Synthesis and biological evaluation of vancomycin dimers with potent activity against vancomycin-resistant bacteria: target-accelerated combinatorial synthesis

Based on the notion that dimerization and/or variation of amino acid 1 of vancomycin could potentially enhance biological activity, a series of synthetic and chemical biology studies were undertaken in order to discover potent antibacterial agents. Herein we describe two ligation methods (disulfide formation and olefin metathesis) for dimerizing vancomycin derivatives and applications of target-accelerated combinatorial synthesis (e.g. combinatorial synthesis in the presence of vancomycin's target Ac2-L-Lys-D-Ala-D-Ala) to generate libraries of vancomycin dimers. Screening of these compound libraries led to the identification of a number of highly potent antibiotics effective against vancomycin-suspectible, vancomycin-intermediate resistant and, most significantly, vancomycin-resistant bacteria.     

A redesigned vancomycin engineered for dual D-Ala-D-ala And D-Ala-D-Lac binding exhibits potent antimicrobial activity against vancomycin-resistant bacteria

The emergence of bacteria resistant to vancomycin, often the antibiotic of last resort, poses a major health problem. Vancomycin-resistant bacteria sense a glycopeptide antibiotic challenge and remodel their cell wall precursor peptidoglycan terminus from d-Ala-d-Ala to d-Ala-d-Lac, reducing the binding of vancomycin to its target 1000-fold and accounting for the loss in antimicrobial activity. Here, we report [Ψ[C(═NH)NH]Tpg(4)]vancomycin aglycon designed to exhibit the dual binding to d-Ala-d-Ala and d-Ala-d-Lac needed to reinstate activity against vancomycin-resistant bacteria. Its binding to a model d-Ala-d-Ala ligand was found to be only 2-fold less than vancomycin aglycon and this affinity was maintained with a model d-Ala-d-Lac ligand, representing a 600-fold increase relative to vancomycin aglycon. Accurately reflecting these binding characteristics, it exhibits potent antimicrobial activity against vancomycin-resistant bacteria (MIC = 0.31 μg/mL, VanA VRE). Thus, a complementary single atom exchange in the vancomycin core structure (O → NH) to counter the single atom exchange in the cell wall precursors of resistant bacteria (NH → O) reinstates potent antimicrobial activity and charts a rational path forward for the development of antibiotics for the treatment of vancomycin-resistant bacterial infections.     

Conformational studies of resin-bound vancomycin and the complex of vancomycin and Ac2-L-Lys-D-Ala-D-Ala

The molecular target of vancomycin, a commonly used glycopeptide antibiotic, is the D-Ala-D-Ala dipeptide subunit on the bacterial cell wall. The molecular basis of interaction between vancomycin and D-Ala-D-Ala in solution is well-known. However, there is no structural data on vancomycin, and its interaction with D-Ala-D-Ala when the drug is tethered to a solid support. In this Article, vancomycin was directly coupled onto TentaGel or PEGA resin through its C terminus. High-resolution magic angle spinning NMR studies indicated that conformation of PEGA bead-bound vancomycin is identical to that of the free drug. Broadening and shifts of the same proton resonances were observed in solution-phase vancomycin or PEGA-bound vancomycin when complexed with Ac(2)-L-Lys-D-Ala-D-Ala. This study demonstrates that bead-bound molecules can behave the same as solution-phase molecules in terms of molecular interaction with its target molecule, thus validating the on-bead screening approach of the "one-bead-one-compound" combinatorial library method.     

Multistep solid-phase synthesis of an antibiotic and receptor tyrosine kinase inhibitors using the traceless phenylhydrazide linker

The hydrazide group is an oxidatively cleavable traceless linker for solid-phase chemistry. This linker technology was used to develop a multistep solid-phase synthesis of an antibiotic that is active against Mycobacterium tuberculosis. Furthermore, we describe an efficient method for the traceless synthesis of 2-aminothiazoles that display dual inhibitory activity against the receptor tyrosine kinases VEGFR-2 and Tie-2. The synthesis method proceeds through 9 steps on the solid phase and should give access to a much larger library of 2-aminothiazoles, from which a new class of anti-angiogenesis drugs may be developed.     

