Liquid Chromatographic Enantioseparation of Newly Synthesized Fluorinated Tryptophan Analogs Applying Macrocyclic Glycopeptides-Based Chiral Stationary Phases Utilizing Core-Shell Particles.

Due to the favorable features obtained through the incorporation of fluorine atom(s), fluorinated drugs are a group with emerging pharmaceutical importance. As their commercial availability is still very limited, to expand the range of possible candidates, new fluorinated tryptophan analogs were synthesized. Control of enantiopurity during the synthesis procedure requires that highly efficient enantioseparation methods be available. In this work, the enantioseparation of seven fluorinated tryptophans and tryptophan was studied and compared systematically to (i) develop analytical methods for enantioselective separations and (ii) explore the chromatographic features of the fluorotrytophans. For enantioresolution, macrocyclic glycopeptide-based selectors linked to core-shell particles were utilized, applying liquid chromatography-based methods. Application of the polar-ionic mode resulted in asymmetric and broadened peaks, while reversed-phase conditions, together with mobile-phase additives, resulted in baseline separation for all studied fluorinated tryptophans. The marked differences observed between the methanol and acetonitrile-containing eluent systems can be explained by the different solvation abilities of the bulk solvents of the applied mobile phases. Among the studied chiral selectors, teicoplanin and teicoplanin aglycone were found to work effectively. Under optimized conditions, baseline separations were achieved within 6 min. Ionic interactions were semi-quantitatively characterized and found to not influence enantiorecognition. Interestingly, fluorination of the analytes does not lead to marked changes in the chromatographic characteristics of the methanol-containing eluents, while larger differences were noticed when the polar but aprotic acetonitrile was applied. Experiments conducted on the influence of the separation temperature indicated that the separations are enthalpically driven, with only one exception. Enantiomeric elution order was found to be constant on both teicoplanin and teicoplanin aglycone-based chiral stationary phases (L < D) under all applied chromatographic conditions.

Click preparation of triazole-bridged teicoplanin-bound chiral stationary phases for efficient separating amino acid enantiomers.

Enantioseparation of amino acids is considered as a challenging task due to the extreme structural similarity of their enantiomers. Herein, teicoplanin was modified with different chemical equivalents of azide groups and attached to silica particles by employing Click Chemistry for resolution of chiral amino acids for the first time. Interestingly, teicoplanin modified with 5-fold the chemical equivalent of azide groups (TK-2 CSP) exhibited superior amino acid separation ability compared to two other columns: one modified with only 1-fold the chemical equivalent of azide groups (TK-1 CSP), and the other modified with excess azide groups (TK-3 CSP). Additionally, the TK-2 CSP exhibited superior enantioselectivity when separating amino acids containing hydrophobic alkyl side chains in comparison to other teicoplanin-based CSPs. The TK-2 CSP column allows the baseline separation of 7 native amino acids. Molecular docking demonstrates that effective enantioseparation arises from distinct patterns of interaction between the host and guest molecules. Moreover, (p-methyl) phenylcarbaminoylated-teicoplanin CSP (TK-4, TK-5 CSP) were prepared by post-modification from TK-1 CSP and TK-2 CSP to isolate Fmoc-modified amino acids. This work explores the impact of various modification methods on the enantioseparation effects of host molecules and paves the way for expanding the potential applications of teicoplanin and macrocyclic glycopeptide molecules.

Resurrecting ancestral antibiotics: unveiling the origins of modern lipid II targeting glycopeptides.

Antibiotics are central to modern medicine, and yet they are mainly the products of intra and inter-kingdom evolutionary warfare. To understand how nature evolves antibiotics around a common mechanism of action, we investigated the origins of an extremely valuable class of compounds, lipid II targeting glycopeptide antibiotics (GPAs, exemplified by teicoplanin and vancomycin), which are used as last resort for the treatment of antibiotic resistant bacterial infections. Using a molecule-centred approach and computational techniques, we first predicted the nonribosomal peptide synthetase assembly line of paleomycin, the ancestral parent of lipid II targeting GPAs. Subsequently, we employed synthetic biology techniques to produce the predicted peptide and validated its antibiotic activity. We revealed the structure of paleomycin, which enabled us to address how nature morphs a peptide antibiotic scaffold through evolution. In doing so, we obtained temporal snapshots of key selection domains in nonribosomal peptide synthesis during the biosynthetic journey from ancestral, teicoplanin-like GPAs to modern GPAs such as vancomycin. Our study demonstrates the synergy of computational techniques and synthetic biology approaches enabling us to journey back in time, trace the temporal evolution of antibiotics, and revive these ancestral molecules. It also reveals the optimisation strategies nature has applied to evolve modern GPAs, laying the foundation for future efforts to engineer this important class of antimicrobial agents.

