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.

Fast, sensitive LC-MS resolution of α -hydroxy acid biomarkers via SPP-teicoplanin and an alternative UV detection approach.

Enantioseparation of α -hydroxy acids is essential since specific enantiomers of these compounds can be used as disease biomarkers for diagnosis and prognosis of cancer, brain diseases, kidney diseases, diabetes, etc., as well as in the food industry to ensure quality. HPLC methods were developed for the enantioselective separation of 11 α -hydroxy acids using a superficially porous particle-based teicoplanin (TeicoShell) chiral stationary phase. The retention behaviors observed for the hydroxy acids were HILIC, reversed phase, and ion-exclusion. While both mass spectrometry and UV spectroscopy detection methods could be used, specific mobile phases containing ammonium formate and potassium dihydrogen phosphate, respectively, were necessary with each approach. The LC-MS mode was approximately two orders of magnitude more sensitive than UV detection. Mobile phase acidity and ionic strength significantly affected enantioresolution and enantioselectivity. Interestingly, higher ionic strength resulted in increased retention and enantioresolution. It was noticed that for formate-containing mobile phases, using acetonitrile as the organic modifier usually resulted in greater enantioresolution compared to methanol. However, sometimes using acetonitrile with high ammonium formate concentrations led to lengthy retention times which could be avoided by using methanol as the organic modifier. Additionally, the enantiomeric purities of single enantiomer standards were determined and it was shown that almost all standards contained some levels of enantiomeric impurities.

Metal-Assisted Carbohydrate Assembly.

The sequence-controlled assembly of nucleic acids and amino acids into well-defined superstructures constitutes one of the most revolutionary technologies in modern science. The elaboration of such superstructures from carbohydrates, however, remains elusive and largely unexplored on account of their intrinsic constitutional and configurational complexity, not to mention their inherent conformational flexibility. Here, we report the bottom-up assembly of two classes of hierarchical superstructures that are formed from a highly flexible cyclo-oligosaccharide─namely, cyclofructan-6 (CF-6). The formation of coordinative bonds between the oxygen atoms of CF-6 and alkali metal cations (i) locks a myriad of flexible conformations of CF-6 into a few rigid conformations, (ii) bridges adjacent CF-6 ligands, and (iii) gives rise to the multiple-level assembly of three extended frameworks. The hierarchical superstructures present in these frameworks have been shown to modulate their nanomechanical properties. This research highlights the unique opportunities of constructing convoluted superstructures from carbohydrates and should encourage future endeavors in this underinvestigated field of science.

Occurrence of D-amino acids in natural products.

Since the identified standard genetic code contains 61 triplet codons of three bases for the 20 L-proteinogenic amino acids (AAs), no D-AA should be found in natural products. This is not what is observed in the living world. D-AAs are found in numerous natural compounds produced by bacteria, algae, fungi, or marine animals, and even vertebrates. A review of the literature indicated the existence of at least 132 peptide natural compounds in which D-AAs are an essential part of their structure. All compounds are listed, numbered and described herein. The two biosynthetic routes leading to the presence of D-AA in natural products are: non-ribosomal peptide synthesis (NRPS), and ribosomally synthesized and post-translationally modified peptide (RiPP) synthesis which are described. The methods used to identify the AA chirality within naturally occurring peptides are briefly discussed. The biological activity of an all-L synthetic peptide is most often completely different from that of the D-containing natural compounds. Analyzing the selected natural compounds showed that D-Ala, D-Val, D-Leu and D-Ser are the most commonly encountered D-AAs closely followed by the non-proteinogenic D-allo-Thr. D-Lys and D-Met were the least prevalent D-AAs in naturally occurring compounds.

Variable fragmentation and ionization of amyloid-beta epimers and isomers.