Unraveling the protein targets of vancomycin in living S. aureus and E. faecalis cells

Vancomycin is a potent glycopeptide antibiotic that has evolved to specifically bind to the D-Ala-D-Ala dipeptide termini of nascent peptidoglycans. Although this mode of action is well established, several studies indicate that vancomycin and analogues exploit noncanonical target sites. In order to address all vancomycin targets in clinically relevant Staphylococcus aureus and Enterococcus faecalis strains we developed a series of small-molecule photoaffinity probes based on vancomycin. Proteomic profiling revealed the specific labeling of two previously unknown vancomycin targets that are likely to contribute to its antibiotic activity. The specific inhibition of the major staphylococcal autolysin Atl confirms previous observations that vancomycin alters S. aureus cell morphology by interaction with the autolytic machinery. Moreover, in E. faecalis the vancomycin photoprobe specifically binds to an ABC transporter protein, which likely impedes the uptake of essential nutrients such as sugars and peptides. The labeling of these two prominent membrane targets in living cells reveals a thus far unexplored mode of vancomycin binding and inhibition that could allow a rational design of variants with improved activity.     

Conformational changes of peptidoglycan fragments during their interactions with vancomycin

Six complexes of vancomycin and peptidoglycan precursors were studied via molecular dynamics simulations. The interactions between the antibiotic and peptidoglycan fragments were identified and described in detail. All six studied modifications of the peptidoglycan precursor resulted in a weakening of the interaction with vancomycin when comparing to the native D-Ala-D-Ala-terminated fragment. It was confirmed that the N-terminus of the vancomycin is directly responsible for peptidoglycan recognition and antimicrobial activity. In simulated systems, the saccharide part of the antibiotic interacts with peptide precursors, thus it could also be important for antimicrobial activity. The complex terminated with D-Lac is the only one in which there is a weak interaction with the sugar moiety in the simulated systems. Analysis of conformational changes is a major scope of this work. The lack of interactions resulting from modification of the peptidoglycan precursors (D-Lac, D-Ser or other substitution) would be counterbalanced by proper modifications of the vancomycin moiety, especially the saccharide part of vancomycin.     

D-Ala-D-lac binding is not required for the high activity of vancomycin dimers against vancomycin resistant enterococci

Covalent dimerization and oligomerization of vancomycin is an important and extensively used strategy to develop analogues active against vancomycin resistant enteroccoci (VRE). Here, we have carried out investigations to probe the role of peptide binding (Lys-d-Ala-d-Lac) in the high anti-VRE activities of covalently linked vancomycin dimers. Covalent dimers of damaged vancomycin (desleucyl) were prepared, and their anti-VRE activities and binding affinities toward various model peptides were measured. Despite the dramatic loss in affinity toward several model peptides in comparison to the corresponding intact vancomycin dimers, these damaged dimers maintained good activity against VRE. These results strongly suggest that the high anti-VRE activities of covalent vancomycin dimers are conferred from mechanisms other than Lys-d-Ala-d-Lac binding.     

Modification and inhibition of vancomycin group antibiotics by formaldehyde and acetaldehyde

It is shown that several vancomycin group antibiotics (vancomycin, eremomycin, and avoparcin) undergo spontaneous chemical modifications when kept at room temperature at neutral pH in aqueous solutions containing traces of formaldehyde or acetaldehyde. This chemical modification predominantly results in a mass increase of 12 Da in the reaction with formaldehyde and 26 Da in the case of acetaldehyde. By using tandem mass spectrometry the modification can unambiguously be identified as originating from the formation of a ring-closed 4-imidazolidinone moiety at the N-terminus of the glycopeptide antibiotics, that is, near the receptor binding pocket of the glycopeptide antibiotics. Bioaffinity mass spectrometry shows that this ring-closure results in a dramatically decreased affinity for the peptidoglycan-mimicking D-alanyl-D-alanine receptor. Additionally, in vitro inhibition measurements on two different strains of bacteria have revealed that the modified antibiotics display reduced antibacterial activity. The ring-closure is also shown to have a dissociative effect on the dimerization of the vancomycin-analogue eremomycin. The spontaneous reaction of vancomycin with formaldehyde or acetaldehyde may have implications not only for the clinical use of this class of antibiotics, but also for the effectiveness of these antibiotics when they are used in chiral separation chromatography or capillary electrophoresis.     