Retention mechanisms of rasagiline and its analogues on superficially porous particle vancomycin- and teicoplanin-based chiral stationary phases.

Retention and separation of enantiomers of amine derivatives of indane and tetralin (rasagiline and its analogues) on chiral stationary phases (CSPs) Chiral-T and Chiral-V with teicoplanin and vancomycin antibiotics grafted onto superficially porous silica particles under conditions of reversed-phase and polar organic chromatography were studied. The mobile phases (MP) were water-methanol and acetonitrile-methanol solvents modified with triethylamine-acetic acid buffer. The effects of molecular structure and physical properties of the analytes on enantioselective retention are discussed. The retention mechanism is hypothesized to involve the ion-ion attraction between the positively charged amino group of an analyte and the carboxylate anion of either antibiotic. The binding occurs outside of the antibiotic's aglycon basket that accounts for relatively low enantioselectivity observed. The presence of a large substitute at the analyte's amino group complicates enantiorecognition. The effect of the MP solvent composition on retention and enantioseparation was investigated. It is a complex phenomenon combined of different oppositely directed influences that resulted in different shapes, increasing, decreasing, or U-shaped, of the retention factor vs. composition dependences. A model taking into account the interaction of both solvents of a binary MP with both an analyte and an adsorption site was successfully applied to approximate a majority of the studied systems. Pros and cons of the model are discussed.

Enantioseparation of a-substituted proline analogs with macrocyclic glycopeptide-based chiral stationary phases immobilized on superficially porous particles of silica applying liquid chromatography with ultraviolet and mass spectrometric detection.

In this study, the liquid chromatography-based direct enantioseparation of the stereoisomers of α-substituted proline analogs has been investigated utilizing chiral stationary phases with UV and/or mass spectrometric (MS) detection. Macrocyclic antibiotics, such as vancomycin, teicoplanin, modified teicoplanin, and teicoplanin aglycone, all covalently immobilized to 2.7 µm superficially porous silica particles have been applied as stationary phases. Mobile phases utilizing mixtures of methanol and acetonitrile with different additives (polar-ionic mode) were optimized during method development. Best separations were achieved with mobile phases of 100% MeOH containing either 20 mM acetic acid or 20 mM triethylammonium acetate. Special attention was given to the applicability of MS-compatible mobile phases. Acetic acid was found to be advantageous as a mobile phase additive for MS detection. Enantioselective chromatographic behaviors are interpreted based on the explored correlations between the analytes' structural features and those of the applied chiral stationary phases. For the thermodynamic characterization, separations were studied in the temperature range of 5-50 °C. Generally, retention and selectivity decreased with increasing temperature, and in most cases, enthalpy-driven enantiorecognition was observed, but entropic contributions also were present. Unexpectedly, unusual shapes for the van Deemter curves were registered in the kinetic evaluations. General trends could be observed in the enantiomeric elution orders: S < R on VancoShell and NicoShell, and opposite R < S on TeicoShell and TagShell columns.

The Enantioselective Potential of NicoShell and TeicoShell Columns for Basic Pharmaceuticals and Forensic Drugs in Sub/Supercritical Fluid Chromatography.

