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Muraymycins are peptidyl nucleoside antibiotics that contain two Cβ-modified amino acids, (2S,3S)-capreomycidine and (2S,3S)-β-OH-Leu. The former is also a component of chymostatins, which are aldehyde-containing peptidic protease inhibitors that─like muraymycin─are derived from nonribosomal peptide synthetases (NRPSs). Using feeding experiments and in vitro characterization of 12 recombinant proteins, the biosynthetic mechanism for both nonproteinogenic amino acids is now defined. The formation of (2S,3S)-capreomycidine is shown to involve an FAD-dependent dehydrogenase:cyclase that requires an NRPS-bound pathway intermediate as a substrate. This cryptic dehydrogenation strategy is both temporally and mechanistically distinct in comparison to the biosynthesis of other capreomycidine diastereomers, which has previously been shown to proceed by Cβ-hydroxylation of free l-Arg catalyzed by a member of the nonheme Fe2+- and α-ketoglutarate (αKG)-dependent dioxygenase family and (eventually) a dehydration-mediated cyclization process catalyzed by a distinct enzyme(s). Contrary to our initial expectation, the sole nonheme Fe2+- and αKG-dependent dioxygenase candidate Mur15 encoded within the muraymycin gene cluster is instead demonstrated to catalyze specific Cβ hydroxylation of the Leu residue to generate (2S,3S)-β-OH-Leu that is found in most muraymycin congeners. Importantly, and in contrast to known l-Arg-Cβ-hydroxylases, the Mur15-catalyzed reaction occurs after the NRPS-mediated assembly of the peptide scaffold. This late-stage functionalization affords the opportunity to exploit Mur15 as a biocatalyst, proof of concept of which is provided.

To overcome bacterial resistances, the need for novel antimicrobial agents is urgent. The class of so‐called nucleoside antibiotics furnishes promising candidates for the development of new antibiotics, as these compounds block a clinically unexploited bacterial target: the integral membrane protein MraY, a key enzyme in cell wall (peptidoglycan) biosynthesis. Nucleoside antibiotics exhibit remarkable structural diversity besides their uridine‐derived core motifs. Some sub‐classes also show specific selectivities towards different Gram‐positive and Gram‐negative bacteria, which are poorly understood so far. Herein, the synthesis of a novel hybrid structure is reported, derived from the 5′‐defunctionalized uridine core moiety of muraymycins and the peptide chain of sansanmycin B, as a new scaffold for the development of antimicrobial agents. The reported muraymycin–sansanmycin hybrid scaffold showed nanomolar activity against the bacterial target enzyme MraY, but displayed no significant antibacterial activity against S. aureus, E. coli, and P. aeruginosa., Two become one: Nucleoside antibiotics are natural products that show remarkable structural diversity. Herein, the synthesis of a novel non‐natural hybrid structure is reported, derived from the simplified uridine core moiety of muraymycins and the peptide chain of sansanmycin B. This muraymycin–sansanmycin hybrid scaffold showed nanomolar activity against the bacterial target enzyme MraY, thus suggesting it may be used for the development of antibacterial agents.

Oligonucleotides (ON) are promising therapeutic candidates, for instance by blocking endogenous mRNA (antisense mechanism). However, ON usually require structural modifications of the native nucleic acid backbone to ensure satisfying pharmacokinetic properties. One such strategy to design novel antisense oligonucleotides is to replace native phosphate diester units by positively charged artificial linkages, thus leading to (partially) zwitterionic backbone structures. Herein, we report a “gapmer” architecture comprised of one zwitterionic central segment (“gap”) containing nucleosyl amino acid (NAA) modifications and two outer segments of locked nucleic acid (LNA). This NAA/LNA‐gapmer approach furnished a partially zwitterionic ON with optimised properties: i) the formation of stable ON‐RNA duplexes with base‐pairing fidelity and superior target selectivity at 37 °C; and ii) excellent stability in complex biological media. Overall, the NAA/LNA‐gapmer approach is thus established as a strategy to design partially zwitterionic ON for the future development of novel antisense agents., Mind the gap! One approach towards backbone‐modified oligonucleotides is the introduction of (partially) zwitterionic structural motifs. In this work, we report a “gapmer” architecture comprising a zwitterionic central “gap” (with cationic NAA linkages) and two outer segments of locked nucleic acid (LNA). This NAA/LNA‐gapmer approach furnished a partially zwitterionic oligonucleotide with promising properties for potential biomedical applications.

