Characterization of PglJ, a Glycosyltransferase in the Campylobacter concisus N-Linked Protein Glycosylation Pathway that Expands Glycan Diversity.

The Campylobacter genus of Gram-negative bacteria is characterized by the expression of N-linked protein glycosylation (pgl) pathways. As Campylobacter concisus is an emerging human pathogen, a better understanding of the variation of the biosynthetic pathways across the genus is necessary to identify the relationships between protein glycosylation and disease. The pgl pathways of C. concisus strains have been reported to diverge from other Campylobacter in steps after the biosynthesis of N-acetylgalactosamine-α1,3-N,N'-diacetylbacillosamine-α-1-diphosphate undecaprenyl (GalNAc-diNAcBac-PP-Und), which is catalyzed by PglC and PglA, a phosphoglycosyltransferase (PGT) and a glycosyltransferase (GT), respectively. Here we characterize the PglJ GTs from two strains of C. concisus. Chemical synthesis was employed to access the stereochemically defined glycan donor substrates, uridine diphosphate N-acetyl-d-galactosaminuronic acid (UDP-GalNAcA) and uridine diphosphate N-acetyl-d-glucosaminuronic acid (UDP-GlcNAcA), to allow biochemical investigation of PglJ. Evidence for the PglJ substrate specificity structural determinants for the C6″ carboxylate-containing sugar was obtained through variant-based biochemical assays. Additionally, characterization of a UDP-sugar dehydrogenase encoded in the pgl operon, which is similar to the Pseudomonas aeruginosa WbpO responsible for the oxidization of a UDP-HexNAc to UDP-HexNAcA, supports the availability of a UDP-HexNAcA substrate for a GT that incorporates the modified sugar and provides evidence for the presence of a HexNAcA in the N-linked glycan. Utilizing sequence similarity network (SSN) analysis, we identified conserved sequence motifs among PglJ glycosyltransferases, shedding light on substrate preferences and offering predictive insights into enzyme functions across the Campylobacter genus. These studies now allow detailed characterization of the later steps in the pgl pathway in C. concisus strains and provide insights into enzyme substrate specificity determinants for glycan assembly enzymes.

Dual Glycosyltransferases from Campylobacter concisus Diverge from the Canonical Campylobacter N-Linked Glycan Assembly Pathway.

Species within the Campylobacter genus are recognized as emerging human pathogens. Common to all known members of the genus is the presence of an asparagine-linked glycosylation pathway encoded by the pgl operon. Campylobacter species are divided into two major groups, Group I and Group II. To date, most biochemical studies have focused on the Group I species including Campylobacter jejuni. We recently reported that the Group II Campylobacter concisus pathway deviates from that of Group I by the inclusion of a C-6″-oxidized GalNAc (GalNAcA) at the third position installed by PglJ. Herein, we investigate the diversification of the PglH enzymes that act subsequent to installation of GalNAcA. The majority of pgl operons from Group II species, including C. concisus, encode two GT-B fold glycosyltransferases (GTs), PglH1 and PglH2. As the functions of these GTs were not clear by simple comparison of their sequences to that of C. jejuni PglH, further analyses were required. We show that subsequent to the action of PglJ, PglH2 installs the next HexNAc followed by PglH1 adding a single sugar. These steps diverge from the C. jejuni pathway not only in the identity of the sugar donors (UDP-GlcNAc) but also in installing single sugars rather than acting processively. These biochemical studies were extended via bioinformatics to identify sequence signatures that provide predictive capabilities for unraveling the prokaryotic glycan landscape. Phylogenetic analysis showed early divergence between the C. jejuni PglH orthologs and C. concisus PglH1/PglH2 orthologs, leading to diversification of the final glycan.

Deploying solid-phase synthesis to access thymine-containing nucleoside analogs that inhibit DNA repair nuclease SNM1A.

Nucleoside analogs show useful bioactive properties. A versatile solid-phase synthesis that readily enables the diversification of thymine-containing nucleoside analogs is presented. The utility of the approach is demonstrated with the preparation of a library of compounds for analysis with SNM1A, a DNA damage repair enzyme that contributes to cytotoxicity. This exploration provided the most promising nucleoside-derived inhibitor of SNM1A to date with an IC50 of 12.3 µM.

Recent advances in the synthesis of C-terminally modified peptides.

