Analogues of Muraymycin Nucleoside Antibiotics with Epimeric Uridine-Derived Core Structures.

Nucleoside analogues have found widespread application as antiviral and antitumor agents, but not yet as antibacterials. Naturally occurring uridine-derived 'nucleoside antibiotics' target the bacterial membrane protein MraY, an enzyme involved in peptidoglycan biosynthesis and a promising target for the development of novel antibacterial agents. Muraymycins represent a nucleoside-peptide subgroup of such MraY-inhibiting natural products. As part of detailed structure-activity relationship (SAR) studies on muraymycins and their analogues, we now report novel insights into the effects of stereochemical variations in the nucleoside core structure. Using a simplified version of the muraymycin scaffold, it was shown that some formal inversions of stereochemistry led to about one order of magnitude loss in inhibitory potency towards the target enzyme MraY. In contrast, epimers of the core motif with retained inhibitory activity were also identified. These 5',6'-anti-configured analogues might serve as novel chemically tractable variations of the muraymycin scaffold for the future development of uridine-derived drug candidates.

The Biosynthesis of Capuramycin-type Antibiotics: IDENTIFICATION OF THE A-102395 BIOSYNTHETIC GENE CLUSTER, MECHANISM OF SELF-RESISTANCE, AND FORMATION OF URIDINE-5'-CARBOXAMIDE.

A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures.

Total Synthesis of Dansylated Park's Nucleotide for High-Throughput MraY Assays.

The membrane protein translocase I (MraY) is a key enzyme in bacterial peptidoglycan biosynthesis. It is therefore frequently discussed as a target for the development of novel antibiotics. The screening of compound libraries for the identification of MraY inhibitors is enabled by an established fluorescence-based MraY assay. However, this assay requires a dansylated derivative of the bacterial biosynthetic intermediate Park's nucleotide as the MraY substrate. Isolation of Park's nucleotide from bacteria and subsequent dansylation only furnishes limited amounts of this substrate, thus hampering the high-throughput screening for MraY inhibitors. Accordingly, the efficient provision of dansylated Park's nucleotide is a major bottleneck in the exploration of this promising drug target. In this work, we present the first total synthesis of dansylated Park's nucleotide, affording an unprecedented amount of the target compound for high-throughput MraY assays.

Aziridine electrophiles in the functionalisation of peptide chains with amine nucleophiles.

We describe herein the synthesis of aziridine-containing amino acids embedded within tripeptide structures. A range of amine nucleophiles have been shown to open the aziridine amino acid regioselectively at the ß-position under mild conditions and without the requirement for a catalyst, forming new adducts in the process. Amino acid N-termini (or an N-containing sidechain) also served as effective nucleophiles for such aziridines and this concept could be extended to encompass a di- or tripeptide nitrogen as a nucleophile, thus providing new methodology for linking together peptide strands using an amine linker.

Palladium-catalyzed α-arylation of carbonyls in the de novo synthesis of aromatic heterocycles.

Aromatic heterocycles are a very well represented motif in natural products and have found various applications in chemistry and material science, as well as being commonly found in pharmaceutical agents. Thus, new and efficient routes towards this class of compound are always desirable, particularly if they expand the scope of chemical methodology or facilitate more effective pathways to complex substitution patterns. This perspective covers recent developments in the de novo synthesis of aromatic heterocycles via palladium-catalysed α-arylation reactions of carbonyls, which is itself a powerful transformation that has undergone significant development in recent years.

Lead structures for new antibacterials: stereocontrolled synthesis of a bioactive muraymycin analogue.

Naturally occurring muraymycin nucleoside antibiotics represent a promising class of novel antibacterial agents. The structural complexity suggests the investigation of simplified analogues as potential lead structures, which can then be further optimized towards highly potent antimicrobials. Herein we report studies on muraymycin-derived potential lead structures lacking an aminoribose motif found in most naturally occurring muraymycins. We have identified a 5'-defunctionalized motif to be ideal in terms of stability and chemical accessibility and have synthesized a full-length muraymycin analogue based on this structure using a novel fully stereocontrolled route. The obtained 5'-deoxy analogue of the natural product muraymycin C4 showed good inhibitory properties towards the bacterial target protein MraY, sufficient pharmacokinetic stability and no cytotoxicity against human cells, thus making it a promising lead for antibacterial drug development.