The enantioselective potential of two macrocyclic glycopeptide-based chiral stationary phases for analysis of 28 structurally diverse biologically active compounds such as derivatives of pyrovalerone, ketamine, cathinone, and other representatives of psychostimulants and antidepressants was evaluated in sub/supercritical fluid chromatography. The chiral selectors immobilized on 2.7 µm superficially porous particles were teicoplanin (TeicoShell column) and modified macrocyclic glycopeptide (NicoShell column). The influence of the organic modifier and different mobile phase additives on the retention and enantioresolution were investigated. The obtained results confirmed that the mobile phase additives, especially water as a single additive or in combination with basic and acidic additives, improve peak shape and enhance enantioresolution. In addition, the effect of temperature was evaluated to optimize the enantioseparation process. Both columns exhibited comparable enantioselectivity, approximately 90% of the compounds tested were enantioseparated, and 30% out of them were baseline enantioresolved under the tested conditions. The complementary enantioselectivity of the macrocyclic glycopeptide-based chiral stationary phases was emphasized. This work can be useful for the method development for the enantioseparation of basic biologically active compounds of interest.

Branched medium-chain fatty acid profiling and enantiomer separation of anteiso-forms of teicoplanin fatty acyl side chain RS3 using UHPLC-MS/MS with polysaccharide columns.

This work reports on targeted UHPLC-tandem mass spectrometry methods for the chiral separation of anteiso-methyl branched fatty acids (aiFAs). The methods involve precolumn derivatization with 1-naphthylamine and chiral separation on Chiralpak IG-U. anteiso-Methyl branched fatty acids with up to eight carbons can be separated. A method was used for the assignment of the absolute configuration of an aiFA present as fatty acyl residue of the teicoplanin mixture, namely teicoplanin RS3. Furthermore, the excellent methylene selectivity and improved selectivity for constitutional isomers of the polysaccharide columns was exploited for the elucidation and structural confirmation of previously unknown fatty acyl residues in teicoplanin. This shows the versatility and practical applicability of polysaccharide columns as orthogonal stationary phases to reversed-phase for structural elucidation of natural compounds. The developed methods are useful tools for related subdisciplines such as targeted metabolomics and lipidomics.

Chiral chromatography and surface chirality of carbon nanoparticles.

Chiral carbon nanoparticles (CNPs) represent a rapidly evolving area of research for optical and biomedical technologies. Similar to small molecules, applications of CNPs as well as fundamental relationships between their optical activity and structural asymmetry would greatly benefit from their enantioselective separations by chromatography. However, this technique remains in its infancy for chiral carbon and other nanoparticles. The possibility of effective separations using high performance liquid chromatography (HPLC) with chiral stationary phases remains an open question whose answer can also shed light on the components of multiscale chirality of the nanoparticles. Herein, we report a detailed methodology of HPLC for successful separation of chiral CNPs and establish a path for its future optimization. A mobile phase of water/acetonitrile was able to achieve chiral separation of CNPs derived from L- and D-cysteine denoted as L-CNPs and D-CNPs. Molecular dynamics simulations show that the teicoplanin-based stationary phase has a higher affinity for L-CNPs than for D-CNPs, in agreement with experiments. The experimental and computational findings jointly indicate that chiral centers of chiral CNPs are present at their surface, which is essential for the multiple applications of these chiral nanostructures and equally essential for interactions with biomolecules and circularly polarized photons.

Boosting the enantioresolution of zwitterionic-teicoplanin chiral stationary phases by moving to wide-pore core-shell particles.

A novel zwitterionic-teicoplanin chiral stationary phase (CSP), based on superficially porous particles (SPPs) of 2.7 µm particle diameter and 160 Å pore size, has been prepared and evaluated towards the enantioseparation of important classes of compounds, including chiral drugs, pesticides, and N-derivatized amino acids. The comparison with two analogous CSPs prepared on SPPs with 2.7 and 2.0 µm particle diameter and 90 Å pore size has revealed that the use of large-pore particles allows to dramatically improve both the enantioselectivity and the resolution-per-analysis-time, at the point that the column prepared with the new CSP outperformed the one packed with the finest particles. On the novel wide-pore CSP, the separation of fifteen racemates of pratical importance was significantly improved in terms of both enantioselectivity and resolution-per-analysis time-compared to the CSPs based on SPPs with smaller pores (90 Å). Such a CSP would be suitable for very fast enantioseparations allowing the saving of solvent for greener high-efficiency/high-throughput applications.

Lipid liquid-crystalline nanoparticles sustained teicoplanin delivery for treatment of chronic osteomyelitis: in vitro and in vivo studies.

