Sugar Precursors Research Articles

Novel glycosaminoglycan precursors as anti-amyloid agents part II.

In vivo amyloids consist of two classes of constituents. The first is the disease defining protein, e.g., A beta in Alzheimer's disease. The second is a set of common structural components that usually are the building blocks of basement membrane (BM), a tissue structure that serves as a scaffold onto which cells normally adhere. In vitro binding interactions between one of these BM components and amyloidogenic proteins rapidly change the conformation of the amyloidogenic protein into amyloid fibrils. The offending BM component is a heparan sulfate (HS) proteoglycan (HSPG), part of which is protein and the remainder a specific linear polysaccharide, which is the portion responsible for binding, and imparting the typical amyloid structure, to the amyloid precursor protein/peptide. Our past work has demonstrated that agents that inhibit the binding between HS and the amyloid precursor are effective anti-amyloid compounds both in vitro and in vivo. The present work is concerned with the design and synthesis of modified sugar precursors of HS, which, when incorporated into the polysaccharide, will alter its structure so that it loses its amyloid precursor protein/peptide-binding and fibril-inducing properties. As part of our continuing study, since our previous report, 17 additional compounds have been designed and synthesized based primarily on the known steps involved in HS biosynthesis. In addition to the 4 reported last year, 10 more have been assessed in tissue culture for their inhibitory effect on heparan sulfate synthesis, and one of these has been assessed for its AA-amyloid inhibitory properties. The majority of the novel sugars are analogues of N-acetylglucosamine. They have been modified either at the 4-OH, 3-OH, or 2-N positions. The majority of the 2-N analogues provide data suggesting that hepatocyte N-demethylases remove the N-substituents converting the 2-N analogues into the natural sugar, a process that dilutes the D-[3H] glucosamine tracer used to track heparan sulfate synthesis and thereby gives the impression that biosynthetic inhibition is occurring. To date 3-deoxy analogues have failed to affect heparan sulfate synthesis significantly. Compounds incorporating the 3,4-dideoxy structural feature are currently being assessed. Using primary hepatocyte cultures, we reported previously that a 4-deoxy analogue is incorporated into HS and terminates its elongation. From the 4-deoxy series, one of the compounds has now been assessed in an in vivo model of AA-amyloid induction. This 4-deoxy analogue inhibited splenic AA amyloid deposition by at least 50%, and liver AA amyloid deposition by 85% when measured as amyloid/unit area of tissue. Furthermore, the spleen weights of the treated group were 1/2-1/3 of that in the untreated group indicating that the total splenic amyloid was 1/4-1/6 of that in the untreated group. The results provide further evidence that heparan sulfate is a critical factor in amyloidogenesis and modifications of sugar precursors of heparan sulfate synthesis may provide leads for therapeutic intervention in amyloidogenesis.

Synthesis and studies of 3′- C-trifluoromethyl nucleoside analogues bearing adenine or cytosine as the base

3′-Deoxy-3′- C-CF 3, 2′,3′-dideoxy-3′- C-CF 3 and 2′,3′-unsaturated-3′- C-CF 3 nucleoside derivatives of adenosine and cytidine have been synthesized. All these derivatives were prepared by glycosylation of adenine and uracil with a suitable peracylated 3-trifluoromethyl sugar precursor. The resulting protected nucleosides were subject to appropriate chemical modifications to afford the target nucleoside derivatives. Additionally, the chemical stability in acidic and neutral media of the 2′,3′-dideoxy-3′- C-CF 3 and 2′,3′-unsaturated-3′- C-CF 3 nucleoside derivatives of adenosine was compared to that of their parent nucleosides 2′,3′-dideoxyadenosine (ddA) and 2′,3′-dideoxy-2′,3′-didehydroadenosine (d 4A). Our results confirm that addition of a trifluoromethyl group at C-3′ on such nucleoside derivatives appears to confer increased chemical stability toward acid-catalyzed cleavage of the glycosidic bond comparatively to their parent counterparts. When evaluated for their antiviral activity in cell culture experiments, two compounds, namely, 2′,3′-dideoxy-3′- C-CF 3-adenosine and 2′,3′-dideoxy-2′,3′-didehydro-3′- C-CF 3-cytidine exhibited moderate anti-HBV activity with EC 50 values of 10 and 5 μM, respectively.