While the existence of D-amino acids in peptides and proteins has recently been accepted in higher forms of life, their roles and importance are yet to be understood. The lack of analytical methods present for such epimeric and/or isomeric analyses often limits developments in the field. Studies have shown the elevated presence of epimeric and isomeric modifications to amyloid-beta (Aß) peptides extracted from Alzheimer's disease patients. These modifications most frequently occur through aspartic acid and serine residues. Because such peptides are indistinguishable by mass alone, selective liquid chromatography tandem mass spectrometry analysis is required to differentiate such peptides. Herein, we examine MS/MS of tryptic fragments of Aß peptides containing D-Asp, L-iso-Asp, D-iso-Asp, and/or D-Ser modifications. Peptide ionizability and fragmentation are explored through selected reaction monitoring, selected ion monitoring, and product ion scan. The results show the variability of ionization and fragmentation for many "identical mass peptides" and how these differences can affect the analysis of isomeric and epimeric peptides.

Antibody binding of amyloid beta peptide epimers/isomers and ramifications for immunotherapies and drug development.

Extracellular deposition of amyloid beta (Aß) peptide is a contributing factor of Alzheimer's disease (AD). Considerable effort has been expended to create effective antibodies, or immunotherapies, targeting Aß peptides. A few immunotherapies are thought to provide some benefit. It is possible that a contributing factor to the responses of such therapies may be the presence of modified, or aberrant, Aß peptides found in AD patients. These aberrations include the isomerization and epimerization of L-Asp and L-Ser residues to form D-Asp, L/D-isoAsp, and D-Ser residues, respectively. An effective methodology is essential to isolate all Aß peptides and then to quantify and locate the aberrant amino acids. Modifications to Aß peptides may elevate the deposition of Aß plaques and/or contribute to the neurodegeneration in AD patients, and may alter the binding affinity to antibodies. Herein, we used immunoprecipitation to examine the binding affinity of four antibodies against 18 epimeric and/or isomeric Aß peptides compared to wild type (all L) Aß peptide. Tandem mass spectrometry was used as a detection method, which also was found to produce highly variable results for epimeric and/or isomeric Aß.

Effect of position of deuterium atoms on gas chromatographic isotope effects.

Deuterium substitution provides various benefits in drug molecules, including improvement in pharmacokinetic properties, reduction of toxicity, reduction of epimerization, etc. Also, it has been shown that the position of deuterium substitution affects the properties of drug molecules. Therefore, it is important to study low molecular weight deuterated isotopologues which constitute the deuterated pool and are building blocks of larger deuterated molecules. The effect of the position and number of deuterium atoms on the retention of 23 deuterated isotopologues on two gas chromatography stationary phases of different polarities was evaluated. It was observed that the ratio of calculated chromatographic isotope effects resulting from a deuterium atom connected to an sp2 vs. an sp3 hybridized carbon was more on the polar IL-111i stationary phase compared to the nonpolar PDMS-5, for each group of isotopologues. Also, a compound with a deuterium atom connected to an sp2 hybridized carbon always had greater retention than the analogous compound where deuterium was connected to an sp3 hybridized carbon. The van't Hoff plots for all analytes showed that the effect of entropy was almost negligible in the separation of deuterated vs. protiated isotopologues, thus these separations were mainly enthalpy driven.

Automated Regularized Deconvolution for Eliminating Extra-Column Effects in Fast High-Efficiency Separations.

With the introduction of ultrahigh efficiency columns and fast separations, the need to eliminate peak deformation contributed by the instrument must be effectively solved. Herein, we develop a robust framework to automate deconvolution and minimize its artifacts, such as negative dips, wild noise oscillations, and ringing, by combining regularized deconvolution and Perona-Malik (PM) anisotropic diffusion methods. A asymmetric generalized normal (AGN) function is proposed to model the instrumental response for the first time. With no-column data at various flow rates, the interior point optimization algorithm extracts the parameters describing instrumental distortion. The column-only chromatogram was reconstructed using the Tikhonov regularization technique with minimal instrumental distortion. For illustration, four different chromatography systems are used in fast chiral and achiral separations with 2.1 and 4.6 mm i.d. columns. Ordinary HPLC data can approach highly optimized UHPLC data. Similarly, in fast HPLC-circular dichroism (CD) detection, 8000 plates were gained for a fast chiral separation. Moment analysis of deconvolved peaks confirms correction of the center of mass, variance, skew, and kurtosis. This approach can be easily integrated and used with virtually any separation and detection system to provide enhanced analytical data.

