Clickable Galactose Analogues for Imaging Glycans in Developing Zebrafish.

Galactose is one of only nine monosaccharide precursors used to build complex glycans in vertebrates. Defects in galactose metabolism cause galactosemia and lysosomal storage diseases, and the ability to visualize metabolic flux through these pathways would help to understand mechanisms underlying disease pathogenesis. Bioorthogonal metabolic reporters are widely used tools to image glycan biosynthesis; however, to date, no galactose analogues have capitalized on this strategy. We demonstrate that the galactose salvage pathway is remarkably intolerant of unnatural galactose and galactose-1-phosphate analogues. Subtle modifications to uridine diphosphate galactose (UDP-Gal), which is the universal donor for galactosyltransferases, however, yielded effective metabolic probes for labeling glycans in vivo. We applied 6-alkynyl UDP-Gal, followed by click chemistry tagging, to visualize glycosylation during zebrafish development, revealing a striking accumulation into glycan-rich ridges within the organism's enveloping layer. UDP-Gal analogues represent a new class of glycan metabolic probes for revealing physiological and pathological changes in glycosylation in vivo.

Base-modified donor analogues reveal novel dynamic features of a glycosyltransferase.

Glycosyltransferases (GTs) are enzymes that are involved, as Nature's "glycosylation reagents," in many fundamental biological processes including cell adhesion and blood group biosynthesis. Although of similar importance to that of other large enzyme families such as protein kinases and proteases, the undisputed potential of GTs for chemical biology and drug discovery has remained largely unrealized to date. This is due, at least in part, to a relative lack of GT inhibitors and tool compounds for structural, mechanistic, and cellular studies. In this study, we have used a novel class of GT donor analogues to obtain new structural and enzymological information for a representative blood group GT. These analogues interfere with the folding of an internal loop and the C terminus, which are essential for catalysis. Our experiments have led to the discovery of an entirely new active site folding mode for this enzyme family, which can be targeted in inhibitor development, similar to the DFG motif in protein kinases. Taken together, our results provide new insights into substrate binding, dynamics, and utilization in this important enzyme family, which can very likely be harnessed for the rational development of new GT inhibitors and probes.

One-pot three-enzyme synthesis of UDP-Glc, UDP-Gal, and their derivatives.

A UTP-glucose-1-phosphate uridylyltransferase (SpGalU) and a galactokinase (SpGalK) were cloned from Streptococcus pneumoniae TIGR4 and were successfully used to synthesize UDP-galactose (UDP-Gal), UDP-glucose (UDP-Glc), and their derivatives in an efficient one-pot reaction system. The reaction conditions for the one-pot multi-enzyme synthesis were optimized and nine UDP-Glc/Gal derivatives were synthesized. Using this system, six unnatural UDP-Gal derivatives, including UDP-2-deoxy-Galactose and UDP-GalN3 which were not accepted by other approach, can be synthesized efficiently in a one pot fashion. More interestingly, this is the first time it has been reported that UDP-Glc can be synthesized in a simpler one-pot three-enzyme synthesis reaction system.

Synthesis of 2-fluoro and 4-fluoro galactopyranosyl phosphonate analogues of UDP-Gal.

Two novel nonisosteric UDP-Gal analogues, (2-deoxy-2-fluoro- and 4-deoxy-4-fluoro-α-D-galactopyranosyl) phosphonoyl phosphates, were synthesized by optimized multistep procedures starting from 3,4,6-tri-O-benzyl-D-galactal and allyl 2,3,6-tri-O-benzyl-α-D-glucopyranoside, respectively. The key steps were a Michaelis-Arbuzov reaction of respective deoxy-fluoro-D-galactopyranosyl acetate with triethyl phosphite followed by a Moffatt-Khorana coupling reaction with UMP-morpholidate. The structure of all new compounds was confirmed by NMR and mass spectroscopies..

Inhibition of galactosyltransferases by a novel class of donor analogues.

Galactosyltransferases (GalT) are important molecular targets in a range of therapeutic areas, including infection, inflammation, and cancer. GalT inhibitors are therefore sought after as potential lead compounds for drug discovery. We have recently discovered a new class of GalT inhibitors with a novel mode of action. In this publication, we describe a series of analogues which provide insights, for the first time, into SAR for this new mode of GalT inhibition. We also report that a new C-glycoside, designed as a chemically stable analogue of the most potent inhibitor in this series, retains inhibitory activity against a panel of GalTs. Initial results from cellular studies suggest that despite their polarity, these sugar-nucleotides are taken up by HL-60 cells. Results from molecular modeling studies with a representative bacterial GalT provide a rationale for the differences in bioactivity observed in this series. These findings may provide a blueprint for the rational development of new GalT inhibitors with improved potency.