The aim of our work is to formulate teicoplanin-loaded lipid liquid-crystalline (cubosomes) nanoparticles laden gel to sustain the release of teicoplanin for effective treatment of infected bone. Cubosomal gels were prepared by emulsification technique. The batches were characterised for morphology, size, entrapment efficacy, viscosity, in-vitro flux, in-vivo drug release and histopathological studies. Transmission electron microscopy images confirmed the bi-continuous liquid crystalline phase. The size (61-202 nm), viscosity (12 138-13 132 cp), and entrapment efficacy (69.0-81.8% w/w) increase with the level of glycerol monooleate. The in-vitro flux data showed sustain teicoplanin release from the cubosomal gels for 36 days, compared to 48 h from the control gel. The in-vivo teicoplanin release study (osteomyelitis induced by S. aureus) showed low serum drug-concentration from the gel (up to 14 days) compared to high-serum drug-concentration using intravenous injections. In conclusion the study demonstrated the potential of cubosomes for effective delivery of teicoplanin to replace injections.

Characterization of Teicoplanin-Specific T-Cells from Drug Naïve Donors Expressing HLA-A*32:01.

Teicoplanin is a glycopeptide antibiotic deployed to combat Gram-positive bacterial infection and has recently been associated with development of adverse drug reactions, particularly following previous exposure to vancomycin. In this study, we generated teicoplanin-specific monoclonal T-cell populations from healthy volunteers expressing HLA-A*32:01 and defined pathways of T-cell activation and HLA allele restriction. Teicoplanin-responsive T-cells were CD8+, HLA class I-restricted, and cross-reacted with the lipoglycopeptide daptomycin in proliferation and cytokine/cytolytic molecule (granzyme B, Perforin, and FasL) release assays. These data show that teicoplanin activates T-cells, which may play a role in the pathogenesis of teicoplanin-induced adverse events, in HLA-A*32:01 positive donors.

Enantioseparation of ß2-amino acids by liquid chromatography using core-shell chiral stationary phases based on teicoplanin and teicoplanin aglycone.

Enantioseparation of nineteen ß2-amino acids has been performed by liquid chromatography on chiral stationary phases based on native teicoplanin and teicoplanin aglycone covalently bonded to 2.7 µm superficially porous silica particles. Separations were carried out in unbuffered (water/methanol), buffered [aqueous triethylammonium acetate (TEAA)/methanol] reversed-phase (RP) mode, and in polar-ionic (TEAA containing acetonitrile/methanol) mobile phases. Effects of pH in the RP mode, acid and salt additives, as well as counter-ion concentrations on chromatographic parameters have been studied. The structure of selectands (ß2-amino acids possessing aliphatic or aromatic side chains) and selectors (native teicoplanin or teicoplanin aglycone) was found to have a considerable influence on separation performance. Analysis of van Deemter plots and determination of thermodynamic parameters were performed to further explore details of the separation performance.

Self-assembled micelles of the (lipo) glycopeptides, teicoplanin, as taxane nanocarriers.

The use of nanoparticles is one of the strategies currently studied to minimize the toxicity and lack of tissue specificity of many cancer drugs used in chemotherapy. In this research the physicochemical and biological behavior of a novel self-assembled nanostructure of the antibiotic Teicoplanin (Teico) was characterized as a nanocarrier system for solubilizing highly hydrophobic drugs like Paclitaxel (Ptx) in aqueous media. The Teico micelles were loaded with Ptx in DMSO or PEG-400. The interaction between the loaded micelles and Albumin human serum albumin (HSA) was then studied by size exclusion chromatography. Transmission electron microscopy, dynamic light scattering and high-resolution liquid chromatography were also used to characterize the physicochemical and structural properties of the micelles to form the Teico/Ptx and Teico/Ptx/HSA micelles. Cellular uptake of Ptx was evaluated by fluorescent microscopy. Thein vitrocytotoxicity of the complexes was studied on Hep-2 tumor cells, by a Crystal Violet assay. Teico cosolvent-free micelles can solubilize up to 20 mg.ml-1of Ptx dissolved in PEG, increasing four times the solubility of Ptx in water compared to Abraxane, and 20 000 times the intrinsic solubility of Ptx in water. In addition, Teico/Ptx micelles binds spontaneously HSA through hydrophobic interaction. Teico and Teico/HSA micelles as a Ptx transporter does not affect its release or biological activity. Therefore, Teico/Ptx or Teico/Ptx/HSA complexes appear as new alternatives for transporting larger amounts of hydrophobic drugs that offer advantages, turning it an interesting option for further study.