Unusual binding ability of vancomycin towards Cu2+ ions

Vancomycin, a "last chance" antibiotic, is a glycopeptide consisting of an oligopeptide unit being potentially the effective binder of Cu2+ ions. The potentiometric and spectroscopic studies (UV-Vis, CD, EPR, NMR) have shown that, indeed, the peptide unit binds cupric ions very effectively forming almost instantly the 3N complex involving the N-terminal nitrogen donors in the metal ion coordination. The comparison of the binding ability of vancomycin with other peptide chelators clearly shows the efficiency of this antibiotic in metal ion coordination. It is very likely that Cu2+ ions may play a crucial role in the pharmacology of vancomycin, particularly when administered in high doses.     

Considerations concerning interaction characterization of oligopeptide mixtures with vancomycin using affinity capillary electrophoresis-electrospray mass spectrometry.

In the past few years affinity capillary electrophoresis (ACE) has proven to be a powerful tool to study molecular interactions. In ACE the change in electrophoretic mobility between a free and a complexed ligand with a receptor dissolved in the background electrolyte is observed. It provides an accurate way to calculate binding or dissociation constants and, when coupled to mass spectrometry, it forms a promising method to analyze solution-based combinatorial libraries. We report a model study on the macrocyclic antibiotic vancomycin using a 36-component library of tetrapeptides of the type 9-fluorenylmethoxycarbonyl (Fmoc)-L-Asp-L-Asp-D-Xaa-D-Xaa. The mass spectrometry conditions were optimized by fine-tuning the background electrolyte and sheath flow composition to achieve optimal sensitivity in the negative ionization mode. Different types of capillaries were also evaluated on their potential to screen combinatorial libraries. The library components that show the strongest interaction were identified. The dissociation constants of a mixture of six compounds with a broad affinity range were simultaneously established by Scatchard analysis on ACE-MS.     

From polymer to small organic molecules: a tight relationship between radical chemistry and solid-phase organic synthesis

Since Gomberg's discovery of radicals as chemical entities, the interest around them has increased through the years. Nowadays, radical chemistry is used in the synthesis of 75% of all polymers, inevitably establishing a close relationship with Solid-Phase Organic Synthesis. More recently, the interest of organic chemists has shifted towards the application of usual "in-solution" radical chemistry to the solid-phase, ranging from the use of supported reagents for radical reactions, to the development of methodologies for the synthesis of small molecules or potential libraries. The aim of this review is to put in perspective radical chemistry, moving it away from its origin as a synthetic means for solid supports, to becoming a useful tool for the synthesis of small molecules.     

Total synthesis and evaluation of [Psi[CH2NH]Tpg4]vancomycin aglycon: reengineering vancomycin for dual D-Ala-D-Ala and D-Ala-D-Lac binding

An effective synthesis of [Psi[CH(2)NH]Tpg(4)]vancomycin aglycon (5) is detailed in which the residue 4 amide carbonyl of vancomycin aglycon has been replaced with a methylene. This removal of a single atom was conducted to enhance binding to D-Ala-D-Lac, countering resistance endowed to bacteria that remodel their D-Ala-D-Ala peptidoglycan cell wall precursor by a similar single atom change (ester O for amide NH). Key elements of the approach include a synthesis of the modified vancomycin ABCD ring system featuring a reductive amination coupling of residues 4 and 5 for installation of the deep-seated amide modification, the first of two diaryl ether closures for formation of the modified CD ring system (76%, 2.5-3:1 kinetic atropodiastereoselectivity), a Suzuki coupling for installation of the hindered AB biaryl bond (90%) on which the atropisomer stereochemistry could be thermally adjusted, and a macrolactamization closure of the AB ring system (70%). Subsequent DE ring system introduction enlisted a room-temperature aromatic nucleophilic substitution reaction for formation of the remaining diaryl ether (86%, 6-7:1 kinetic atropodiastereoselectivity), completing the carbon skeleton of 5. Consistent with expectations and relative to the vancomycin aglycon, 5 exhibited a 40-fold increase in affinity for D-Ala-D-Lac (K(a) = 5.2 x 10(3) M(-1)) and a 35-fold reduction in affinity for D-Ala-D-Ala (K(a) = 4.8 x 10(3) M(-1)), providing a glycopeptide analogue with balanced, dual binding characteristics. Beautifully, 5 exhibited antimicrobial activity (MIC = 31 microg/mL) against a VanA-resistant organism that remodels its D-Ala-D-Ala cell wall precursor to d-Ala-d-Lac upon glycopeptide antibiotic challenge, displaying a potency that reflects these binding characteristics.     