Muraymycins are a subclass of naturally occurring nucleoside antibiotics with promising antibacterial activity. They inhibit the bacterial enzyme translocase I (MraY), a clinically yet unexploited target mediating an essential intracellular step of bacterial peptidoglycan biosynthesis. Several structurally simplified muraymycin analogues have already been synthesized for structure&ndash, activity relationship (SAR) studies. We now report on novel derivatives with unprecedented variations in the nucleoside unit. For the synthesis of these new muraymycin analogues, we employed a bipartite approach facilitating the introduction of different nucleosyl amino acid motifs. This also included thymidine- and 5-fluorouridine-derived nucleoside core structures. Using an in vitro assay for MraY activity, it was found that the introduction of substituents in the 5-position of the pyrimidine nucleobase led to a significant loss of inhibitory activity towards MraY. The loss of nucleobase aromaticity (by reduction of the uracil C5-C6 double bond) resulted in a ca. tenfold decrease in inhibitory potency. In contrast, removal of the 2'-hydroxy group furnished retained activity, thus demonstrating that modifications of the ribose moiety might be well-tolerated. Overall, these new SAR insights will guide the future design of novel muraymycin analogues for their potential development towards antibacterial drug candidates.

Their unique ability to selectively bind specific nucleic acid sequences makes oligonucleotides promising bioactive agents. However, modifications of the nucleic acid structure are an essential prerequisite for their application in vivo or even in cellulo. The oligoanionic backbone structure of oligonucleotides mainly hampers their ability to penetrate biological barriers such as cellular membranes. Hence, particular attention has been given to structural modifications of oligonucleotides which reduce their overall number of negative charges. One such approach is the site-specific replacement of the negatively charged phosphate diester linkage with alternative structural motifs which are positively charged at physiological pH, thus resulting in zwitterionic or even oligocationic backbone structures. This review provides a general overview of this concept and summarizes research on four according artificial backbone linkages: aminoalkylated phosphoramidates (and related systems), guanidinium groups, S-methylthiourea motifs, and nucleosyl amino acid (NAA)-derived modifications. The synthesis and properties of the corresponding oligonucleotide analogues are described.

Muraymycins belong to a family of nucleoside antibiotics that have a distinctive disaccharide core consisting of 5-amino-5-deoxyribofuranose (ADR) attached to 6'- N-alkyl-5'- C-glycyluridine (GlyU). Here, we functionally assign and characterize six enzymes from the muraymycin biosynthetic pathway involved in the core assembly that starts from uridine monophosphate (UMP). The biosynthesis is initiated by Mur16, a nonheme Fe(II)- and α-ketoglutarate-dependent dioxygenase, followed by four transferase enzymes: Mur17, a pyridoxal-5'-phosphate (PLP)-dependent transaldolase; Mur20, an aminotransferase; Mur26, a pyrimidine phosphorylase; and Mur18, a nucleotidylyltransferase. The pathway culminates in glycosidic bond formation in a reaction catalyzed by an additional transferase enzyme, Mur19, a ribosyltransferase. Analysis of the biochemical properties revealed several noteworthy discoveries including that (i) Mur16 and downstream enzymes can also process 2'-deoxy-UMP to generate a 2-deoxy-ADR, which is consistent with the structure of some muraymycin congeners; (ii) Mur20 prefers l-Tyr as the amino donor source; (iii) Mur18 activity absolutely depends on the amine functionality of the ADR precursor consistent with the nucleotidyltransfer reaction occurring after the Mur20-catalyzed aminotransfer reaction; and (iv) the bona fide sugar acceptor for Mur19 is (5' S,6' S)-GlyU, suggesting that ribosyltransfer occurs prior to N-alkylation of GlyU. Finally, a one-pot, six-enzyme reaction was utilized to generate the ADR-GlyU disaccharide core starting from UMP.

