Modular synthesis of diphospholipid oligosaccharide fragments of the bacterial cell wall and their use to study the mechanism of moenomycin and other antibiotics

We present a flexible, modular route to GlcNAc-MurNAc-oligosaccharides that can be readily converted into peptidoglycan (PG) fragments to serve as reagents for the study of bacterial enzymes that are targets for antibiotics. Demonstrating the utility of these synthetic PG substrates, we show that the tetrasaccharide substrate lipid IV (3), but not the disaccharide substrate lipid II (2), significantly increases the concentration of moenomycin A required to inhibit a prototypical PG-glycosyltransferase (PGT). These results imply that lipid IV and moenomycin A bind to the same site on the enzyme. We also show the moenomycin A inhibits the formation of elongated polysaccharide product but does not affect length distribution. We conclude that moenomycin A blocks PG-strand initiation rather than elongation or chain termination. Synthetic access to diphospholipid oligosaccharides will enable further studies of bacterial cell wall synthesis with the long-term goal of identifying novel antibiotics.     

Membrane Anchoring and Intervesicle Transfer of a Derivative of the Antibiotic Moenomycin A

The anchoring of moenomycin A (1) to the bacterial cell cytoplasmic membrane is essential for its biological activity. The first details of the strength of this interaction and the kinetics of the diffusion-mediated intervesicle transfer have been obtained by means of fluorescence spectroscopic methods using a coumarin-labeled moenomycin A derivative.     

Synthesis of modified peptidoglycan precursor analogues for the inhibition of glycosyltransferase

The peptidoglycan glycosyltransferases (GTs) are essential enzymes that catalyze the polymerization of glycan chains of the bacterial cell wall from lipid II and thus constitute a validated antibacterial target. Their enzymatic cavity is composed of a donor site for the growing glycan chain (where the inhibitor moenomycin binds) and an acceptor site for lipid II substrate. In order to find lead inhibitors able to fill this large active site, we have synthesized a series of substrate analogues of lipid I and lipid II with variations in the lipid, the pyrophosphate, and the peptide moieties and evaluated their biological effect on the GT activity of E. coli PBP1b and their antibacterial potential. We found several compounds able to inhibit the GT activity in vitro and cause growth defect in Bacillus subtilis . The more active was C16-phosphoglycerate-MurNAc-(L-Ala-D-Glu)-GlcNAc, which also showed antibacterial activity. These molecules are promising leads for the design of new antibacterial GT inhibitors.     

Synthesis of moenocinol and its analogs using BT-sulfone in Julia-Kocienski olefination

Moenocinol (C₂₅H₄₂O), the acyclic terpenoid unsaturated lipid part of moenomycin antibiotics, was prepared by an expedient method, which comprised organometallic reaction, Julia-Kocienski olefination, and enolate carbon bond formation as the key steps. The starting materials, nerol and 3-butyn-1-ol, were elaborated to the benzothiazole sulfone 2 and aldehyde 3, and the subsequent Julia-Kocienski olefination occurred in a stereospecific manner to give the desired 6E-configuration of moenocinol. Moenocinol (1) was thus synthesized by 10 linear steps in 12% overall yield, and its analogs 23, 24, and 28 with different chain lengths and unsaturation degrees were also realized by the similar reaction sequences.     

The total synthesis of moenomycin A

Moenomycin A is the only known natural antibiotic that inhibits bacterial cell wall synthesis by binding to the transglycosylases that catalyze formation of the carbohydrate chains of peptidoglycan. We report here the total synthesis of moenomycin A using the sulfoxide glycosylation method. A newly discovered byproduct of sulfoxide reactions was isolated that resulted in substantial loss of the glycosyl acceptor. A general method to suppress this byproduct was introduced, which enabled the glycosylations to proceed efficiently. The inverse addition protocol for sulfoxide glycosylations also proved essential in constructing some of the glycosidic linkages. The synthetic route is flexible and will allow for derivatives to be constructed to further analyze moenomycin A's mechanism of action.     

