Interaction of Neisseria meningitidis Group X N-acetylglucosamine-1-phosphotransferase with its donor substrate.

Neisseria meningitidis Group X is an emerging cause of bacterial meningitis in Sub-Saharan Africa. The capsular polysaccharide of Group X is a homopolymer of N-acetylglucosamine α(1-4) phosphate and is a vaccine target for prevention of disease associated with this meningococcal serogroup. We have demonstrated previously that the formation of the polymer is catalyzed by a phosphotransferase which transfers N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the 4-hydroxyl of the N-acetylglucosamine on the nonreducing end of the growing chain. In this study, we use substrate analogs of UDP-GlcNAc to define the enzyme/donor substrate interactions critical for catalysis. Our kinetic analysis of the phosphotransferase reaction is consistent with a sequential mechanism of substrate addition and product release. The use of novel uracil modified analogs designed by Wagner et al. enabled us to assess whether the CsxA-catalyzed reaction is consistent with a donor dependent conformational change. As expected with this model for glycosyltransferases, UDP-GlcNAc analogs with bulky uracil modifications are not substrates but are inhibitors. An analog with a smaller iodo uracil substitution is a substrate and a less potent inhibitor. Moreover, our survey of analogs with modifications on the N-acetylglucosamine residue of the sugar nucleotide donor highlights the importance of substituents at C2 and C4 of the sugar residue. The hydroxyl group at C4 and the structure of the acyl group at C2 are very important for specificity and substrate interactions during the polymerization reaction. While most analogs modified at C2 were inhibitors, acetamido analogs were also substrates suggesting the importance of the carbonyl group.

Enzymatic synthesis of nucleobase-modified UDP-sugars: scope and limitations.

Glucose-1-phosphate uridylyltransferase in conjunction with UDP-glucose pyrophosphorylase was found to catalyse the conversion of a range of 5-substituted UTP derivatives into the corresponding UDP-galactose derivatives in poor yield. Notably the 5-iodo derivative was not converted to UDP-sugar. In contrast, UDP-glucose pyrophosphorylase in conjunction with inorganic pyrophosphatase was particularly effective at converting 5-substituted UTP derivatives, including the iodo compound, into a range of gluco-configured 5-substituted UDP-sugar derivatives in good yields. Attempts to effect 4"-epimerization of these 5-substituted UDP-glucose with UDP-glucose 4"-epimerase from yeast were unsuccessful, while use of the corresponding enzyme from Erwinia amylovora resulted in efficient epimerization of only 5-iodo-UDP-Glc, but not the corresponding 5-aryl derivatives, to give 5-iodo-UDP-Gal. Given the established potential for Pd-mediated cross-coupling of 5-iodo-UDP-sugars, this provides convenient access to the galacto-configured 5-substituted-UDP-sugars from gluco-configured substrates and 5-iodo-UTP.

Exploring the role of the 5-substituent for the intrinsic fluorescence of 5-aryl and 5-heteroaryl uracil nucleotides: a systematic study.

Derivatives of UMP (uridine monophosphate) with a fluorogenic substituent in position 5 represent a small but unique class of fluorophores, which has found important applications in chemical biology and biomolecular chemistry. In this study, we have synthesised a series of derivatives of the uracil nucleotides UMP, UDP and UTP with different aromatic and heteroaromatic substituents in position 5, in order to systematically investigate the influence of the 5-substituent on fluorescence emission. We have determined relevant photophysical parameters for all derivatives in this series, including quantum yields for the best fluorophores. The strongest fluorescence emission was observed with a 5-formylthien-2-yl substituent in position 5 of the uracil base, while the corresponding 3-formylthien-2-yl-substituted regioisomer was significantly less fluorescent. The 5-(5-formylthien-2-yl) uracil fluorophore was studied further in solvents of different polarity and proticity. In conjunction with results from a conformational analysis based on NMR data and computational experiments, these findings provide insights into the steric and electronic factors that govern fluorescence emission in this class of fluorophores. In particular, they highlight the interplay between fluorescence emission and conformation in this series. Finally, we carried out ligand-binding experiments with the 5-(5-formylthien-2-yl) uracil fluorophore and a UDP-sugar-dependent glycosyltransferase, demonstrating its utility for biological applications. The results from our photophysical and biological studies suggest, for the first time, a structural explanation for the fluorescence quenching effect that is observed upon binding of these fluorophores to a target protein.

Optimised chemical synthesis of 5-substituted UDP-sugars and their evaluation as glycosyltransferase inhibitors.

We have investigated the applicability of different chemical methods for pyrophosphate bond formation to the synthesis of 5-substituted UDP-galactose and UDP-N-acetylglucosamine derivatives. The use of phosphoromorpholidate chemistry, in conjunction with N-methyl imidazolium chloride as the promoter, was identified as the most reliable synthetic protocol for the preparation of these non-natural sugar-nucleotides. Under these conditions, the primary synthetic targets 5-iodo UDP-galactose and 5-iodo UDP-N-acetylglucosamine were consistently obtained in isolated yields of 40-43%. Both 5-iodo UDP-sugars were used successfully as substrates in the Suzuki-Miyaura cross-coupling with 5-formylthien-2-ylboronic acid under aqueous conditions. Importantly, 5-iodo UDP-GlcNAc and 5-(5-formylthien-2-yl) UDP-GlcNAc showed moderate inhibitory activity against the GlcNAc transferase GnT-V, providing the first examples for the inhibition of a GlcNAc transferase by a base-modified donor analogue.

One-step synthesis of novel glycosyltransferase inhibitors.

A one-step synthesis of two 5-CF(3) UDP-sugars is reported. These non-natural sugar-nucleotides are micromolar inhibitors of two different galactosyltransferases.

[2+2] photocycloadditions of thiomaleimides.

Thiomaleimides, generated by the addition of bromomaleimides to thiols including cysteine, undergo highly efficient [2+2] photocycloadditions.

Protein modification, bioconjugation, and disulfide bridging using bromomaleimides.

The maleimide motif is widely used for the selective chemical modification of cysteine residues in proteins. Despite widespread utilization, there are some potential limitations, including the irreversible nature of the reaction and, hence, the modification and the number of attachment positions. We conceived of a new class of maleimide which would address some of these limitations and provide new opportunities for protein modification. We report herein the use of mono- and dibromomaleimides for reversible cysteine modification and illustrate this on the SH2 domain of the Grb2 adaptor protein (L111C). After initial modification of a protein with a bromo- or dibromomaleimide, it is possible to add an equivalent of a second thiol to give further bioconjugation, demonstrating that bromomaleimides offer opportunities for up to three points of attachment. The resultant protein-maleimide products can be cleaved to regenerate the unmodified protein by addition of a phosphine or a large excess of a thiol. Furthermore, dibromomaleimide can insert into a disulfide bond, forming a maleimide bridge, and this is illustrated on the peptide hormone somatostatin. Fluorescein-labeled dibromomaleimide is synthesized and inserted into the disulfide to construct a fluorescent somatostatin analogue. These results highlight the significant potential for this new class of reagents in protein modification.

Bromomaleimides: new reagents for the selective and reversible modification of cysteine.

Bromomaleimides react rapidly and selectively with cysteine to afford thiomaleimides which can be cleaved with a phosphine to regenerate the cysteine or treated with a base to afford dehydroalanine.

In situ reduction in photocycloadditions: a method to prevent secondary photoreactions.

Secondary photoreactions are a common cause for the low yields often observed in photochemical reactions, preventing their widespread deployment in synthesis. In situ reductions which remove the chromophore from the product as it is formed provide a convenient method to prevent these secondary photoreactions.