Design and synthesis of a novel class of sugar-peptide hybrids: C-linked glyco beta-amino acids through a stereoselective "acetate" Mannich reaction as the key strategic element.

A new type of sugar-amino acid hybrid, which is comprised of a sugar unit (gluco-, galacto-, or mannopyranose) linked through a C-glycosidic linkage to the beta-position of an alpha-unsubstituted beta-amino acid unit, is presented. It is hypothesized that these new compounds, or the oligomeric peptides derived therefrom, might possess the structural features of beta-amino acid oligomers and the chemical and enzymatic resistance of C-glycosides to hydrolysis. The synthetic strategy is based on a new Mannich-type reaction between a chiral acetate enolate equivalent and alpha-amido sulfones derived from the corresponding sugar-C-glycoside aldehydes. While the sugar-C-glycoside aldehyde partner is prepared from well-established transformations on known sugar precursors, the lithium enolate derived from (1R)-endo-2-acetylisoborneol 3 is employed as the key element. This Mannich approach proceeds with essentially perfect diasteromeric control leading to the new beta-amino carbonyl adducts in good yields. Further, cleavage of the camphor auxiliary is smoothly performed by oxidative treatment with ammonium cerium nitrate (CAN). Complementarily, direct peptide-type coupling of the beta-amino carbonyl Mannich adducts with an alpha- or beta-amino acid residue and subsequent CAN-promoted detachment of the auxiliary yields dipeptide fragments bearing a sugar-containing aliphatic side chain and is a process that can be iterated. A preliminary conformational study based on the combination of experimental NMR data and molecular mechanics and molecular dynamics (MD) of one particular adduct is also provided.

Analyses of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues by fluorophore-assisted carbohydrate electrophoresis.

Lipid-linked oligosaccharides (LLOs) are the precursors of asparagine (N)-linked glycans, which are essential information carriers in many biological systems, and defects in LLO synthesis cause Type I congenital disorders of glycosylation. Due to the low abundance of LLOs and the limitations of the chemical and physical methods previously used to detect them, simple and sensitive nonradioactive methods for LLO analysis are lacking. Thus, almost all studies of LLO synthesis have relied on metabolic labeling of the oligosaccharides with radioactive sugar precursors. We report that LLOs in cell cultures and tissues can be easily detected and quantified with a sensitivity of 1-2 pmol by fluorophore-assisted carbohydrate electrophoresis (FACE). These analyses required efficient removal of contaminants, most likely trace quantities of glycogen breakdown products, that interfered with FACE. Studies with CHO-K1 cells showed that LLOs detected by FACE and by metabolic labeling had similar turnover rates. Glc(3)Man(9)GlcNAc(2)-P-P-dolichol was the most prominent LLO detected by FACE in normal cultured cells and mouse tissues. However, the relative amounts of Glc(0-2)Man(5-9)GlcNAc(2)-P-P-dolichol intermediates in tissues, such as liver and kidney, were unexpectedly greater than for cultured cells. IV injection of D-mannose, raising the circulatory concentration by three- to fourfold, did not affect LLO composition. Thus, the relative accumulation of LLO intermediates in mouse liver and kidney is not likely due to inadequate D-mannose in the circulation. In summary, FACE is a facile, accurate, and sensitive method for LLO analysis, permitting investigations not feasible by metabolic labeling.

Stereomeric Pyrrolidinopentoses Bearing an Imidazole Ring − Synthesis, Chiroptical Properties, and Evaluation as Potential Sugar-Mimic Glycosidase Inhibitors

The syntheses of the imidazolo-pyrrolidino-pentoses ent-2 (L-arabino), 3 (D-xylo), 4 (D-lyxo), ent-4 (L-lyxo), and 5 (D-ribo) are reported, completing the series of all eight possible stereomers. The corresponding five linear imidazolo sugar precursors were prepared by nucleophilic addition of C(4)-metallated imidazole derivatives to the appropriately configured and protected aldotetroses. Cyclisation of the resulting linear imidazolo-carbohydrates was performed by means of intramolecular Walden inversion processes, followed by deprotection to afford the five target imidazolo-sugars. Three of the four D-configured stereomers proved to be good to moderate glycosidase inhibitors, as determined by Michaelis−Menten kinetics.