C-Terminally modified peptides are important for the development and delivery of peptide-based pharmaceuticals because they impact peptide activity, stability, hydrophobicity, and membrane permeability. Additionally, the vulnerability of C-terminal esters to cleavage by endogenous esterases makes them excellent pro-drugs. Methods for post-SPPS C-terminal functionalization potentially enable access to libraries of modified peptides, facilitating tailoring of their solubility, potency, toxicity, and uptake pathway. Apparently minor structural changes can significantly impact the binding, folding, and pharmacokinetics of the peptide. This review summarizes developments in chemical methods for C-terminal modification of peptides published since the last review on this topic in 2003.

Uridine natural products: Challenging targets and inspiration for novel small molecule inhibitors.

Nucleoside derivatives, in particular those featuring uridine, are familiar components of the nucleoside family of bioactive natural products. The structural complexity and biological activities of these compounds have inspired research from organic chemistry and chemical biology communities seeking to develop novel approaches to assemble the challenging molecular targets, to gain inspiration for enzyme inhibitor development and to fuel antibiotic discovery efforts. This review will present recent case studies describing the total synthesis and biosynthesis of uridine natural products, and de novo synthetic efforts exploiting features of the natural products to produce simplified scaffolds. This research has culminated in the development of complementary strategies that can lead to effective uridine-based inhibitors and antibiotics. The strengths and challenges of the juxtaposing methods will be illustrated by examining select uridine natural products. Moreover, structure-activity relationships (SAR) for each natural product-inspired scaffold will be discussed, highlighting the impact on inhibitor development, with the aim of future uridine-based small molecule expansion.

Bringing Macrolactamization Full Circle: Self-Cleaving Head-to-Tail Macrocyclization of Unprotected Peptides via Mild N-Acyl Urea Activation.

We establish herein conditions for the cyclization of unprotected N-acyl urea-linked peptides to form macrocyclic peptides mediated by N-terminal cysteine. We report a detailed investigation of the parameters of the reaction, including variation of the reaction conditions, the C-terminal residue, and the macrocycle size. C-Terminal epimerization was not observed. The synthesis of macrocyclic targets ranging from tetrapeptides to the disulfide-linked 14-mer, sunflower trypsin inhibitor 1 are demonstrated. For most substrates, hydrolysis and head-to-tail dimer formation are avoided.

Sequence Diversification by Divergent C-Terminal Elongation of Peptides.

Sequence diversification at the C terminus is traditionally limited by significant epimerization of the C-terminal residue during its activation toward nucleophilic attack, thus mandating repetition of the peptide synthesis for each targeted variation. Here, we accomplish divergent C-terminal elongation of a single peptide substrate with concomitant resin cleavage via displacement of an N-acyl urea moiety. Sterically hindered amino acids such as Ile and Pro are well-tolerated in this approach, which proceeds reasonable conversion and no detectable epimerization of the starting peptide's C-terminal amino acid.

Exploiting the MeDbz Linker To Generate Protected or Unprotected C-Terminally Modified Peptides.

C-terminally modified peptides are important targets for pharmaceutical and biochemical applications. Known methods for C-terminal diversification are limited mainly in terms of the scope of accessible modifications or by epimerization of the C-terminal amino acid. In this work, we present a broadly applicable approach that enables access to a variety of C-terminally functionalized peptides in either protected or unprotected form. This chemistry proceeds without epimerization of C-terminal Ala and tolerates nucleophiles of varying nucleophilicity. Finally, unprotected peptides bearing nucleophilic side chain groups can be selectively functionalized by strong nucleophiles, whereas macrocyclization is observed for weaker nucleophiles. The potential utility of this method is demonstrated through the divergent synthesis of the conotoxin conopressin G and GLP-1(7-36) and analogs.

Proteome-Wide Bioinformatic Annotation and Functional Validation of the Monotopic Phosphoglycosyl Transferase Superfamily.

Phosphoglycosyl transferases (PGTs) are membrane proteins that initiate glycoconjugate biosynthesis by transferring a phospho-sugar moiety from a soluble nucleoside diphosphate sugar to a membrane-embedded polyprenol phosphate acceptor. The centrality of PGTs in complex glycan assembly and the current lack of functional information make these enzymes high-value targets for biochemical investigation. In particular, the small monotopic PGT family is exclusively bacterial and represents the minimal functional unit of the monotopic PGT superfamily. Here, we combine a sequence similarity network (SSN) analysis with a generalizable, luminescence-based activity assay to probe the substrate specificity of this family of monoPGTs in a bacterial cell-membrane fraction. This strategy allows us to identify specificity on a far more significant scale than previously achievable and correlate preferred substrate specificities with predicted structural differences within the conserved monoPGT fold. Finally, we present the proof-of-concept for a small-scale inhibitor screen (eight nucleoside analogs) with four monoPGTs of diverse substrate specificity, thus building a foundation for future inhibitor discovery initiatives.

