl-(1-->2)-(3,4-di-O-acetyl-beta-d-xylopyranoside) (6), and (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-3beta,22-dihydroxycholest-5-en-1beta-yl O-alpha-l-rhamnopyranosyl-(1-->2)-O-[beta-d-xylopyranosyl-(1-->3)]-beta-d-xylopyranoside (7), respectively. This is the first isolation of a series of cholestane glycosides from a Ruscus species.     

Synthesis and chemical behaviour of D-glucosyl esters of glutamic acid having the side-chain carboxyl group involved in the glycosidic linkage.

Simultaneous and stepwise deprotection of the fully benzylated D-glucosyl esters of 1-benzyl N-benzyloxycarbonyl- and N-tert-butyloxycarbonyl-L-glutamic acid (1 and 5, respectively) have been examined. Catalytic hydrogenation of 1 led to intramolecular aminolysis to give pyroglutamic acid and D-glucose, but similar treatment in the presence of trifluoroacetic acid afforded both anomers of 1-O-(L-gamma-glutamyl)-D-glucopyranose, which were characterized as trifluoroacetates (2alpha and 2beta) and converted into 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(acetyl)-L-glutam-5-oyl]-D-glucopyranose (4) which was also prepared by a definitive method. Hydrogenolysis of 5 gave both anomers of 1-O-[N-(tert-butyloxycarbonyl)-L-gamma-glutamyl]-D-glucopyranose (6), which, upon treatment with trifluoroacetic acid at - 10 degrees, afforded 2alpha and 2beta, respectively. The structure of 6beta was established by its conversion into 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-beta-D-glucopyranose (7beta), whereas similar treatment of 6alpha gave a mixture of 1,3,4,6-tetra-O-acetyl-2-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-alpha-D-glucopyranose (9) and 7alpha. A 1 leads to 2 acyl migration occurred during esterification of the aglycon carboxyl group of 6alpha with diazomethane to give 2-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-alpha-D-glucopyranose (8).     

Synthesis, 3D-QSAR, and docking studies of 1-phenyl-1H-1,2,3-triazoles as selective antagonists for beta3 over alpha1beta2gamma2 GABA receptors

A series of 16 1-phenyl-1H-1,2,3-triazoles with substituents at both the 4- and 5-positions of the triazole ring were synthesized, and a total of 49 compounds, including previously reported 4- or 5-monosubstituted analogues, were examined for their ability to inhibit the specific binding of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist, to human homo-oligomeric beta3 and hetero-oligomeric alpha1beta2gamma2 gamma-aminobutyric acid (GABA) receptors. Among all tested compounds, the 4-n-propyl-5-chloromethyl analogue of 1-(2,6-dichloro-4-trifluoromethylphenyl)-1H-1,2,3-triazole showed the highest level of affinity for both beta3 and alpha1beta2gamma2 receptors, with K(i) values of 659pM and 266nM, respectively. Most of the tested compounds showed selectivity for beta3 over alpha1beta2gamma2 receptors. Among all 1-phenyl-1H-1,2,3-triazoles, the 4-n-propyl-5-ethyl analogue exhibited the highest (>1133-fold) selectivity, followed by the 4-n-propyl-5-methyl analogue of 1-(2,6-dibromo-4-trifluoromethylphenyl)-1H-1,2,3-triazole with a >671-fold selectivity. The 2,6-dichloro plus 4-trifluoromethyl substitution pattern on the benzene ring was found to be important for the high affinity for both beta3 and alpha1beta2gamma2 receptors. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) provided similar contour maps, revealing that an electronegative substituent at the 4-position of the benzene ring, a compact, hydrophobic substituent at the 4-position of the triazole ring, and a small, electronegative substituent at the 5-position of the triazole ring play significant roles for the high potency in beta3 receptors. Molecular docking studies suggested that the putative binding sites for 1-phenyl-1H-1,2,3-triazole antagonists are located in the channel-lining 2'-6' region of the second transmembrane segment of beta3 and alpha1beta2gamma2 receptors. A difference in the hydrophobic environment at the 2' position might underlie the selectivity of 1-phenyl-1H-1,2,3-triazoles for beta3 over alpha1beta2gamma2 receptors. The compounds that had high affinity for beta3 receptors with homology to insect GABA receptors showed insecticidal activity against houseflies with LD(50) values in the pmol/fly range. The information obtained in the present study should prove helpful for the discovery of selective insect control chemicals.     