The rkp-3 gene region of Sinorhizobium meliloti Rm41 contains strain-specific genes that determine K antigen structure.

The rkp-3 region is indispensable for capsular polysaccharide (K antigen) synthesis in Sinorhizobium meliloti Rm41. Strain Rm41 produces a K antigen of strain-specific structure, designated as the KR5 antigen. The data in this report show that the rkp-3 gene region comprises 10 open reading frames involved in bacterial polysaccharide synthesis and export. The predicted amino acid sequences for the rkpL-Q gene products are homologous to enzymes involved in the production of specific sugar moieties, while the putative products of the rkpRST genes show a high degree of similarity to proteins required for transporting polysaccharides to the cell surface. Southern analysis experiments using gene-specific probes suggest that genes involved in the synthesis of the precursor sugars are unique in strain Rm41, whereas sequences coding for export proteins are widely distributed among Sinorhizobium species. Mutations in the rkpL-Q genes result in a modified K antigen pattern and impaired symbiotic capabilities. On this basis, we suggest that these genes are required for the production of the KR5 antigen that is necessary for S. meliloti Rm41 exoB (AK631)-alfalfa (Medicago sativa) symbiosis.

Fastidian gum: the Xylella fastidiosa exopolysaccharide possibly involved in bacterial pathogenicity

The Gram-negative bacterium Xylella fastidiosa was the first plant pathogen to be completely sequenced. This species causes several economically important plant diseases, including citrus variegated chlorosis (CVC). Analysis of the genomic sequence of X. fastidiosa revealed a 12 kb DNA fragment containing an operon closely related to the gum operon of Xanthomonas campestris. The presence of all genes involved in the synthesis of sugar precursors, existence of exopolysaccharide (EPS) production regulators in the genome, and the absence of three of the X. campestris gum genes suggested that X. fastidiosa is able to synthesize an EPS different from that of xanthan gum. This novel EPS probably consists of polymerized tetrasaccharide repeating units assembled by the sequential addition of glucose-1-phosphate, glucose, mannose and glucuronic acid on a polyprenol phosphate carrier.

Fastidian gum: the Xylella fastidiosa exopolysaccharide possibly involved in bacterial pathogenicity.

The Gram-negative bacterium Xylella fastidiosa was the first plant pathogen to be completely sequenced. This species causes several economically important plant diseases, including citrus variegated chlorosis (CVC). Analysis of the genomic sequence of X. fastidiosa revealed a 12 kb DNA fragment containing an operon closely related to the gum operon of Xanthomonas campestris. The presence of all genes involved in the synthesis of sugar precursors, existence of exopolysaccharide (EPS) production regulators in the genome, and the absence of three of the X. campestris gum genes suggested that X. fastidiosa is able to synthesize an EPS different from that of xanthan gum. This novel EPS probably consists of polymerized tetrasaccharide repeating units assembled by the sequential addition of glucose-1-phosphate, glucose, mannose and glucuronic acid on a polyprenol phosphate carrier.

Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells.

Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation.

Reaction of sugar allyltins with aldehydes. A remarkable difference in reactivity between furanose and pyranose organometallic derivatives

The reaction of organometallic derivatives of monosaccharides with aldehydes catalyzed with BF 3·OEt 2 was studied. A significant difference in reactivity between the pyranosidic and furanosidic allyltins was noted. The former reacted readily with aldehydes affording precursors of higher carbon sugars with very high stereoselectivity, while the latter underwent rearrangement with elimination of the stannyl moiety prior to reaction with the aldehyde.

Analysis of the wee gene cluster responsible for the biosynthesis of the polymeric bioemulsifier from the oil-degrading strain Acinetobacter lwoffii RAG-1.

A cluster (27 kbp) of genes responsible for the biosynthesis of the amphipathic, polysaccharide bioemulsifier emulsan from the oil-degrading Acinetobacter lwoffii RAG-1 was isolated and characterized. The complete sequence of this cluster, termed wee, consisted of 20 ORFs. One set of 17 ORFs was transcribed in one direction, while a second set of three ORFs, 607 bp upstream of the first, was transcribed in the opposite direction. Mutations in either of the two regions caused defects in emulsan production, yielding specific activities of 5-14% of parental emulsifying activity. Putative functions could be assigned to proteins involved in production of nucleotide amino sugar precursors, transglycosylation, transacetylation, polymerization and transport. However, no JUMPstart or ops sequences, normally found associated with some polysaccharide biosynthetic gene clusters, were identified. Evidence is presented suggesting that the bioemulsifier may be a member of the group 1 or group 4 polysaccharides.