The first C-glycosidic analogue of a novel galactosyltransferase inhibitor.

Structural analogues and mimics of the natural sugar-nucleotide UDP-galactose (UDP-Gal) are sought after as chemical tools for glycobiology and drug discovery. We have recently developed a novel class of galactosyltransferase (GalT) inhibitors derived from UDP-Gal, bearing an additional substituent at the 5-position of the uracil base. Herein we report the first C-glycosidic derivative of this new class of GalT inhibitors. We describe a practical convergent synthesis of the new UDP-C-Gal derivative, including a systematic study into the use of radical chemistry for the preparation of galactosyl ethylphosphonate, a key synthetic intermediate. The new inhibitor showed activity against a bacterial UDP-Gal 4'-epimerase at micromolar concentrations. This is the first example of a base-modified UDP-sugar as an inhibitor of a UDP-sugar-dependent enzyme which is not a glycosyltransferase, and these results may therefore have implications for the design of inhibitors of these enzymes in the future.

A novel fluorescent probe for retaining galactosyltransferases.

Glycosyltransferases (GTs) are a large class of carbohydrate-active enzymes that are involved, in both pro- and eukaryotic organisms, in numerous important biological processes, from cellular adhesion to carcinogenesis. GTs have enormous potential as molecular targets for chemical biology and drug discovery. For the full realisation of this potential, operationally simple and generally applicable GT bioassays, especially for inhibitor screening, are indispensable tools. In order to facilitate the development of GT high-throughput screening assays for the identification of GT inhibitors, we have developed novel, fluorescent derivatives of UDP-galactose (UDP-Gal) that are recognised as donor analogues by several different retaining galactosyltransferases (GalTs). We demonstrate for one of these derivatives that fluorescence emission is quenched upon specific binding to individual GalTs, and that this effect can be used as the read-out in ligand-displacement experiments. The novel fluorophore acts as an excellent sensor for several different enzymes and is suitable for the development of a new type of GalT bioassay, whose modular nature and operational simplicity will significantly facilitate inhibitor screening. Importantly, the structural differences between the natural donor UDP-Gal and the new fluorescent derivatives are minimal, and the general assay principle described herein may therefore also be applicable to other GalTs and/or proteins that use nucleotides or nucleotide conjugates as their cofactor.

En route to deoxygenated N-acetyllactosamine analogues employing uridyl and galactosyl transferases.

All monodeoxygenated galactoses were treated with galactokinase, and for the 2-, 3-, and 4-deoxy compounds, transformation into the corresponding galactopyranosyl phosphates could be observed. In case of the 2-deoxy derivative, further reaction via UDP-2-deoxy-D-lyxo-hexose (UDP-2-deoxygalactose), which was also obtained chemically, the multiple enzymatic system could be employed to prepare 2'-deoxy-N-acetyllactosamine.

Trypanosoma brucei UDP-galactose-4'-epimerase in ternary complex with NAD+ and the substrate analogue UDP-4-deoxy-4-fluoro-alpha-D-galactose.

The structure of the NAD-dependent oxidoreductase UDP-galactose-4'-epimerase from Trypanosoma brucei in complex with cofactor and the substrate analogue UDP-4-deoxy-4-fluoro-alpha-D-galactose has been determined using diffraction data to 2.7 A resolution. Despite the high level of sequence and structure conservation between the trypanosomatid enzyme and those from humans, yeast and bacteria, the binding of the 4-fluoro-alpha-D-galactose moiety is distinct from previously reported structures. Of particular note is the observation that when bound to the T. brucei enzyme, the galactose moiety of this fluoro-derivative is rotated approximately 180 degrees with respect to the orientation of the hexose component of UDP-glucose when in complex with the human enzyme. The architecture of the catalytic centre is designed to effectively bind different orientations of the hexose, a finding that is consistent with a mechanism that requires the sugar to maintain a degree of flexibility within the active site.

Synthesis of uridine 5'-[2-S-pyridyl-3-thio-alpha-D-galactopyranosyl diphosphate]: precursor of UDP-thiogal sugar nucleotide donor substrate for beta-1,4-galactosyltransferase.

The syntheses of a novel uridine diphosphate galactose (UDP-Gal) analog, (UDP-2,4,6-tri-O-acetyl-3-S-acetyl-3-thio-alpha-D-galactopyranose) (11) and the thiolpyridine protected (Uridine 5'-[3-S-(2-S-pyridyl)-3-thio-alpha-D-galactopyranosyl diphosphate) analog (12) are described. The reported synthesis relies on the novel use of thiolpyridine to generate 12 which is a suitably protected intermediate for generating a UDP-thioGal derivative by reduction prior to enzyme transfer via beta-1,4-galactosyltransferase.