Purification and Preparation of Moenomycin A from Fermentation Broth by Multidimensional Chromatography

Based on various adsorption characteristics of resins, a novel method for purifying and preparing moenomycin A from fermentation broth was established by combining different chromatographic modes into a three-step preparative chromatography process. Fermentation broth of moenomycins was firstly prepurified by macroporous adsorbent XAD7HP to remove most strong polar impurities, then further purified by anion exchange resin FPA98Cl, and finally refined moenomycin A was obtained by use of semi-preparative reversed-phase chromatographic column packed with Chromtorex C8 silica gel. As the main indicators, purity and yield of moenomycin A were examined in order to optimise the chromatographic process for each step. Under optimized chromatographic conditions, the purity and total yield of moenomycin A were 95.0 and 22.2 %, and the biological potency of moenomycin A was 2232 U mg⁻¹, significantly higher than 1395 U mg⁻¹, which is the potency of the standard from Agriculture Ministry of China. Three-step preparative chromatographic mode could gradually and effectively remove impurities. The present method is practical, easy to be operated with less solvent consumption, and provides a new idea for the preparation of moenomycin A with high purity.

     

Studies on the Synthesis of Di‐ and Trisaccharide Analogues of Moenomycin A. Modifications in Unit E and in the Lipid Part

Routes allowing the synthesis of moenomycin analogues with one modified sugar component and with new lipid parts were developed (see 10c, 12c, 16b , and 20b in Schemes 2–4). It is anticipated that such analogues will be useful for studying the mode of action of the moenomycin‐type transglycosylase inhibitors in detail and for preparing analogues with improved pharmacokinetic properties.     

Discovery of a Single Monooxygenase that Catalyzes Carbamate Formation and Ring Contraction in the Biosynthesis of the Legonmycins

Pyrrolizidine alkaloids (PAs) are a group of natural products with important biological activities. The discovery and characterization of the multifunctional FAD‐dependent enzyme LgnC is now described. The enzyme is shown to convert indolizidine intermediates into pyrrolizidines through an unusual ring expansion/contraction mechanism, and catalyze the biosynthesis of new bacterial PAs, the so‐called legonmycins. By genome‐driven analysis, heterologous expression, and gene inactivation, the legonmycins were also shown to originate from non‐ribosomal peptide synthetases (NRPSs). The biosynthetic origin of bacterial PAs has thus been disclosed for the first time.     

Moenomycin a: New chemistry that allows to attach the antibiotic to reporter groups, solid supports, and proteins

Moenomycin A (18) on reaction with the diazonium salt derived from bifunctional (protected) 15 yields the coupling product 19 which on reduction is converted into the moenomycin thiol derivative 21. Thiol 21 has been used to prepare selectively moenomycin dansyl and biotin adducts 26 and 28, respectively. This work was performed with the aim to use moenomycin as a tool for studies of the transglycosylation step in peptidoglycan biosynthesis.     

Characterization of moenomycin antibiotic complex by multistage MALDI-IT/RTOF-MS and ESI-IT-MS

Flavomycin is a commercially available antimicrobial growth promoter and an authorized additive for feeding stuffs in the EU and in the USA. As most antibiotically active products biosynthesized by microorganisms, it contains not only a single active compound but is a complex mixture of structurally closely related substances. Multistage matrix-assisted laser desorption/ionization-ion trap/reflectron time-of-flight mass spectrometry (MALDI-IT/RTOF-MS) and liquid chromatography-electrospray ionization-ion trap-mass spectrometry (LC-ESI-IT-MS) were utilized for a detailed analysis of the constituents of the Flavomycin complex based on low-energy collision induced dissociation (CID). An optimal sample preparation for negative ion vacuum MALDI-MS for this compound class was developed. The MALDI-IT/RTOF-MS2 and -MS3 analysis starting with the precursor [M - H]- ions of these interesting phosphoglycolipids, named moenomycins, yielded a large variety of product ions that facilitated the structural characterization of this class of compounds. Based on the derived CID fragmentation pathway of the five known major constituents, namely moenomycin A, moenomycin A12, moenomycin C4, moenomycin C3. and moenomycin C1, four not yet described moenomycin-type constituents could be characterized. They were assigned as 4F-demethyl-6E-O-de-beta-D-glucopyranosyl-moenomycin A, 6B-N-de(2-hydroxy-5-oxo-1-cyclopenten-1-yl)-moenomycin A, 6B-hydroxy-6B-de[N-(2-hydroxy-5-oxo-1-cyclopenten-1-yl)amino]-moenomycin A, and 6C-hydroxy-moenomycin A. In addition, a moenomycin A carrying an oxygen in the moenocinol-group was found, which is most probably a chemical degradation product. These new compounds were verified by LC-ESI-IT-MS.     

