Todd L. Lowary

Chemistry and biology of galactofuranose-containing polysaccharides.

The thermodynamically less stable form of galactose—galactofuranose (Galf)—is essential for the viability of several pathogenic species of bacteria and protozoa but absent in this form in mammals, so the biochemical pathways by which Galf‐containing glycans are assembled and catabolysed are attractive sites for drug action. This potential has led to increasing interest in the synthesis of molecules containing Galf residues, their subsequent use in studies directed towards understanding the enzymes that process these residues and the identification of potential inhibitors of these pathways. Major achievements of the past several years have included an in‐depth understanding of the mechanism of UDP‐galactopyranose mutase (UGM), the enzyme that produces UDP‐Galf, which is the donor species for galactofuranosyltransferases. A number of methods for the synthesis of galactofuranosides have also been developed, and practitioners in the field now have many options for the initiation of a synthesis of glycoconjugates containing either α‐ or β‐Galf residues. UDP‐Galf has also been prepared by a number of approaches, and it appears that a chemoenzymatic approach is currently the most viable method for producing multi‐milligram amounts of this important intermediate. Recent advances both in the understanding of the mechanism of UGM and in the synthesis of galactofuranose and its derivatives are highlighted in this review.

The mannose cap of mycobacterial lipoarabinomannan does not dominate the Mycobacterium–host interaction

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL−10 production via interaction with the mannose receptor or DC‐SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose‐dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC‐SIGN‐transfected Raji cells, but no differences in IL‐10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose‐capped LAM does not dominate the Mycobacterium–host interaction.

Recent advances in the synthesis of 2-deoxy-glycosides

Glycosides of 2-deoxy-sugars, monosaccharides in which the hydroxyl group at C-2 is replaced with a hydrogen atom, occur widely in natural products and therefore have been the subject of intense synthetic activity. The report summarizes recent advances in this area, with a particular focus on work published since an earlier review on the topic, in 2000 (Marzabadi, C. H.; Franck, R. W. Tetrahedron2000, 56, 8385-8417).

Galactosyl Transferases in Mycobacterial Cell Wall Synthesis

Two galactosyl transferases can apparently account for the full biosynthesis of the cell wall galactan of mycobacteria. Evidence is presented based on enzymatic incubations with purified natural and synthetic galactofuranose (Galf) acceptors that the recombinant galactofuranosyl transferase, GlfT1, from Mycobacterium smegmatis, the Mycobacterium tuberculosis Rv3782 ortholog known to be involved in the initial steps of galactan formation, harbors dual beta-(1-->4) and beta-(1-->5) Galf transferase activities and that the product of the enzyme, decaprenyl-P-P-GlcNAc-Rha-Galf-Galf, serves as a direct substrate for full polymerization catalyzed by another bifunctional Galf transferase, GlfT2, the Rv3808c enzyme.

Recognition of synthetic O-methyl, epimeric, and amino analogues of the acceptor α-L-Fucp-(1 → 2)-β-D-Galp-OR glycosyltransferases

Abstract The disaccharid α-L-Fuc p -(1 → 2)-β-D-Gal p -O-(CH 2 ) 7 CH 3 ( 6 is an acceptor for the glycosyltransferases responsible for the biosynthesis of the A and B blood-group antigens. These enzymes respectively transfer GalNAc and Gal in a α linkage to OH-3 of the Gal residue in (bd6). All eight possible O-methyl, epimeric, and amino analogues of (bd6) having modifications on the target Gal residue were chemically synthesized and kinetically evaluated both as substrates and inhibitors for the A and B glycosyltransferases. The results support earlier findings that both enzymes will tolerate replacement of the hydroxyl groups at the 3 and 6 positions of the Gal residue. Substitution at or replacement of )H-4 of Gal residue, however abolishes recognition. The 6- O -methyl and 6-amino compounds are substrates for both enzymes while the 3-epimeric (bd10) and 3-amino (bd12) compounds are inhibitors. For The B transferase, 10 is a competitive inhibitor with a K i of 7.8 μM. Attempts to determine a K i for 12 with the B transferase were unsuccesful because of a complex mode of inhibiton. Similarly, both 10 and 12 are potent inhibitors of the A trasferase, but the inhibition constants could not be calculated because of a complex mode of inhibition, resembling that for the B transferase, resembling that for the B transferase. With the A transferase, 12 had an estimated K i in the 200 nM range.

ABO(H) blood group A and B glycosyltransferases recognize substrate via specific conformational changes.

The final step in the enzymatic synthesis of the ABO(H) blood group A and B antigens is catalyzed by two closely related glycosyltransferases, an α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and an α-(1→3)-galactosyltransferase (GTB). Of their 354 amino acid residues, GTA and GTB differ by only four “critical” residues. High resolution structures for GTB and the GTA/GTB chimeric enzymes GTB/G176R and GTB/G176R/G235S bound to a panel of donor and acceptor analog substrates reveal “open,” “semi-closed,” and “closed” conformations as the enzymes go from the unliganded to the liganded states. In the open form the internal polypeptide loop (amino acid residues 177-195) adjacent to the active site in the unliganded or H antigen-bound enzymes is composed of two α-helices spanning Arg180-Met186 and Arg188-Asp194, respectively. The semi-closed and closed forms of the enzymes are generated by binding of UDP or of UDP and H antigen analogs, respectively, and show that these helices merge to form a single distorted helical structure with alternating α-310-α character that partially occludes the active site. The closed form is distinguished from the semi-closed form by the ordering of the final nine C-terminal residues through the formation of hydrogen bonds to both UDP and H antigen analogs. The semi-closed forms for various mutants generally show significantly more disorder than the open forms, whereas the closed forms display little or no disorder depending strongly on the identity of residue 176. Finally, the use of synthetic analogs reveals how H antigen acceptor binding can be critical in stabilizing the closed conformation. These structures demonstrate a delicately balanced substrate recognition mechanism and give insight on critical aspects of donor and acceptor specificity, on the order of substrate binding, and on the requirements for catalysis.