Molecular modeling insights into the catalytic mechanism of the retaining galactosyltransferase LgtC.

The bacterial enzyme lipopolysaccharyl alpha-galactosyltransferase C (EC 2.4.1.x, LgtC) is involved in the synthesis of lipooligosaccharides displayed on the cell surfaces of Neisseria meningitidis. LgtC catalyzes the transfer of a galactosyl residue from UDP-Gal to the terminal galactose residue of glycoconjugates with an overall retention of stereochemistry at the anomeric center. Several hypothetical catalytic mechanisms of the LgtC enzyme were examined herein using DFT quantum chemical methods up to the B3LYP/6-311++G**//B3LYP/6-31G* level. The computational model used to follow the reaction is based on the crystallographic structure of LgtC in complex with both the nucleotide-galactose donor and the oligosaccharide-acceptor analogues. The 136 atoms included in this model represent fragments of residues critical for the substrate binding and catalysis. From our calculations, the preferred pathway is predicted to be a one step mechanism with the nucleophilic attack of the acceptor oxygen onto the anomeric carbon and the proton transfer to a phosphate oxygen occurring simultaneously. This mechanism has an A(N)D(N)A(H)D(H) character, with the unique transition state structure in which the attacking galactose group is more closely bound to the anomeric carbon than to the UDP leaving group and where the hydrogen bond between the nucleophile and the leaving group oxygens facilitates the attack of the acceptor O4(') from the same side of the transferred galactose.

Thiosugar nucleotide analogs: synthesis of 5'-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-alpha-D-galactopyranosyl diphosphate).

The synthesis of a novel analog of uridine diphosphate galactose (UDP-Gal) is described. A sulfur atom was inserted into the 6-position of galactose to give uridine 5'-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-alpha-D-galactopyranosyl diphosphate). This peracetylated thiol analogue of UDP-Gal has been synthesized in nine steps starting from methyl alpha-D-galactopyranoside in an overall yield of 3%.

Thiosugar nucleotide analogues: synthesis of uridine 5'-(2,3,6-tri-O-acetyl-4-S-acetyl-4-thio-alpha-D-galactopyranosyl diphosphate).

The synthesis of a novel uridine diphosphate galactose (UDP-Gal) analog, (UDP-2,3,6-tri-O-acetyl-4-S-acetyl-4-thio-alpha-D-galactopyranose) (10) is described. Compound 10 contains a sulfur in the place of oxygen at the 4-position of the galactose moiety. Compound 10 represents a protected form of a novel sugar nucleotide analog that can potentially be used during chemoenzymatic synthesis to modify complex oligosaccharides.

Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs.

Many bacterial pathogens express lipooligosaccharides that mimic human cell surface glycoconjugates, enabling them to attach to host receptors and to evade the immune response. In Neisseria meningitidis, the galactosyltransferase LgtC catalyzes a key step in the biosynthesis of lipooligosaccharide structure by transferring alpha-d-galactose from UDP-galactose to a terminal lactose. The product retains the configuration of the donor sugar glycosidic bond; LgtC is thus a retaining glycosyltranferase. We report the 2 A crystal structures of the complex of LgtC with manganese and UDP 2-deoxy-2-fluoro-galactose (a donor sugar analog) in the presence and absence of the acceptor sugar analog 4'-deoxylactose. The structures, together with results from site-directed mutagenesis and kinetic analysis, give valuable insights into the unique catalytic mechanism and, as the first structure of a glycosyltransferase in complex with both the donor and acceptor sugars, provide a starting point for inhibitor design.

Enzymatic synthesis of UDP-(3-deoxy-3-fluoro)-D-galactose and UDP-(2-deoxy-2-fluoro)-D-galactose and substrate activity with UDP-galactopyranose mutase.

The novel UDP-sugar uridine 5'-(3-deoxy-3-fluoro-D-galactopyranosyl diphosphate) (1) and UDP-(2-deoxy-2-fluoro)-D-galactose (2) have been prepared enzymatically and tested as substrate analogues for the enzyme UDP-galactopyranose mutase (UDP-Galp mutase EC 5.4.99.9). Turnover of both 1 and 2 by UDP-Galp mutase was observed by HPLC and 19F NMR. The HPLC elution profile and 19F chemical shift of the products are consistent with the formation of the predicted furanose forms of 1 and 2. The Km values for compounds 1 and 2 were similar to those of the natural substrate UDP-Galp (0.26 mM for 1, 0.2 mM for 2, and 0.6 mM for UDP-Galp), but the values for kcat were substantially different (1.6/min for 1, 0.02/min for 2, and 1364/min for UDP-Galp). A correlation was also observed between the equilibrium yield of product formed during turnover of UDP-sugar by UDP-Galp mutase (UDP-Galp, compound 1 or compound 2), and the amount of furanose present for the free sugar at thermal equilibrium in aqueous solution, using 1H and 19F NMR spectroscopy. The implications of these results to the mechanism of the unusual enzymatic reaction are discussed.