Bacterial resistance to vancomycin: five genes and one missing hydrogen bond tell the story

A plasmid-borne transposon encodes enzymes and regulator proteins that confer resistance of enterococcal bacteria to the antibiotic vancomycin. Purification and characterization of individual proteins encoded by this operon has helped to elucidate the molecular basis of vancomycin resistance. This new understanding provides opportunities for intervention to reverse resistance.     

Polymer-supported synthesis of pyridone-focused libraries as inhibitors of anaplastic lymphoma kinase

The optimization of screening hits on a promising new target for therapy of certain cancers involving anaplastic lymphoma kinase (ALK) inspired the development of this efficient solid-phase chemistry. A series of novel pyridones have been recently discovered as inhibitors of ALK, which led to the design of focused libraries around the pyridone scaffold. A stepwise process involving iterative template modification based on both medicinal chemistry insights and computational ranking of virtual libraries was employed in the design. The unique solid-phase chemistry has addressed the need for rapid optimization of this "early lead" series. Herein the methodology and scope of the chemistry, as well as its application for library synthesis, are discussed.     

Direct interaction of a vancomycin derivative with bacterial enzymes involved in cell wall biosynthesis.

BACKGROUND: The glycopeptide antibiotic vancomycin complexes DAla-DAla termini of bacterial cell walls and peptidoglycan precursors and interferes with enzymes involved in murein biosynthesis. Semisynthetic vancomycins incorporating hydrophobic sugar substituents exhibit efficacy against DAla-DLac-containing vancomycin-resistant enterococci, albeit by an undetermined mechanism. Contrasting models that invoke either cooperative dimerization and membrane anchoring or direct inhibition of bacterial transglycosylases have been proposed to explain the bioactivity of these glycopeptides. RESULTS: Affinity chromatography has revealed direct interactions between a semisynthetic hydrophobic vancomycin (DCB-PV), and select Escherichia coli membrane proteins, including at least six enzymes involved in peptidoglycan assembly. The N(4)-vancosamine substituent is critical for protein binding. DCB-PV inhibits transglycosylation in permeabilized E. coli, consistent with the observed binding of the PBP-1B transglycosylase-transpeptidase. CONCLUSIONS: Hydrophobic vancomycins interact directly with a select subset of bacterial membrane proteins, suggesting the existence of discrete protein targets. Transglycosylase inhibition may play a role in the enhanced bioactivity of semisynthetic glycopeptides.     

Modern separation techniques for the efficient workup in organic synthesis

The shift of paradigm in combinatorial chemistry, from large compound libraries (of mixtures) on a small scale towards defined compound libraries where each compound is prepared in an individual well, has stimulated the search for alternative separation approaches. The key to a rapid and efficient synthesis is not only the parallel arrangement of reactions, but simple work-up procedures so as to circumvent time-consuming and laborious purification steps. During the initial development stages of combinatorial synthesis it was believed that rational synthesis of individual compounds could only be achieved by solid-phase strategies. However, there are a number of problems in solid-phase chemistry: most notably there is the need for a suitable linker unit, the limitation of the reaction conditions to certain solvents and reagents, and the heterogeneous reaction conditions. Further disadvantages are: the moderate loading capacities of the polymeric support and the limited stability of the solid support. In the last few years several new separation techniques have been developed. Depending on the chemical problem or the class of compounds to be prepared, one can choose from a whole array of different approaches. Most of these modern separation approaches rely on solution-phase chemistry, even though some of them use solid-phase resins as tools (for example, as scavengers). Several of these separation techniques are based on liquid-liquid phase separation, including ionic liquids, fluorous phases, and supercritical solvents. Besides being benign with respect to their environmental aspects, they also show a number of advantages with respect to the work-up procedures of organic reactions as well as simplicity in the isolation of products. Another set of separation strategies involves polymeric supports (for example, as scavengers or for cyclative cleavage), either as solid phases or as soluble polymeric supports. In contrast to solid-phase resins, soluble polymeric supports allow reactions to be performed under homogeneous conditions, which can be an important factor in catalysis. At the same time, a whole set of techniques has been developed for the separation of these soluble polymeric supports from small target molecules. Finally, miscellaneous separation techniques, such as phase-switchable tags for precipitation by chemical modification or magnetic beads, can accelerate the separation of compounds in a parallel format.     