Defining early steps in Bacillus subtilis biofilm biosynthesis.

IMPORTANCE: Biofilms are the communal way of life that microbes adopt to increase survival. Key to our ability to systematically promote or ablate biofilm formation is a detailed understanding of the biofilm matrix macromolecules. Here, we identify the first two essential steps in the Bacillus subtilis biofilm matrix exopolysaccharide (EPS) synthesis pathway. Together, our studies and approaches provide the foundation for the sequential characterization of the steps in EPS biosynthesis, using prior steps to enable chemoenzymatic synthesis of the undecaprenyl diphosphate-linked glycan substrates.

Defining Early Steps in B. subtilis Biofilm Biosynthesis.

The Bacillus subtilis extracellular biofilm matrix includes an exopolysaccharide that is critical for the architecture and function of the community. To date, our understanding of the biosynthetic machinery and the molecular composition of the exopolysaccharide of B. subtilis remains unclear and incomplete. This report presents synergistic biochemical and genetic studies built from a foundation of comparative sequence analyses targeted at elucidating the activities of the first two membrane-committed steps in the exopolysaccharide biosynthetic pathway. By taking this approach, we determined the nucleotide sugar donor and lipid-linked acceptor substrates for the first two enzymes in the B. subtilis biofilm exopolysaccharide biosynthetic pathway. EpsL catalyzes the first phosphoglycosyl transferase step using UDP-di- N -acetyl bacillosamine as phospho-sugar donor. EpsD is a GT-B fold glycosyl transferase that facilitates the second step in the pathway that utilizes the product of EpsL as an acceptor substrate and UDP- N -acetyl glucosamine as the sugar donor. Thus, the study defines the first two monosaccharides at the reducing end of the growing exopolysaccharide unit. In doing so we provide the first evidence of the presence of bacillosamine in an exopolysaccharide synthesized by a Gram-positive bacterium. IMPORTANCE: Biofilms are the communal way of life that microbes adopt to increase survival. Key to our ability to systematically promote or ablate biofilm formation is a detailed understanding of the biofilm matrix macromolecules. Here we identify the first two essential steps in the Bacillus subtilis biofilm matrix exopolysaccharide synthesis pathway. Together our studies and approaches provide the foundation for the sequential characterization of the steps in exopolysaccharide biosynthesis, using prior steps to enable chemoenzymatic synthesis of the undecaprenol diphosphate-linked glycan substrates.

Backbone-Anchoring, Solid-Phase Synthesis Strategy To Access a Library of Peptidouridine-Containing Small Molecules.

Nucleoside diphosphate sugar (NDP-sugar) substrates provide the inspiration for nucleoside analogue inhibitor scaffolds. By employing solid-phase synthesis, we provide a method to access a library of peptidouridine inhibitors with both minimal compound handling and purification steps. Specifically, this strategy is exemplified by generating uridine diphosphate sugar (UDP-sugar) mimics, which allow for compound elaboration by altering the dipeptide composition, the N-terminal linkage, and a pendant aryl group. To exemplify the versatility, 41 unique nucleoside analogues are presented.

Probing Monotopic Phosphoglycosyl Transferases from Complex Cellular Milieu.

Monotopic phosphoglycosyl transferase enzymes (monoPGTs) initiate the assembly of prokaryotic glycoconjugates essential for bacterial survival and proliferation. MonoPGTs belong to an expansive superfamily with a diverse and richly annotated sequence space; however, the biochemical roles of most monoPGTs in glycoconjugate biosynthesis pathways remain elusive. To better understand these critical enzymes, we have implemented activity-based protein profiling (ABPP) probes as protein-centric, membrane protein compatible tools that lay the groundwork for understanding the activity and regulation of the monoPGT superfamily from a cellular proteome. With straightforward gel-based readouts, we demonstrate robust, covalent labeling at the active site of various representative monoPGTs from cell membrane fractions using 3-phenyl-2H-azirine probes.

Epimerization-free access to C-terminal cysteine peptide acids, carboxamides, secondary amides, and esters via complimentary strategies.

C-Terminal cysteine peptide acids are difficult to access without epimerization of the cysteine α-stereocenter. Diversification of the C-terminus after solid-phase peptide synthesis poses an even greater challenge because of the proclivity of the cysteine α-stereocenter to undergo deprotonation upon activation of the C-terminal carboxylic acid. We present herein two general strategies to access C-terminal cysteine peptide derivatives without detectable epimerization, diketopiperazine formation, or piperidinylalanine side products.