Employment of chiral columns with superficially porous particles in chiral separations of cobalt bis (dicarbollide) and nido-7,8-C2 B9 H12 (1-) derivatives.

Derivatives of the nido-7,8-C2 B9 H12 (1-) (dicarbollide ion) and [3,3'-Co-(1,2-C2 B9 H11 )2 ](1-) cobalt sandwich (COSAN) ion represent groups of extremely chemically and thermally stable abiotic compounds. They are being investigated in many research areas, that is, medicinal chemistry, material sciences, analytical chemistry, and electrochemistry. The chirality of these compounds remains still grossly overlooked, what is also reflected in limited number of reports on their chiral separations. Continued progress depends on reliable, fast, and cost-effective methods for such separations. Recently, chiral separations of COSAN derivatives were achieved in liquid chromatography and supercritical fluid chromatography. Only five anionic derivatives of nido-7,8-C2 B9 H12 (1-) were successfully enantioseparated in liquid chromatography. Efforts to separate anionic nido-7,8-C2 B9 H12 (1-) in supercritical chromatography have failed, and only a few dicarbollide ions were separated using liquid chromatography. Generally, all chiral separations in liquid chromatography took about 30 min. Herein, we identify a versatile column capable of separating both COSAN and nido-7,8-C2 B9 H12 (1-) derivatives and achieve faster analyses times employing commercially available superficially porous chiral stationary phases. The semisynthetic hydroxypropyl ß-cyclodextrin-based column (CDShell-RSP) is identified as the column of choice from the tested columns by separating 19 of 27 compounds from each structural motifs tested mainly in less than 10 min. The dihydroxyalkyl, oxygen-bridged hydroxyalkyl, and bisphenylene-bridged COSAN derivatives were baseline separated in less than 5 min exceeding the results of supercritical fluid chromatography. Methods developed herein will aid synthetic chemists without the possession of a supercritical fluid chromatograph to achieve fast chiral separations of COSAN and derivatives of nido-7,8-C2 B9 H12 (1-) on a common liquid chromatograph without the need of dedicated instrumentation.

d-Amino acids in biological systems.

Investigations on the occurrence and biochemical roles of free D-amino acids and D-amino acid-containing peptides and proteins in living systems have increased in frequency and significance. Their occurrence and roles may vary substantially with progression from microbiotic to evermore advanced macrobiotic systems. We now understand many of the biosynthetic and regulatory pathways, which are outlined herein. Important uses for D-amino acids in plants, invertebrates, and vertebrates are reviewed. Given its importance, a separate section on the occurrence and role of D-amino acids in human disease is presented.

Detection and analysis of chiral molecules as disease biomarkers.

The chirality of small metabolic molecules is important in controlling physiological processes and indicating the health status of humans. Abnormal enantiomeric ratios of chiral molecules in biofluids and tissues occur in many diseases, including cancers and kidney and brain diseases. Thus, chiral small molecules are promising biomarkers for disease diagnosis, prognosis, adverse drug-effect monitoring, pharmacodynamic studies and personalized medicine. However, it remains difficult to achieve cost-effective and reliable analysis of small chiral molecules in clinical procedures, in part owing to their large variety and low concentration. In this Review, we describe current and emerging techniques that detect and quantify small-molecule enantiomers and their biological importance.

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.

Unusual enantiomeric D,L-N-acyl homoserine lactones in Pectobacterium atrosepticum and Pseudomonas aeruginosa.