Genomic-Led Discovery of a Novel Glycopeptide Antibiotic by Nonomuraea coxensis DSM 45129.

Glycopeptide antibiotics (GPAs) are last defense line drugs against multidrug-resistant Gram-positive pathogens. Natural GPAs teicoplanin and vancomycin, as well as semisynthetic oritavancin, telavancin, and dalbavancin, are currently approved for clinical use. Although these antibiotics remain efficient, emergence of novel GPA-resistant pathogens is a question of time. Therefore, it is important to investigate the natural variety of GPAs coming from so-called "rare" actinobacteria. Herein we describe a novel GPA producer-Nonomuraea coxensis DSM 45129. Its de novo sequenced and completely assembled genome harbors a biosynthetic gene cluster (BGC) similar to the dbv BGC of A40926, the natural precursor to dalbavancin. The strain produces a novel GPA, which we propose is an A40926 analogue lacking the carboxyl group on the N-acylglucosamine moiety. This structural difference correlates with the absence of dbv29-coding for an enzyme responsible for the oxidation of the N-acylglucosamine moiety. Introduction of dbv29 into N. coxensis led to A40926 production in this strain. Finally, we successfully applied dbv3 and dbv4 heterologous transcriptional regulators to trigger and improve A50926 production in N. coxensis, making them prospective tools for screening other Nonomuraea spp. for GPA production. Our work highlights genus Nonomuraea as a still untapped source of novel GPAs.

Determination of teicoplanin in human plasma by reverse micelle mediated dispersive liquid-liquid microextraction with high performance liquid chromatography.

A reverse micelle mediated dispersive liquid-liquid microextraction (RM-DLLME) combined with high performance liquid chromatography-ultraviolet detector (HPLC-UV) was developed for extraction and determination of 5 A2 components of teicoplanin (TA2-1, TA2-2, TA2-3, TA2-4, TA2-5) in human plasma, and the mechanism of RM-DLLME was analysed and explored. In this method, 80 µL of the reverse micelle solution of cetylpyridinium chloride/n-hexanol (15 mmol/L) was used as the extraction solvent for the separation, extraction and enrichment of the teicoplanin in plasma sample. All factors affecting the extraction efficiencies of the target analytes, such as the amounts of acetonitrile and chloroform, the type and volume of reverse micelle solution, pH and volume of sample phase, dispersant, salt addition, extraction mode and time, centrifugation rate and time, were investigated and optimized. Under the optimum conditions, the 5 A2 components of teicoplanin achieved effective enrichment with the enrichment factors of 228-347 and obtained good linearity in the range of 0.8375-100.5 µg/mL with correlation coefficients higher than 0.9960. The limits of detection were ranged between 0.5025-3.015 µg/mL. Relative standard deviation values of the method precisions were lower than 10.6% and the average recoveries were in the range of 82.7-111.3%. The determination results of the method were demonstrated with favorable characteristics, such as high enrichment, good selectivity and sensitivity, satisfactory precision and accuracy, and this method could be employed to analysis of the teicoplanin in human plasma samples.

Understanding the early stages of peptide formation during the biosynthesis of teicoplanin and related glycopeptide antibiotics.

The biosynthesis of the glycopeptide antibiotics (GPAs) demonstrates the exceptional ability of nonribosomal peptide (NRP) synthesis to generate diverse and complex structures from an expanded array of amino acid precursors. Whilst the heptapeptide cores of GPAs share a conserved C terminus, including the aromatic residues involved cross-linking and that are essential for the antibiotic activity of GPAs, most structural diversity is found within the N terminus of the peptide. Furthermore, the origin of the (D)-stereochemistry of residue 1 of all GPAs is currently unclear, despite its importance for antibiotic activity. Given these important features, we have now reconstituted modules (M) 1-4 of the NRP synthetase (NRPS) assembly lines that synthesise the clinically relevant type IV GPA teicoplanin and the related compound A40926. Our results show that important roles in amino acid modification during the NRPS-mediated biosynthesis of GPAs can be ascribed to the actions of condensation domains present within these modules, including the incorporation of (D)-amino acids at position 1 of the peptide. Our results also indicate that hybrid NRPS assembly lines can be generated in a facile manner by mixing NRPS proteins from different systems and that uncoupling of peptide formation due to different rates of activity seen for NRPS modules can be controlled by varying the ratio of NRPS modules. Taken together, this indicates that NRPS assembly lines function as dynamic peptide assembly lines and not static megaenzyme complexes, which has significant implications for biosynthetic redesign of these important biosynthetic systems.