SYNTHESIS AND ANTIVIRAL EVALUATION OF 3′-C-TRIFLUOROMETHYL NUCLEOSIDE DERIVATIVES BEARING ADENINE AS THE BASE

3′-deoxy-3′-C-trifluoromethyl- (3), 2′,3′-dideoxy-3′-C-trifluoromethyl- (5) and 2′,3′-dideoxy-2′,3′-didehydro-3′-C-trifluoromethyladenosine (6) derivatives have been synthesized and their antiviral properties examined. All these derivatives were stereospecifically prepared by glycosylation of adenine with a trifluoromethyl sugar precursor (1), followed by appropriate chemical modifications. The prepared compounds were tested for their activity against HIV, but they did not show an antiviral effect.

Stereoisomeric Imidazolo-Pentoses − Synthesis, Chiroptical Properties, and Evaluation as Glycosidase Inhibitors

The syntheses of all four imidazolo-piperidino-pentoses in the L-series ent-2 to ent-5, and of three out of the four possible stereomers in the D-series 3, 4, and 5, are reported. The linear imidazolo sugar precursors were prepared, either by double condensation of formamidine with protected aldohexoses, or by nucleophilic addition of a lithiated imidazole derivative to protected aldotetroses. Cyclisation of these linear imidazolo-carbohydrates was performed by intramolecular SN2 reactions. These were followed by deprotection to the target molecules. The four pairs of opposite enantiomers showed pronounced mirror-image-type Cotton effects in their CD spectra. All stereomers of the D-series show a negative rotatory power ([α]D), while the stereomers of the L-series show a positive one. None of the eight imidazolo sugars inhibited the replication of HIV-1. Some of them proved to be rather selective but only moderately potent inhibitors of α-glycosidases, as determined by Michaelis-Menten kinetics.

Novel scintillation proximity assay for measuring membrane-associated steps of peptidoglycan biosynthesis in Escherichia coli.

We have developed a novel, high-throughput scintillation proximity assay to measure the membrane-associated steps (stages 2 and 3) of peptidoglycan synthesis in Escherichia coli. At least five enzymes are involved in these two stages, all of which are thought to be essential for the survival of the cell. The individual enzymes are difficult to assay since the substrates are lipidic and difficult to isolate in large quantities and analysis is done by paper chromatography. We have assayed all five enzymes in a single mixture by monitoring synthesis of cross-linked peptidoglycan, which is the final product of the pathway. E. coli membranes are incubated with the two sugar precursors, UDP-N-acetyl muramylpentapeptide and UDP-[(3)H]-N-acetylglucosamine. The radiolabel is incorporated into peptidoglycan, which is captured using wheat germ agglutinin-coated scintillation proximity assay beads. The assay monitors the activity of the translocase (MraY), the transferase (MurG), the lipid pyrophosphorylase, and the transglycosylase and transpeptidase activities of the penicillin-binding proteins. Vancomyin, tunicamycin, nisin, moenomycin, bacitracin, and penicillin inhibit the assay, and these inhibitors have been used to validate the assay. The search for new antimicrobial agents that act via the late stages of peptidoglycan biosynthesis can now be performed in high throughput in a microtiter plate.

Enzymes Leading to the Nucleotide Sugar Precursors for Exopolysaccharide Synthesis in Burkholderia cepacia

Based on the chemical composition of the exopolysaccharide produced by the cystic fibrosis bacterial isolate Burkholderia cepacia IST408, we postulated and confirmed, based on the specificity of enzymes detected in crude cell-free extracts, the pathway leading to the presumptive activated sugar precursors: UDP-D-glucose, UDP-D-galactose, UDP-D-glucuronic acid, GDP-D-mannose, and GDP-D-rhamnose. Results also suggest that regulation of the expression of the mucoid phenotype in B. cepacia does not occur at the level of synthesis of any of these enzymes.

Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin.

The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.