Muraymycins are nucleoside antibiotics isolated from Streptomyces sp. NRRL 30471 and several mutant strains thereof that were generated by random, chemical mutagenesis. Reinvestigation of two mutant strains using new media conditions led to the isolation of three new muraymycin congeners, named B8, B9, and C6 (1-3), as well as a known muraymycin, C1. Structures of the compounds were elucidated by HRMS and 1D and 2D NMR spectroscopic analyses. Complete 2D NMR assignments for the known muraymycin C1 are also provided for the first time. Compounds 1 and 2, which differ from other muraymycins by having an elongated, terminally branched fatty acid side chain, had picomolar IC50 values against Staphylococcus aureus and Aquifex aeolicus MraY and showed good antibacterial activity against S. aureus (MIC = 2 and 6 μg/mL, respectively) and Escherichia coli Δ tolC (MIC = 4 and 2 μg/mL, respectively). Compound 3, which is characterized by an N-acetyl modification of the primary amine of the dissacharide core that is shared among nearly all of the reported muraymycin congeners, greatly reduced its inhibitory and antibacterial activity compared to nonacylated muraymycin C1, which possibly indicates this modification is used for self-resistance.

Naturally occurring nucleoside antibiotics (e.g., muraymycins and caprazamycins) represent attractive lead structures for the development of urgently needed novel antibacterial agents. One major challenge in the total synthesis of muraymycins, caprazamycins, and their analogues is the efficient construction of the densely functionalized aminoribosylated uridine-derived core unit. In order to avoid tedious protecting-group manipulations, we have aimed to conduct the aminoribosylation step with an acid-labile glycosyl acceptor. Therefore, different glycosylation approaches have been studied, with pentenyl glycosides giving the best results.

Muraymycins are a promising class of antimicrobial natural products. These uridine-derived nucleoside-peptide antibiotics inhibit the bacterial membrane protein translocase I (MraY), a key enzyme in the intracellular part of peptidoglycan biosynthesis. This review describes the structures of naturally occurring muraymycins, their mode of action, synthetic access to muraymycins and their analogues, some structure–activity relationship (SAR) studies and first insights into muraymycin biosynthesis. It therefore provides an overview on the current state of research, as well as an outlook on possible future developments in this field.

Naturally occurring nucleoside-peptide antibiotics such as muraymycins or caprazamycins are of major interest for the development of novel antibacterial agents. However, the synthesis of new analogues of these natural products for structure–activity relationship (SAR) studies is challenging. In our synthetic efforts towards a muraymycin-derived nucleoside building block suitable for attachment to a solid support, we came across an interesting side product. This compound resulted from an undesired Fmoc deprotection with subsequent cyclization, thus furnishing a remarkable caprazamycin-like seven-membered diazepanone ring.

Deficient stability towards nuclease-mediated degradation is one of the most relevant tasks in the development of oligonucleotide-derived biomedical agents. This hurdle can be overcome through modifications to the native oligonucleotide backbone structure, with the goal of simultaneously retaining the unique hybridization properties of nucleic acids. The nucleosyl amino acid (NAA)-modification is a recently introduced artificial cationic backbone linkage. Partially zwitterionic NAA-modified oligonucleotides had previously shown hybridization with DNA strands with retained base-pairing fidelity. In this study, we report the significantly enhanced stability of NAA-modified oligonucleotides towards 3&prime, and 5&prime, exonuclease-mediated degradation as well as in complex biological media such as human plasma and whole cell lysate. This demonstrates the potential versatility of the NAA-motif as a backbone modification for the development of biomedically active oligonucleotide analogues.

Muraymycins are a subclass of antimicrobially active uridine-derived natural products. Biological data on several muraymycin analogues have already been reported, including some inhibitory in vitro activities towards their target protein, the bacterial membrane enzyme MraY. However, a structure-activity relationship (SAR) study on naturally occurring muraymycins based on such in vitro data has been missing so far. In this work, we report a detailed SAR investigation on representatives of the four muraymycin subgroups A–D using a fluorescence-based in vitro MraY assay. For some muraymycins, inhibition of MraY with IC(50) values in the low pM range was observed. These inhibitory potencies were compared with antibacterial activities and were correlated to modelling data derived from a previously reported X-ray crystal structure of MraY in complex with a muraymycin inhibitor. Overall, these results will pave the way for the development of muraymycin analogues with optimized properties as antibacterial drug candidates.