Two distinct mechanisms for TIM barrel prenyltransferases in bacteria

The reactions of two bacterial TIM barrel prenyltransferases (PTs), MoeO5 and PcrB, were explored. MoeO5, the enzyme responsible for the first step in moenomycin biosynthesis, catalyzes the transfer of farnesyl to 3-phosphoglyceric acid (3PG) to give a product containing a cis-allylic double bond. We show that this reaction involves isomerization to a nerolidyl pyrophosphate intermediate followed by bond rotation prior to attack by the nucleophile. This mechanism is unprecedented for a prenyltransferase that catalyzes an intermolecular coupling. We also show that PcrB transfers geranyl and geranylgeranyl groups to glycerol-1-phosphate (G1P), making it the first known bacterial enzyme to use G1P as a substrate. Unlike MoeO5, PcrB catalyzes prenyl transfer without isomerization to give products that retain the trans-allylic bond of the prenyl donors. The TIM barrel family of PTs is unique in including enzymes that catalyze prenyl transfer by distinctly different reaction mechanisms.     

On Penicillin‐Binding Protein 1b Affinity‐Labeling Reagents

The synthesis of some 3‐aryl‐3‐(trifluoromethyl)3H‐diazirine and benzophenone‐based photoaffinity labels is reported. The photolabile group is bound to a scaffold that also accommodates functional groups to which an indicator unit (biotin) and the bioactive ligand can be attached orthogonally. To three of the labels, moenomycin was conjugated with the aim to provide tools for the identification of the moenomycin binding site within the transglycosylase domain of the enzyme PBP 1b. Some preliminary photoaffinity‐labeling experiments were carried out.     

Membrane activity of biomimetic facially amphiphilic antibiotics

Membranes are a central feature of all biological systems, and their ability to control many cellular processes is critically important. As a result, a better understanding of how molecules bind to and select between biological membranes is an active area of research. Antimicrobial host defense peptides are known to be membrane-active and, in many cases, exhibit discrimination between prokaryotic and eukaryotic cells. The design of synthetic molecules that capture the biological activity of these natural peptides has been shown. In this report, the interaction between our biomimetic structures and different biological membranes is reported using both model vesicle and in vitro bacterial cell experiments. Compound 1 induces 12% leakage at 20 microg/mL against phosphatidylglycerol (PG)-phosphatidylethanolamine (PE) vesicles vs only 3% leakage at 200 microg/mL against phosphatidyl-L-serine (PS)-phosphatidylcholine (PC) vesicles. Similarly, a 40% reduction in fluorescence is measured in lipid movement experiments for PG-PE compared to 10% for PS-PC at 600 s. A 30 degrees C increase in the phase transition of stearoyl-oleoyl-phosphatidylserine is observed in the presence of 1. These results show that lipid composition is more important for selectivity than overall net charge. Additionally, the overall concentration of a given lipid is another important factor. An effort is made to connect model vesicle studies with in vitro data and naturally occurring lipid compositions.     

Effects of lysophospholipids on membrane order of phosphatidylcholine

Lysophospholipids are known to play a role in a wide range of cellular processes involving membrane–protein or membrane–membrane interactions; however lysolipids–lamellar lipids interactions remain unclear. The effects of lysolipids on membrane order and dynamics were examined using optical birefringence and fluorescence techniques. We found that lysophosphatidic acid (LPA) induces a considerable disorder in chain orientation for synthetic lipid of dimyristoyl-phosphatidylcholines (DMPC), whereas a slight order for natural lipid of egg yolk phosphatidylcholine (Egg-PC), e.g. the chain order decreases by 10% at 0.1 mole ratio for DMPC in comparison with the membranes without LPA and increases by 3.4% at 0.09 mole ratio for Egg-PC. Also, membrane fluidity corresponds with the change in the chain disorder, namely, the fluidity increases for DMPC membranes, while decreases for Egg-PC membranes by addition of LPA. The difference in the effects of LPA is interpreted by a difference in the chain packing between the synthetic and the natural lipid bilayers. LPA can be incorporated into natural lipid membranes without disturbance, and readjusts itself to a more favorable hydrophobic match with the bilayers. Lysophophatidylcholine (LPC) also induces a disorder in DMPC membranes, but the decrease in chain order is only half compared with that for LPA.     

SAR Studies of the Leupyrrins: Design and Total Synthesis of Highly Potent Simplified Leupylogs

Leupyrrins are highly potent antifungal agents. A structure–activity-relationship study of natural and synthetic derivatives is reported which reveals important insights into the biological relevance of several structural subunits leading to the discovery of highly potent but drastically simplified leupylogs that incorporate a stable and readily available aromatic side chain. For their synthesis a concise strategy is described that enables a short and versatile access.     