Synthesis of deuterium-labeled tetrahydrocortisol and tetrahydrocortisone for study of cortisol metabolism in humans

A method is described for the preparation of multi-labeled tetrahydrocortisol (3alpha,11beta,17alpha,21-tetrahydroxy-5beta-[1, 2,3,4,5-2H5]pregnan-20-one, THF-d5), allo-tetrahydrocortisol (3alpha,11beta,17alpha,21-tetrahydroxy-5alpha-[1 ,2,3,4,5-2H5]pregnan-20-one, allo-THF-d5), and tetrahydrocortisone (3alpha,17alpha,21-trihydroxy-5beta-[1,2,3,4,5-2H5]pre gnane-11,20-dione, THE-d5) containing five non-exchangeable deuterium atoms in the steroid ring A. Reductive deuteration at C-1, C-2, C-3, C-4, and C-5 of prednisolone or prednisone was performed in CH3COOD with rhodium (5%) on alumina under the deuterium atmosphere. The isotopic purities of the labeled compounds as [2H5]-form were estimated to be 86.17 atom%D for THF-d5, 74.46 atom%D for allo-THF-d5 and 81.90 atom%D for THE-d5, based on the ion intensities in the region of the molecular ion of methoxime-trimethylsilyl (MO-TMS) derivatives measured by GC-MS.     

Characterization and specific assay for a galactoside beta-3-galactosyltransferase of human kidney

Two galactosyltransferases identified as UDP-galactose:lactose (lactosylceramide) alpha-4- and beta-3-galactosyltransferases [Bailly P. et al. (1986) Biochem. Biophys. Res. Commun. 141, 84-91] have been characterized in human kidney microsomes. Using methyl beta-D-galactoside as acceptor substrate, we have determined the experimental conditions (pH 5.0, 4 mM Cd2+) in which only the beta-3-galactosyltransferase activity is detectable. The reaction product has been characterized by chemical methods and glycosidase studies. Under these experimental conditions, some of the enzyme properties have been further investigated. Apparent Km values are for UDP-galactose, 0.170 mM; for lactose, 242 mM; and for lactosylceramide, 2.5 mM. Acceptor specificity studies suggest that the beta-3-galactosyltransferase is specific for terminal Gal beta 1-4Glc(NAc) residues and responsible for elongation of oligosaccharide chains in glycolipids. Competition studies with lactose and N-acetylgalactosamine as acceptor substrates indicate that the transferase described here can be distinguished from the UDP-galactose:2-acetamide-2-deoxy-D-galactose beta-3-galactosyltransferase and therefore represents a novel enzyme capable of synthesizing unusual carbohydrate structures similar to those which accumulate in certain neurological diseases.     

Biosynthesis of terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide alpha 1----3-galactosyltransferase from calf thymus

A UDP-Gal:Gal beta 1----4GlcNAc-R alpha 1----3- and a UDP-Gal:GlcNAc-R beta 1----4-galactosyltransferase have been purified 44,000- and 101,000-fold, respectively, from a Triton X-100 extract of calf thymus by affinity chromatography on UDP-hexanolamine-Sepharose and alpha-lactalbumin-Sepharose in a yield of 25-40%. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a major polypeptide species with a molecular weight of 40,000 and a minor form at Mr 42,000 for the alpha 1----3-galactosyltransferase and a major polypeptide with Mr 51,000 for the beta 1----4-galactosyltransferase. Analytical gel filtration on Sephadex G-100 yielded a monomeric form for each of the galactosyltransferases with Mr 43,000 and 59,000 respectively, in addition to peaks of activity at higher molecular weights. Isoelectric focussing of the alpha 1----3-galactosyltransferase revealed a significant charge heterogeneity with forms varying in pI values between 5.0 and 6.5. Acceptor specificity studies indicated that the purified alpha 1----3-galactosyltransferase was free from contaminating galactosyltransferase activities such as those involved in the synthesis of Gal beta 1----4GlcNAc-R and Gal beta 1----3GalNAc-R sequences, the blood group B determinant, the Pk antigen, trihexosylceramide, and ganglioside GM1. The alpha 1----3-galactosyltransferase appeared to be highly active with glycoproteins, oligosaccharides, and glycolipids having a terminal Gal beta 1----4GlcNAc beta 1----unit such as asialo-alpha 1-acid glycoprotein (Km = 1.25 mM), Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3Man beta 1----4GlcNAc (Km = 0.57 mM), and paragloboside. The action of the alpha 1----3-galactosyltransferase was found to be mutually exclusive with that of the NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase from bovine colostrum. In addition alpha 1----3-fucosylation of the N-acetylglucosamine residue in the preferred disaccharide acceptor structure completely blocked galactosylation of the alpha 1----3-galactosyltransferase.     