Quorum Sensing allows bacteria to sense their population density via diffusible N-acyl homoserine lactone (N-HL) signaling molecules. Upon reaching a high enough cell density, bacteria will collectively exhibit a phenotype. Until recently, methods used for detection of N-HLs have not considered the chirality of these molecules and it was assumed that only the L-enantiomer was produced by bacteria. The production and effects of D-N-HLs have rarely been studied. In this work, the temporal production of D-N-HLs by the plant pathogen Pectobacterium atrosepticum and the human pathogen Pseudomonas aeruginosa are reported. Both bacteria produced D-N-HLs in significant amounts and in some cases their concentrations were higher than other low abundance L-N-HLs. Previously unreported D-enantiomers of N-3-oxoacyl and N-3-hydroxyacyl homoserine lactones were detected in P. atrosepticum. Interestingly, L-N-HLs produced in the lowest concentrations had relatively higher amounts of their corresponding D-enantiomers. Potential sources of D-N-HLs and their significance are considered.

Teicoplanin aglycone media and carboxypeptidase Y: Tools for finding low-abundance D-amino acids and epimeric peptides.

D-amino acids and epimeric peptides/proteins can play crucial biological roles and adversely affect protein folding and oligopeptide aggregation in age-related pathologies in humans. This has ignited interest in free D-amino acids as well as those incorporated in peptides/proteins and their effects in humans. However, such stereoisomeric analytes are often elusive and in low abundance with few existing methodologies capable of scouting for and identifying them. In this work, we examine the feasibility of using teicoplanin aglycone, a macrocyclic antibiotic, which has been reported to strongly retain D-amino acids and peptides with a D-amino acid on the C-terminus, for use as a solid phase extraction (SPE) medium. The HPLC retention factors of L-/D-amino acids and C-terminus modified D-amino acid-containing peptides and their L-amino acid exclusive counterparts on teicoplanin aglycone are presented. Retention curve differences between amino acids and peptides highlight regions of solvent composition that can be utilized for their separation. This approach is particularly useful when coupled with enzymatic hydrolysis via carboxypeptidase Y to eliminate all L-amino acid exclusive peptides. The remaining peptides with carboxy-terminal D-amino acids are then more easily concentrated and identified.

Investigating chirality in quorum sensing by analysis of Burkholderia cepacia and Vibrio fischeri with comprehensive chiral LC-MS/MS and GC-MS/MS methods.

N-acyl homoserine lactones (N-HLs) are signaling molecules used by Gram-negative bacteria in a phenomenon called quorum sensing. Bacteria will detect N-HLs as a way of monitoring their population which, upon reaching a critical level, will express a specific phenotype. An example is the expression of bioluminescence by Vibrio fischeri. Most studies have not considered the chirality of these molecules nor have they used highly sensitive detection methods. Here, the production of d,l-N-HLs are monitored for V. fischeri, Burkholderia cepacia, Pseudomonas fluorescens, and P. putida, using highly sensitive tandem mass spectrometry analysis. Novel N-HLs are reported for both V. fischeri and B. cepacia, including a plethora of previously unknown d-N-HLs, including the first d-N-HLs containing oxo and hydroxy functionalities. Anomalously, N-HLs were not detected in any cultures of P. fluorescens and P. putida, which are species that previously were reported to produce N-HLs. However, it is apparent that differences in the reported occurrence and levels of N-HLs can result from (a) different strains of bacteria, (b) different growth media and environmental conditions, and (c) sometimes false-positive results from detection methodologies. Time studies of V. fischeri suggest the possibility that separate synthetic and elimination pathways exist between d- and l-N-HLs. Possible biological processes that could be the source of d-N-HL production are considered.

Comparison of core-shell versus fully porous particle packings for chiral liquid chromatography productivity.