Mechanism of Type 3 Capsular Polysaccharide Synthesis inStreptococcus pneumoniae

The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae ([3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->](n)) are formed by the membrane-associated type 3 synthase (Cps3S), which is capable of synthesizing polymer from UDP sugar precursors. Using membrane preparations of S. pneumoniae in an in vitro assay, we observed type 3 synthase activity in the presence of either Mn(2+) or Mg(2+) with maximal levels seen with 10-20 mM Mn(2+). High molecular weight polymer synthesized in the assay was composed of Glc and glucuronic acid and could be degraded to a low molecular weight product by a type 3-specific depolymerase from Bacillus circulans. Additionally, the polymer bound specifically to an affinity column made with a type 3 polysaccharide-specific monoclonal antibody. The polysaccharide was rapidly synthesized from smaller chains and remained associated with the enzyme-containing membrane fraction throughout its synthesis, indicating a processive mechanism of synthesis. Release of the polysaccharide was observed, however, when the level of one of the substrates became limiting. Finally, addition of sugars to the growing type 3 polysaccharide was shown to occur at the nonreducing end of the polysaccharide chain.

Synthesis of A Novel Sialyl Lewis X Analogue Containing A Pyrrolidine in Place of N-Acetyl Glucosamine

ABSTRACT The synthesis of the new sialyl Lewis X analogue, 4-O-(α-L-fucopyranosyl)-3-O-(3-O-sodium sulfonato-β-D-galactopyranosyl)-(2S,3R, 4R)-2-ethyl-3,4-dihydroxypyrrolidine 2 has been achieved. The N-acetyl glucosamine unit of natural Lewis X has been replaced by a rigid 3R/4R-dihydroxylated pyrrolidine 12. This one has been synthezised from the methyl 4-O-benzoyl-2,3-di-O-benzyl-6-deoxy-6-iodo-α-D-altropyranoside sugar precursor 10 using the Ganem/Bernotas one-pot elimination-reductive amination ring contraction reaction. The (2S, 3R, 4R)-1-benzyloxycarbonyl-3,4-dihydroxy-2-ethylpyrrolidine 12 obtained was subsequently regioselectively glycosylated, using 2,3,4-tri-O-benzyl-α-L-fucopyranosyl fluoride and 2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl bromide as glycosyl donors. Disaccharide containing pyrrolidine 21 was finally transformed into the target O-sulfated analog 2, after regioselective sulfation and usual deprotection.

Molecular characterization of the locus encoding biosynthesis of the lipopolysaccharide O antigen of Escherichia coli serotype O113.

Shiga toxigenic Escherichia coli (STEC) strains are a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, eae-positive STEC strains, particularly those belonging to serogroups O157 and O111, appear to have greater virulence for humans. However, in spite of being eae negative, STEC strains belonging to serogroup O113 have frequently been associated with cases of severe STEC disease, including hemolytic-uremic syndrome (HUS). Western blot analysis with convalescent-phase serum from a patient with HUS caused by an O113:H21 STEC strain indicated that human immune responses were directed principally against lipopolysaccharide O antigen. Accordingly, the serum was used to isolate a clone expressing O113 O antigen from a cosmid library of O113:H21 DNA constructed in E. coli K-12. Sequence analysis indicated that the O113 O-antigen biosynthesis (rfb) locus contains a cluster of nine genes which may be cotranscribed. Comparison with sequence databases identified candidate genes for four glycosyl transferases, an O-acetyl transferase, an O-unit flippase, and an O-antigen polymerase, as well as copies of galE and gnd. Two additional, separately transcribed genes downstream of the O113 rfb region were predicted to encode enzymes involved in synthesis of activated sugar precursors, one of which (designated wbnF) was essential for O113 O-antigen synthesis, and so is clearly a part of the O113 rfb locus. Interestingly, expression of O113 O antigen by E. coli K-12 significantly increased in vitro adherence to both HEp-2 and Henle 407 cells.

Homologues of Isomeric Dideoxynucleosides as Potential Antiviral Agents: Synthesis of Isodideoxy-Nucleosides with a Furanethanol Sugar Moiety

The synthesis of a homologues series of compounds related to (R, S)-isodideoxynucleosides has been completed by coupling a variety of natural purine and pyrimidine bases with a modified sugar intermediate. This sugar precursor was prepared regiospecifically and stereospecifically from D-glucose.