Complete enzymic synthesis of the mucin-type sialyl Lewis x epitope, involved in the interaction between PSGL-1 and P-selectin

Sialyl Lewis x (sLe(x)) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-alpha-D-galactosaminide (GalNAc(alpha1-pNp as core substrate, the sLe(x)-oligosaccharide Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)[Gal(bet a1-3)]GalNAc(alpha1-pNp, representing the O-linked form, was synthesized in an overall yield of 32%. In a first step, Gal(beta1-3)GalNAc(alpha1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of beta3-galactosyltransferase (EC 2.4.1.122) from rat liver. UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 beta6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc(alpha1-pNp in a yield of >85%. The core 2 structure was galactosylated using UDP-Gal and purified human milk beta4-galactosyltransferase 1 (EC 2.4.1.38) (yield of >85%), then sialylated using CMP-Neu5Ac and purified recombinant alpha3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human alpha3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%). Overall 1.5 micromol of product was prepared. MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY 1H NMR analysis confirmed the obtained structure.     

Modeling of deoxy- and dideoxyaldohexopyranosyl ring puckering with MM3(92)

Extensive variations of the ring structures of three deoxyaldohexopyranoses, L-fucose, D-quinovose, and L-rhamnose, and four dideoxyaldohexopyranoses, D-digitoxose, abequose, paratose, and tyvelose, were studied by energy minimization with the molecular mechanics algorithm MM3(92). Chair conformers, 4C(1) in D-quinovose and the equivalent 1C(4) in L-fucose and L-rhamnose, overwhelmingly dominate in the three deoxyhexoses; in the D-dideoxyhexoses, 4C(1) is again dominant, but with increased amounts of 1C(4) forms in the alpha anomers of the three 3,6-dideoxyhexoses, abequose, paratose, and tyvelose and in both alpha and beta anomers of the 2,6-dideoxyhexose D-digitoxose. In general, modeled proton-proton coupling constants agreed well with experimental values. Computed anomeric ratios strongly favor the beta configuration except for D-digitoxose, which is almost equally divided between alpha and beta configurations, and L-rhamnose, where the beta configuration is somewhat favored. MM3(92) appears to overstate the prevalence of the equatorial beta anomer in all three deoxyhexoses, as earlier found with fully oxygenated aldohexopyranoses.     

UDP-6-deoxy-6-fluoro-alpha-D-galactose binds to two different galactosyltransferases, but neither can effectively catalyze transfer of the modified galactose to the appropriate acceptor.

The effect of substitution of the HO-6 of D-galactose with fluorine on the ability of alpha-(1-->3)-galactosyltransferase (EC 2.4.1.151) and beta-(1-->4)-galactosyltransferase (EC 2.4.1.22) to catalyze its transfer from UDP to an appropriate acceptor was determined. HPLC analyses indicated that each transferase properly catalyzed formation of the expected product [beta-D-Gal-(1-->4)-D-GlcNAc] for the beta-(1-->4)-galactosyltransferase and alpha-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-D-GlcNAc for the alpha-(1-->3)-D-galactosyltransferase] when UDP-alpha-D-Gal was the substrate. When UDP-6-deoxy-6-fluoro-alpha-D-galactose (6) was used in conjunction with each transferase, no product indicative of transfer of 6-deoxy-6-fluoro-D-galactose to its respective acceptor sugar was identified. 6-Deoxy-6-fluoro-D-galactose (3) was obtained by hydrolysis of methyl 6-deoxy-6-fluoro-alpha-D-galactopyranoside, synthesized by the selective fluorination of methyl alpha-D-galactopyranoside with diethylaminosulfur trifluoride (DAST), with aqueous trifluoroacetic acid. Acetylation of 3 gave crystalline 1,2,3,4-tetra-O-acetyl-6-deoxy-6-fluoro-beta-D-galactopyranose, which was converted to the corresponding 1-alpha-phosphate and used for the synthesis of 6.