Non‐natural oligonucleotides represent important (bio)chemical tools and potential therapeutic agents. Backbone modifications altering hybridization properties and biostability can provide useful analogues. Here, we employ an artificial nucleosyl amino acid (NAA) motif for the synthesis of oligonucleotides containing a backbone decorated with primary amines. An oligo‐T sequence of this cationic DNA analogue shows significantly increased affinity for complementary DNA. Notably, hybridization with DNA is still governed by Watson–Crick base pairing. However, single base pair mismatches are tolerated and some degree of sequence‐independent interactions between the cationic NAA backbone and fully mismatched DNA are observed. These findings demonstrate that a high density of positive charges directly connected to the oligonucleotide backbone can affect Watson–Crick base pairing. This provides a paradigm for the design of therapeutic oligonucleotides with altered backbone charge patterns.

The β-hydroxy-α-amino acid 3-hydroxy-L-arginine is an important intermediate in biosynthetic pathways involving 2-oxoglutarate (2-OG) dependent FeII oxygenases. It also plays an essential role in post-translational modifications of ribosomal proteins. For profound studies on arginine-hydroxylating enzymes, synthetic reference compounds are of utmost importance. However, the synthesis of β-hydroxy-α-amino acids is often not trivial. In particular, the preparation of a densely functionalized compound such as 3-hydroxy-L-arginine poses a challenge in terms of a protecting-group strategy. We have previously established a concise synthesis of 3-hydroxy-L-arginine that is suitable for the preparation of both 3-epimers of this β-hydroxy-α-amino acid. However, we have since observed a remarkable isomerization reaction upon removal of the hydrogenolytically cleavable protecting groups, thus limiting the reliability of our previously described route. In this paper, we report a comparative study of protecting-group strategies for this synthetic route, including a newly developed, more robust pathway to the target amino acid. The versatility of this improved route is demonstrated by the synthesis of the deuterium-labelled derivatives (3R)- and (3S)-3-hydroxy-[5-2H]-L-arginine. These new isotope-labelled arginine derivatives represent important tools for the elucidation of biosynthetic pathways, such as the formation of the arginine-derived amino acid epicapreomycidine in the biosynthesis of muraymycin nucleoside antibiotics.

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.

Stem cell research is one of the most promising fields of modern biomedical research and regenerative medicine. Limited availability and ethical concerns suggest the renouncement of embryonic stem cells (ESCs), thus raising the need for more efficient procedures for the generation of stem cells, ideally through reprogramming of mammalian cells. The small molecule N-benzyl-2-(pyrimidin-4′-ylamino)-thiazole-4-carboxamide (thiazovivin) is known to improve the generation of human induced pluripotent stem cells (iPSCs) from human fibroblasts. We herein describe a highly efficient procedure for the synthesis of thiazovivin over just five steps, which should be suitable for a large-scale application, and the first x-ray crystal structure of the target compound. [Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications® for the following free supplemental resource: Full experimental and spectral details.]

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.

Gram-negative bacteria possess a unique and complex cell envelope, composed of an inner and outer membrane separated by an intermediate cell wall-containing periplasm. This tripartite structure acts intrinsically as a significant biological barrier, often limiting the permeation of anti-infectives, and so preventing such drugs from reaching their target. Furthermore, identification of the specific permeation-limiting envelope component proves difficult in the case of many anti-infectives, due to the challenges associated with isolation of individual cell envelope structures in bacterial culture. The development of an in vitro permeation model of the Gram-negative inner membrane, prepared by repeated coating of physiologically-relevant phospholipids on Transwell(®) filter inserts, is therefore reported, as a first step in the development of an overall cell envelope model. Characterization and permeability investigations of model compounds as well as anti-infectives confirmed the suitability of the model for quantitative and kinetically-resolved permeability assessment, and additionally confirmed the importance of employing bacteria-specific base materials for more accurate mimicking of the inner membrane lipid composition - both advantages compared to the majority of existing in vitro approaches. Additional incorporation of further elements of the Gram-negative bacterial cell envelope could ultimately facilitate model application as a screening tool in anti-infective drug discovery or formulation development.