The effect of solvent composition on vancomycin hydrochloride and free base vancomycin release from in situ forming implants

In situ forming drug delivery systems that are formed by solvent‐induced phase inversion have attracted extensive attention in sustained delivery of peptides and proteins. Based on the findings of our previous studies, N‐methyl‐2‐pyrrolidone (NMP) and acetone are two solvents that could improve the release profile of vancomycin from in situ forming systems based on poly(D,L‐lactide‐co‐glycolic acid). In this study, the effect of different compositions of these solvents on the release profile of hydrochloride and free base forms of vancomycin was investigated. To this end, several formulations with vancomycin (either hydrochloride or free base form) and different proportions of NMP and acetone were prepared. The cumulative drug release at specified time was determined and tested against conventional kinetic models. The surface and cross‐sectional morphology of implants were investigated by SEM. The experimental results showed that as solvent composition changed, the amount of vancomycin release during the first 12 h changed, too. The use of free base vancomycin resulted in an extended vancomycin release profile with less initial burst release. The formulation containing free base vancomycin and mixed solvents of acetone and NMP in 2:1 ratio released 70% of loaded drug in 6 weeks with near zero‐order kinetic. The best kinetic model to fit the in vitro release profiles was found to be Peppas–Sahlin model. Copyright © 2016 John Wiley & Sons, Ltd.     

An improved solid-phase methodology for the synthesis of putative hexa- and heptapeptide intermediates in vancomycin biosynthesis

The biosynthesis of the vancomycin aglycone involves three oxidative phenol coupling reactions, each catalyzed by a discrete cytochrome P450-like enzyme. Studies on the mechanism and specificity of the enzyme (called OxyB) catalyzing the first coupling, require access to suitable linear peptide precursors, each conjugated as a thioester to a peptide carrier domain of the vancomycin non-ribosomal peptide synthetase. An efficient route to representative free linear peptides is described here. The method makes use of Alloc-chemistry during solid-phase assembly of the peptide backbone, but importantly and in contrast to earlier efforts, largely avoids the use of amino acid side chain protecting groups. In this way, the target linear peptides can be released directly from the solid support under very mild conditions.     

Synthesis and evaluation of poly(oxyethylene glycol) polymer (POP) supports

Mono- and alpha,omega-bis-styryl-oligo(oxyethylene glycol) ethers have been constructed in an efficient two-step synthesis. From these precursors, poly(oxyethylene glycol) polymer (POP) supports of varying monomer and cross-linker composition have been produced. The swelling properties and mass-solvent uptake of these novel materials have been evaluated in a variety of solvents, demonstrating that POP supports exhibit enhanced solvent compatibilities over the commercial resins TENTA-GEL, ARGO-GEL, and Merrifield's resin. The utility of POP supports in solid-phase organic chemistry has also been demonstrated successfully. It is anticipated that these high-loading polymeric supports will have generic application in the solid-phase synthesis of combinatorial libraries and the in situ screening of these libraries in the aqueous environment of a bioassay.     

Evaluating measurement uncertainty in the microbiological assay of vancomycin from methodology validation data

Methodology validation and measurement uncertainty estimation are fundamental to obtain reliable results. The microbiological methods are widely used to determine antibiotic assay, as they permit evaluation of the analyzed antibiotic activity. A microbiological assay of vancomycin was performed with adoption of experimental design 5 x 1 (interpolation in 5-point standard curve assay) with final concentrations from 6.4 to 15.6 microg/mL (standards) and 10.0 microg/mL (sample). Bacillus subtitlis (ATCC 6633) was the microorganism used, with antibiotic medium No. 8 as base layer and inoculated layer. The plates were incubated at 37 degrees C for 18 h. The method adopted for the microbiological assay of vancomycin through agar diffusion was validated according to statistic results demonstrated for suitability of the method concerning linearity, precision, and accuracy. The estimated relative expanded uncertainty (4.3%) was considered adequate for this method purpose.