Synthesis of Galactofuranose-Containing Acceptor Substrates for Mycobacterial Galactofuranosyltransferases

The major structural component of the cell wall in Mycobacterium tuberculosis, infection by which causes tuberculosis, is the mycolyl-arabinogalactan (mAG) complex. This large glycoconjugates has at its core a backbone of approximately 30 D-galactofuranose (Gal(f)) residues that are linked to peptidoglycan by way of a linker disaccharide containing L-rhamnose and 2-acetamido-2-deoxy-D-glucose. Recent studies have supported a model of galactan biosynthesis in which the entire structure is assembled by the action of two bifunctional galactofuranosyltransferases. These biochemical investigations were made possible, in part, by access to a panel of oligosaccharide fragments of the mAG complex (1-12), the synthesis of which we describe here. An early key finding in this study was that the iodine-promoted cyclization of galactose diethyl dithioacetal (19) in the presence of an alcohol solvent led to the formation Gal(f) glycosides contaminated with no pyranoside isomer, thus allowing the efficient preparation of furanoside derivatives of this monosaccharide. The synthesis of disaccharide targets 1, 2, 11 and 12 proceeded without difficulty through the use of thioglycoside donors and octyl glycoside acceptors, both carrying benzoyl protection. In the synthesis of the tri- and tetrasaccharides 3-6, we explored routes in which the molecule was assembled from the reducing to nonreducing end, and the reverse. The latter approach was found to be preferable for the preparation of 6, and in the case of 3 and 4, this strategy allowed the development of efficient one-pot methods for their synthesis. We have also carried out the first synthesis of three mAG fragments (8-10) consisting of the linker disaccharide further elaborated with one, two or three Gal(f) residues. A key step in the synthesis of these target compounds was the coupling of a protected linker disaccharide derivative (58) with a mono-, di-, or trigalactofuranosyl thioglycoside (17, 54, or 53, respectively).

Biosynthesis of mycobacterial arabinogalactan: identification of a novel α(1→3) arabinofuranosyltransferase

The cell wall mycolyl‐arabinogalactan–peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis and is the target of several antitubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt‐EmbA and Mt‐EmbB. A bioinformatics approach identified putative integral membrane proteins, MSMEG2785 in Mycobacterium smegmatis, Rv2673 in Mycobacterium tuberculosis and NCgl1822 in Corynebacterium glutamicum, with 10 predicted transmembrane domains and a glycosyltransferase motif (DDX), features that are common to the GT‐C superfamily of glycosyltransferases. Deletion of M. smegmatis MSMEG2785 resulted in altered growth and glycosyl linkage analysis revealed the absence of AG α(1→3)‐linked arabinofuranosyl (Araf) residues. Complementation of the M. smegmatis deletion mutant was fully restored to a wild‐type phenotype by MSMEG2785 and Rv2673, and as a result, we have now termed this previously uncharacterized open reading frame, arabinofuranosyltransferase C (aftC). Enzyme assays using the sugar donor β‐d‐arabinofuranosyl‐1‐monophosphoryl‐decaprenol (DPA) and a newly synthesized linear α(1→5)‐linked Ara5 neoglycolipid acceptor together with chemical identification of products formed, clearly identified AftC as a branching α(1→3) arabinofuranosyltransferase. This newly discovered glycosyltransferase sheds further light on the complexities of Mycobacterium cell wall biosynthesis, such as in M. tuberculosis and related species and represents a potential new drug target.

Recognition of synthetic deoxy and deoxyfluoro analogs of the acceptor α-L-Fucp-(1→2)-β-D-Galp-OR by the blood-group A and B gene-specified glycosyltransferases

Abstract The disaccharide α- l -Fuc p -O-(CH 2 ) 7 CH 3 (6) , is an acceptor for both glycosyltransferases responsible for the biosynthesis of the A and B blood-group antigens. These enzymes transfer GalNAc and Gal, respectively, with an α-linkage to OH-3 of the Gal residue in 6 . All six possible deoxy and deoxyfluoro analogs of 6 , with modifications on the target Gal residue, were chemically synthesized and kinetically evaluated as both substrates and inhibitors for the A and B glycosyltransferases. Both enzymes will tolerate replacement of the hydroxyl groups at the 3 and 6 positions of the Gal residue. Substitution of OH-4 of the Gal residue, however, abolishes recognition by these glycosyltransferases. The 6-deoxy and 6-fluoro compounds are substrates for both enzymes while the 3-deoxy and 3-flouro compounds are competitive inhibitors, with K i values in the range 14–110 μM. Kinetic constants have been determined for the 6-deoxy and 6-fluoro derivatives.

Recent progress towards the identification of inhibitors of mycobacterial cell wall polysaccharide biosynthesis.

Mycobacterial infections have recently attracted significant attention from international health agencies due to the resurgence of these diseases worldwide. This review summarizes the recent work directed towards the identification of new anti-tuberculosis agents that act by inhibiting mycobacterial cell wall polysaccharide biosynthesis.