Hydration properties of symmetric chain and asymmetric chain sphingomyelin bilayers

Hydration properties of lipid bilayer systems are compared for symmetric chain sphingomyelin (N-palmitoylsphingomyelin) and asymmetric chain sphingomyelin (N-lignoceroylsphingomyelin). These sphingomyelins were semisynthesized by a deacylation- reacylation process with a natural sphingomyelin used as a starting material. The number of differently bound water molecules was estimated by a deconvolution analysis of the ice-melting curves obtained by a differential scanning calorimetry (DSC) and was used to construct a water distribution diagram for these water molecules. Similarly to a natural sphingomyelin used for comparison, the asymmetric chain sphingomyelin was found to form small size vesicles having an internal cavity and incorporate 15 water molecules per molecule of lipid into its cavity, in contrast with 5 H₂O/lipid for freezable interlamellar water observed for large size multilamellar vesicles formed by the symmetric chain sphingomyelin.     

Synthesis of a carbohydrate-derived hydroxamic acid inhibitor of the bacterial enzyme (LpxC) involved in lipid A biosynthesis

[reaction: see text] The enzyme LpxC (UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc deacetylase) catalyzes the second step of lipid A biosynthesis and is essential for bacterial growth. A GlcNAc-derived hydroxamic acid inhibitor 8 of this enzyme was synthesized using two different routes. Compound 8 exhibits activity toward LpxC enzymes from a wider spectrum of bacterial species than any of the previously reported hydroxamic acid inhibitors.     

Synthesis and biological evaluation of analogues of the antibiotic pantocin B

Strains of the bacteria Erwinia herbicola produce antibiotics that effectively control E. amylovora, the bacterial pathogen responsible for the plant disease fire blight. Pantocin B was the first of these antibiotics to be characterized, and a flexible synthesis of various analogues is reported. Embedded in the "pseudo-tripeptide" backbone of pantocin B are a methylenediamine and a methyl sulfone, both unusual structural features in natural products. The peptidic nature of pantocin B facilitated a series of structure-activity relationship studies that probed the roles of these functional groups in determining the biological activity of pantocin B. A clear demarcation of the roles between the N- and C-terminal portions of the antibiotic was determined as a result of the structure-activity relationship studies. The N-terminal L-alanyl group is needed for cellular import but not for interaction with the intracellular target, the arginine biosynthetic enzyme N-acetylornithine aminotransferase. The methylenediamine and methyl sulfone portions were found to be essential for antibiotic activity, presumably due to extensive interactions with N-acetylornithine aminotransferase.     

Vertical and directional insertion of helical peptide into lipid bilayer membrane

A novel helical hexadecapeptide carrying a poly(ethylene glycol) (PEG) chain at the N terminal was synthesized. The N and C terminals of the compound are labeled with a fluorescein isothiocyanate (FITC) group and an N-ethylcarbazolyl group (ECz), respectively. An octapeptide carrying the same groups and a hexadecapeptide without a PEG chain were also synthesized and used as control. A mixture of the peptide and dimyristoylphosphatidylcholine was sonicated in a buffer to prepare the liposome. The orientation as well as direction of the helical segment in the lipid bilayer were analyzed by quenching experiments of the FITC and the ECz fluorescence. The results clearly indicated that the helical segment of the peptide penetrated into the lipid bilayer with vertical orientation in both the gel and liquid crystalline states of the lipid bilayer. Notably, the bulky N terminal was left behind in the outer aqueous phase of liposome, meaning that the C terminal of the peptide points to the inner aqueous phase of liposome. The insertion mode of the helical peptide into a bilayer membrane is therefore well-regulated in terms of the orientation and the directionality by designing the balance between the PEG chain and the helix length. The methodology presented here will initiate a way to construct artificial functional molecular systems that can induce vectorial transport phenomena as seen in biological systems.     

Synthesis and bioactivity of fluorescence- and biotin-labeled lipid A analogues for investigation of recognition mechanism in innate immunity

Fluorescence- and biotin-labeled lipid A analogues were synthesized for the investigation of bacterial lipopolysaccharide (LPS)/lipid A recognition in the innate immune system. For the introduction of the labeling moiety, a hydrophilic glutaryl-glucose linker was used for maintaining the bioactivity and also for preventing self-aggregation, which causes quenching of the fluorescence. We also observed the biological activity of the labeled compounds.