Natural(ly) fit: The X-ray crystal structure of the bacterial membrane protein MraY in complex with its natural product inhibitor muraymycin D2 is discussed. MraY catalyzes one of the membrane-associated steps in peptidoglycan biosynthesis and, therefore, represents a promising target for novel antibiotics. Structural insights derived from the protein-inhibitor complex might now pave the way for the development of new antimicrobial drugs.

Nucleoside analogues have found widespread application as antiviral and antitumor agents, but not yet as antibacterials. Naturally occurring uridine-derived &lsquo, nucleoside antibiotics&rsquo, 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&prime, 6&prime, anti-configured analogues might serve as novel chemically tractable variations of the muraymycin scaffold for the future development of uridine-derived drug candidates.

The reaction of protected uridine 5′-aldehydes with sulfur ylides has been reinvestigated. Further transformation of the resulting epoxide product provided a compound of which a single crystal for X-ray diffraction was obtained. As a consequence from the elucidated structure, the stereochemical configuration of the epoxide furnished by the sulfur ylide reaction was revised. Based on these results, an efficient synthesis of the core structure of the naturally occurring muraymycin and caprazamycin nucleoside antibiotics was developed.

The hydroxylated amino acid 3-hydroxy- l -arginine is an intermediate in the biosynthesis of the non-proteinogenic amino acid capreomycidine and possibly also of its epimer epicapreomycidine. The novel concise synthesis of 3-hydroxy- l -arginine presented here allows the efficient preparation of both 3-epimers of this β-hydroxy amino acid. It also offers the potential to obtain suitably isotope-labelled derivatives for the elucidation of epicapreomycidine assembly in the biosynthesis of complex natural products.

Functional insights into bioactive natural products with medicinal potential are often hindered by their structural complexity. We herein report a simplified model system to investigate the functional significance of a structural motif of biologically potent muraymycin antibiotics of the A-series. These compounds have a highly unusual ω-guanidinylated fatty acid moiety, which has been proposed to mediate membrane penetration, thus enabling the interaction of A-series muraymycins with their intracellular target MraY. Our assay was based on a synthetic conjugate of this fatty acid structure with a negatively charged fluorophore lacking membrane permeability. Using this conjugate, immobilised giant unilamellar lipid vesicles and confocal laser scanning fluorescence microscopy, we demonstrated that the attachment of the ω-N-hydroxyguanidinyl fatty acid unit led to an enhanced uptake of the fluorophore into the vesicles. This represents the first experimental evidence of this unusual structural motif's functional relevance for the parent natural product, which may support the future design of novel muraymycin analogues. peerReviewed

The non-proteinogenic amino acids capreomycidine and epicapreomycidine are constituents of antibiotically active natural products, but the synthesis of these unusual cyclic guanidine derivatives is challenging. The biosynthesis of capreomycidine has therefore been employed as a guideline to develop a concise biomimetic synthesis of both epimeric amino acids. The resulting domino-guanidinylation-aza-Michael-addition reaction provides the most convenient access to these amino acids in racemic form. Attempts to dissect the domino reaction into two separate transformations for a stereocontrolled version of this synthetic approach have also been made. The synthesized didehydro-arginine derivatives with urethane-protected guanidine moieties did not undergo the aza-Michael-addition anymore. These results may have wider implications for the 1,4-addition of guanidines to α,β-unsaturated carbonyl compounds, particularly to didehydro amino acids. Electronic supplementary material The online version of this article (doi:10.1007/s00726-012-1309-8) contains supplementary material, which is available to authorized users.

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.

(Chemical Equation Presented) Enzyme in action: Labeling studies and the finding that carboxymethylproline synthase catalyzes production of deuterated (2S,5S)-6,6′-dimethyl-trans-carboxymethylproline (3) from dimethylmalonyl-CoA (1) and labeled l-pyrroline-5-carboxylate (2) limit possible mechanisms of C-C bond formation and thioester hydrolysis. A key feature in the catalysis is that intermediates are stabilized by hydrogen bonds in the "oxy-anion hole" of the enzyme (dark curve in scheme). © 2008 Wiley-VCH Verlag GmbH and Co. KGaA.

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.