A loading and productivity study was done using three racemates on vancomycin and teicoplanin-bonded chiral stationary phases of different particle formats. Two columns were packed with 2.7 µm superficially porous particles and two columns were packed with identically bonded 5 µm fully porous particles. The last two columns were packed with specially synthesized 4.5 µm vancomycin and teicoplanin superficially porous particles. The loading of different chiral compounds showed that the columns filled with 2.7-µm chiral stationary phases were inappropriate for preparative separations due to their very low permeability which precluded high flow rates. However, columns containing 4.5 µm superficially porous (core-shell) particles were as effective for small-scale preparative chiral separations as columns filled with classical 5 µm fully porous particles. Comparing the 4.5 µm superficially porous particles and 5 µm fully porous particles teicoplanin columns, the observed respective productivities of 270 and 265 mg/g chiral phase/h for 5-methyl-5-phenyl hydantoin enantiomers were obtained. Particular attention was given to the peculiar case of the mianserin enantiomeric separation on vancomycin columns that gave observed productivities of 200 and 205 mg/g chiral phase/h on the 4.5 µm superficially porous particles and 5 µm fully porous particles, respectively.

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.

Total synthesis of haploscleridamine, villagorgin A and an approach towards lissoclin C.

An investigation of asymmetric total syntheses of three indole-imidazole alkaloids from histidine are described. A common advanced piperidinone was contructed via a ring-closing metathesis which was then subjected to a modified Fischer indole synthesis. Deprotection of an N-tosyl group via a dissolving metal reduction affords haploscleridamine which upon reaction with aqueous formaldehyde in trifluoroethanol provided villagorgin A. On closer examination, it was found that villagorgin A was produced as a byproduct during the reductive detosylation in the presence of magnesium and methanol. Attempts to obtain the brominated haploscleridamine congener, lissoclin C through use of bromophenyl hydrazone were thwarted by reductive debromination during deprotection efforts. Investigation of the enantiopurity of the synthetic natural products revealed production of almost racemic materials in some batches as the result of partial racemization of an early stage intermediate. A revised approach routinely provided scalemic haploscleridamine and villagorgin in 30% ee. Analysis of the enantiomer composition of all intermediates by HPLC using columns with chiral stationary phases; this analysis revealed several steps where erosion of enantiomer composition occurred.

Comprehensive chiral GC-MS/MS and LC-MS/MS methods for identification and determination of N-acyl homoserine lactones.

N-acyl homoserine lactones (N-HLs) are signaling molecules synthesized by gram-negative bacteria to communicate in a process called quorum sensing. Most reported methods for the analysis of N-HLs, which are chiral molecules, do not distinguish between enantiomers. Typical examples include biosensors, liquid chromatography with UV detection, gas chromatography coupled with a mass spectrometer (GC-MS) and liquid chromatography coupled with mass spectrometer (LC-MS). Recently, the production of both D,L-N-HLs have been reported in Vibrio fischeri and Burkholderia cepacia. Concentrations of the D-N-HLs were found at the limit of quantification for the employed method. Therefore, for further studies of the role of the D-N-HLs in bacterial physiology, more sensitive, reliable, and selective analytical methods are necessary. In this work, such comprehensive chiral analytical methods for the identification and determination of 18 N-HLs using solid phase extraction followed by GC-MS/MS and LC-MS/MS analyses were developed. Extraction recoveries for the more hydrophilic C4 N-HLs were <10% of all other N-HLs, thus offering a possible explanation as to their lack of detection in previous studies. The chiral separations of all 18 N-HLs derivatives were accomplished by the complementary GC-MS/MS and LC-MS/MS methods. The limit of detection for LC-MS/MS method was as low as 1 ppb. The limit of detection for the GC-MS/MS method was found to be one to three orders of magnitude higher than the LC-MS/MS method. Due to the high extraction recovery and a preconcentration factor of 100, concentrations as low as 10 ppt can be detected by LC-MS/MS in biological samples. The LC-MS/MS approach provided greater enantioselectivity for the larger, more hydrophobic N-HLs while GC-MS/MS provided better enantioselectivity for the smaller N-HLs.