Structure-based gene targeting discovery of sphaerimicin, a bacterial translocase I inhibitor.

Rise and shine: Using a gene-targeting approach aimed at identifying potential L-threonine:uridine-5'-transaldolases that catalyze the formation of (5'S,6'S)-C-glycyluridine, a new bacterial translocase I inhibitor was discovered from an actinomycete following fermentation optimization.

Amalgamation of nucleosides and amino acids in antibiotic biosynthesis: discovery of an L-threonine:uridine-5'-aldehyde transaldolase.

The lipopeptidyl nucleoside antibiotics represented by A-90289, caprazamycin, and muraymycin are structurally highlighted by a nucleoside core that contains a nonproteinogenic ß-hydroxy-α-amino acid named 5'-C-glycyluridine (GlyU). Bioinformatic analysis of the biosynthetic gene clusters revealed a shared open reading frame encoding a protein with sequence similarity to serine hydroxymethyltransferases, resulting in the proposal that this shared enzyme catalyzes an aldol-type condensation with glycine and uridine-5'-aldehyde to furnish GlyU. Using LipK involved in A-90289 biosynthesis as a model, we now functionally assign and characterize the enzyme responsible for the C-C bond-forming event during GlyU biosynthesis as an l-threonine:uridine-5'-aldehyde transaldolase. Biochemical analysis revealed this transformation is dependent upon pyridoxal-5'-phosphate, the enzyme has no activity with alternative amino acids, such as glycine or serine, as aldol donors, and acetaldehyde is a coproduct. Structural characterization of the enzyme product is consistent with stereochemical assignment as the threo diastereomer (5'S,6'S)-GlyU. Thus this enzyme orchestrates C-C bond breaking and formation with concomitant installation of two stereocenters to make a new l-α-amino acid with a nucleoside side chain.

Stereoselective synthesis of uridine-derived nucleosyl amino acids.

Novel hybrid structures of 5'-deoxyuridine and glycine were conceived and synthesized. Such nucleosyl amino acids (NAAs) represent simplified analogues of the core structure of muraymycin nucleoside antibiotics, making them useful synthetic building blocks for structure-activity relationship (SAR) studies. The key step of the developed synthetic route was the efficient and highly diastereoselective asymmetric hydrogenation of didehydro amino acid precursors toward protected NAAs. It was anticipated that the synthesis of unprotected muraymycin derivatives via this route would require a suitable intermediate protecting group at the N-3 of the uracil base. After initial attempts using PMB- and BOM-N-3 protection, both of which resulted in problematic deprotection steps, an N-3 protecting group-free route was envisaged. In spite of the pronounced acidity of the uracil-3-NH, this route worked equally efficient and with identical stereoselectivities as the initial strategies involving N-3 protection. The obtained NAA building blocks were employed for the synthesis of truncated 5'-deoxymuraymycin analogues.

Novel 5'-deoxy nucleosyl amino acid scaffolds for the synthesis of muraymycin analogues.

Naturally occurring nucleoside antibiotics such as muraymycins represent promising lead structures for the development of novel antibacterial agents. A concise synthesis of 5'-deoxy muraymycin derivatives has been developed. The key step was the highly stereoselective asymmetric hydrogenation of suitable didehydro amino acid precursors, providing unique nucleosyl amino acid structures.

Synthesis and properties of DNA oligonucleotides with a zwitterionic backbone structure.

The nucleosyl amino acid (NAA)-modification of oligonucleotides is introduced, which enables the preparation of oligonucleotides with zwitterionic backbone structures. It is demonstrated that partially zwitterionic NAA-modified DNA oligonucleotides are capable of duplex formation with native polyanionic counterstrands and show retained sensitivity towards base-pairing mismatches.