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.

Specific inhibition of an alpha-galactosyltransferase from Trypanosoma brucei by synthetic substrate analogues.

Since the alpha-D-galactose-(1-->3)-D-galactose epitope has been identified to be the major target in the process of hyperacute rejection of xenografts transplanted from nonprimate donors to humans, specific inhibitors of alpha-galactosyltransferases are of broad interest. Using Trypanosoma brucei, a protozoan parasite causing sleeping sickness and Nagana, we have a very useful model system for the investigation of alpha-galactosyltransferase inhibitors, since the variant surface glycoprotein (VSG) accounts for about 10% of the total cell protein an this parasite expresses many different galactosyltransferases including the one catalysing the formation of the Galalpha1-->3Gal epitope. In order to study inhibition of galactosylation on the VSG from Trypanosoma brucei, we designed, synthesized and tested substrate analogues of trypanosomal alpha-galactosyltransferases. Effective inhibitors were a pair of diastereoisomeric UDP-galactose analogs, in which the galactose residue is linked to UDP via a methylene bridge rather than an ester linkage. Hence, galactose cannot be transferred to the respective acceptor substrate VSG or the synthetic acceptor substrate Manalpha1-->6Manalpha1S-(CH2)7-CH3, which was previously proven to replace VSG effectively [Smith et al. (1996) J Biol Chem 271:6476-82]. Inhibitors have been prepared starting from 1-formyl galactal. The final condensation was performed using UMP morpholidate leading to a pair of diastereomeric compounds in 39% or 30% yield, respectively. These compounds were tested using alpha-galactosyltransferases prepared from T. brucei membranes and lactose synthetase from bovine milk. While the K(M)-value for UDP-galactose was determined as 59 microM on bovine lactose synthetase, the K(I)-values for both inhibitors were 0.3 mM and 1.1 mM respectively, showing that these inhibitors are unable to inhibit enzyme activity significantly. However, using the N-glycan specific alpha-galactosyltransferase from trypanosomes, the K(M)-value was determined as 20 microM, while the K(I)-values were 34 microM and 21 microM respectively. Interestingly, other trypanosomal alpha-galactosyltransferases, which modify the GPI membrane anchor, are 2 orders of magnitude less effected by the inhibitor.

Quantitative electrospray mass spectrometry for the rapid assay of enzyme inhibitors.

BACKGROUND: Combinatorial chemistry has become an important method for identifying effective ligand-receptor binding, new catalysts and enzyme inhibitors. In order to distinguish the most active component of a library or to obtain structure-activity relationships of compounds in a library, an efficient quantitative assay is crucial. Electrospray mass spectrometry has become an indispensable tool for qualitatively screening combinatorial libraries and its use for quantitative analysis has recently been demonstrated. RESULTS: This paper describes the use of quantitative electrospray mass spectrometry for screening libraries of inhibitors of enzymatic reactions, specifically the enzymatic glycosylation by beta-1,4-galactosyltransferase, which catalyzes the transfer of galactose from uridine-5'-diphosphogalactose to the 4-position of N-acetylglucosamine beta OBn (Bn: benzene) to form N-acetyllactosamine beta OBn. Our mass spectrometric screening approach showed that both nucleoside diphosphates and triphosphates inhibited galactosyltransferase while none of the nucleoside monophosphates, including uridine-5'-monophosphate, showed any inhibition. Additional libraries were generated in which the concentrations of the inhibitors were varied and, using mass spectrometry, uridine-5'-diphosphate-2-deoxy-2-fluorogalactose was identified as the best inhibitor. CONCLUSIONS: This report introduces quantitative electrospray mass spectrometry as a rapid, sensitive and accurate quantitative assaying tool for inhibitor libraries that does not require a chromophore or radiolabeling. A viable alternative to existing analytical techniques is thus provided. The new technique will greatly facilitate the discovery of novel inhibitors against galactosyltransferase, an enzyme for which there are few potent inhibitors.

A chemoenzymatic synthesis of UDP-(2-deoxy-2-fluoro)-galactose and evaluation of its interaction with galactosyltransferase.

Uridine 5'-diphospho-(2-deoxy-2-fluoro)galactose (UDP-2FGal), prepared and characterized for the first time by a chemoenzymatic method, was found to be a competitive inhibitor of beta-1,4-galactosyltransferase with a Ki value of 149 microM. This study supports that the glycosyltransferase reaction mechanism proceeds through a glycosidic cleavage transition state with sp2 character developed at the anomeric center.