Structural Basis for the Binding Mechanism of Human Serum Albumin Complexed with Cyclic Peptide Dalbavancin.

Cyclic peptides, with unique structural features, have emerged as new candidates for drug discovery; their association with human serum albumin (HSA; long blood half-life) is crucial to improve drug delivery and avoid renal clearance. Here, we present the crystal structure of HSA complexed with dalbavancin, a clinically used cyclic peptide. Small-angle X-ray scattering and isothermal titration calorimetry experiments showed that the HSA-dalbavancin complex exists in a monomeric state; dalbavancin is only bound to the subdomain IA of HSA in solution. Structural analysis and MD simulation revealed that the swing of Phe70 and movement of the helix near dalbavancin were necessary for binding. The flip of Leu251 promoted the formation of the binding pocket with an induced-fit mechanism; moreover, the movement of the loop region including Glu60 increased the number of noncovalent interactions with HSA. These findings may support the development of new cyclic peptides for clinical use, particularly the elucidation of their binding mechanism to HSA.

Simultaneous Quantification and Pharmacokinetic Study of Five Homologs of Dalbavancin in Rat Plasma Using UHPLC-MS/MS.

Dalbavancin is a novel semisynthetic glycopeptide antibiotic that comprises multiple homologs and isomers of similar polarities. However, pharmacokinetic studies have only analyzed the primary components of dalbavancin, namely B0 and B1. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to simultaneously determinate and investigate the five homologous components of dalbavancin, namely, A0, A1, B0, B1, and B2, in rat plasma. In this method, methanol was used to precipitate plasma, and a triple-bonded alkyl chromatographic column was used for molecule separation, using 0.1% formic acid-acetonitrile as the mobile phase for gradient elution. Targeted homologs were analyzed by a triple quadrupole mass spectrometer using positive electrospray ionization in multiple reaction monitoring mode. The linearity range was 50-2500 ng/mL with a high correlation coefficient (r2 > 0.998). This method was successfully applied in the pharmacokinetic analysis of dalbavancin hydrochloride to investigate dalbavancin components in rats.

Screening and evaluation of approved drugs as inhibitors of main protease of SARS-CoV-2.

The COVID-19 pandemic caused by SARS-CoV-2 has emerged as a global catastrophe. The virus requires main protease for processing the viral polyproteins PP1A and PP1AB translated from the viral RNA. In search of a quick, safe and successful therapeutic agent; we screened various clinically approved drugs for the in-vitro inhibitory effect on 3CLPro which may be able to halt virus replication. The methods used includes protease activity assay, fluorescence quenching, surface plasmon resonance (SPR), Thermofluor® Assay, Size exclusion chromatography and in-silico docking studies. We found that Teicoplanin as most effective drug with IC50 ~ 1.5 µM. Additionally, through fluorescence quenching Stern-Volmer quenching constant (KSV) for Teicoplanin was estimated as 2.5 × 105 L·mol-1, which suggests a relatively high affinity between Teicoplanin and 3CLPro protease. The SPR shows good interaction between Teicoplanin and 3CLPro with KD ~ 1.6 µM. Our results provide critical insights into the mechanism of action of Teicoplanin as a potential therapeutic against COVID-19. We found that Teicoplanin is about 10-20 fold more potent in inhibiting protease activity than other drugs in use, such as lopinavir, hydroxychloroquine, chloroquine, azithromycin, atazanavir etc. Therefore, Teicoplanin emerged as the best inhibitor among all drug molecules we screened against 3CLPro of SARS-CoV-2.