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.

Influence of theoretical and semi-empirical peak models on the efficiency calculation in chiral chromatography.

Height equivalent to theoretical plate (H) equations, such as the van Deemter or Knox-Saleem equations, and other efficiency vs. linear velocity equations (u), provide kinetic insights into chromatographic separations phenomena and column performance. In enantioselective separations, the peak shape of the two enantiomers can differ significantly and are often asymmetric. The peak efficiency calculations heavily impact these efficiency-flow profiles, leading to erroneous estimations of eddy diffusion, longitudinal diffusion, and mass transfer terms. In this work, new asymmetric peak functions are employed for modeling enantiomer peaks based on the Haarhoff-Van der Linde function, its generalized variant (GHVL), once Generalized Asymmetric Gaussian (AGN), and Twice Generalized Gaussian (TGN). The new models (AGN, TGN, and GHVL) incorporate higher statistical moments besides the zeroth, first, and second moments to account for two-sided asymmetry (fronting or tailing). The fit results are compared with the traditional efficiency calculation methods endorsed by official pharmacopeia and numerical estimation of moments from the raw data. Enantiomeric separations of ibuprofen and dl-homophenylalanine were chosen as probe molecules. The results demonstrate that non-linear least squares fitted functions provide better estimations of peak efficiency data even in the presence of high noise. In particular, the generalized models consistently offered the best quality fits for various peak shapes in chiral separations. Conversely, the half-height Gaussian method greatly overpredicted skewed peak efficiencies. This investigation reveals that the commonly held assumptions of peak shape and numerical integration of raw data are highly insufficient for chiral chromatography. The impact of asymmetry on plate height should not be overlooked when accurate data from efficiency-flow rate curves is derived. We advocate for the broader adoption of these new generalized peak (AGN, TGN, GHVL) models because they provide robustness at various SNRs that account for right or left asymmetry while accurately representing peak geometry.

Discovery of 9H-pyrimido[4,5-b]indole derivatives as dual RET/TRKA inhibitors.

Aberrant RET kinase signaling is activated in numerous cancers including lung, thyroid, breast, pancreatic, and prostate. Recent approvals of selective RET inhibitors, pralsetinib and selpercatinib, has shifted the focus of RET kinase drug discovery programs towards the development of selective inhibitors. However, selective inhibitors invariably lose efficacy as the selective nature of the inhibitor places Darwinian-like pressure on the tumor to bypass treatment through the selection of novel oncogenic drivers. Further, selective inhibitors are restricted for use in tumors with specific genetic backgrounds that do not encompass diverse patient classes. Here we report the identification of a pyrimido indole RET inhibitor found to also have activity against TRK. This selective dual RET/TRK inhibitor can be utilized in tumors with both RET and TRK genetic backgrounds and can also provide blockade of NTRK-fusions that are selected for from RET inhibitor treatments. Efforts towards developing dual RET/TRK inhibitors can be beneficial in terms of encompassing more diverse patient classes while also achieving blockade against emerging resistance mechanisms.

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.

Determining whether biomechanical variables that describe common 'safe lifting' cues are associated with low back loads.

Lift technique training programs have been implemented to help reduce injury risk, but the underlying content validity of cues used within these programs is not clear. The objective of this study was to determine whether biomechanical variables, that commonly used lifting cues aim to elicit, are associated with resultant low back extensor moment exposures. A sample of 72 participants were recruited to perform 10 repetitions of a floor-to-waist height barbell lift while whole-body kinematics and ground reaction forces were collected. Kinematic, kinetic, and energetic variables representative of characteristics commonly targeted by lifting cues were calculated as predictor variables, while peak and cumulative low back moments were calculated as dependent measures. Multiple regression revealed that 56.6-59.2% of variance in low back moments was explained by predictor variables. From these regression models, generating motion with the legs (both greater hip and knee work), minimizing the horizontal distance of the body to the load, maintaining a stable body position, and minimizing lift time were associated with lower magnitudes of low back moments. These data support that using cues targeting these identified variables may be more effective at reducing peak low back moment exposures via lift training.

Kinase inhibitor macrocycles: a perspective on limiting conformational flexibility when targeting the kinome with small molecules.

Methods utilized for drug discovery and development within the kinome have rapidly evolved since the approval of imatinib, the first small molecule kinase inhibitor. Macrocycles have received increasing interest as a technique to improve kinase inhibitor drug properties evident by the FDA approvals of lorlatinib, pacritinib, and repotrectinib. Compared to their acyclic counterparts, macrocycles can possess improved pharmacodynamic and pharmacokinetic properties. This review highlights clinical success stories when implementing macrocycles in kinase-based drug discovery and showcases that macrocyclization is a clinically validated drug discovery strategy when targeting the kinome.

The Influence of Contextual and Theoretical Expertise on Generic and Occupation-Specific Lifting Strategy.

OBJECTIVE: To determine whether (i) low back loads and/or (ii) kinematic coordination patterns differed across theoretical expert, contextual expert and novice groups when completing both generic and occupation-specific lifts. BACKGROUND: Experience has been proposed as a factor that could reduce biomechanical exposures in lifting, but the literature reports mixed effects. The inconsistent relationship between experience and exposures may be partially attributable to the broad classification of experience and experimental lifting protocols not replicating the environment where experience was gained. METHODS: Purposive sampling was used to recruit 72 participants including theoretical experts (formal training on lifting mechanics), contextual experts (paramedics), and novices. Participants performed 10 barbell and crate (generic) lifts, as well as backboard and stretcher (occupation-specific) lifts while whole-body kinematics and ground reaction forces were collected. Peak low back compression and anteroposterior shear loads normalized to body mass, as well as kinematic coordination patterns, were calculated as dependent variables. RESULTS: No significant differences in low back loads were observed across expertise groups. However, significant differences were seen in kinematic coordination patterns across expertise groups in occupation-specific lifts, but not in generic lifts. CONCLUSION: Increasing expertise is unlikely to minimize low back loads in lifting. However, contextual expertise did influence lifting kinematics, but only when performing occupationally specific lifts. APPLICATION: Contextual expertise may help lifters adopt lifting kinematics that enhance the tolerance of their musculoskeletal system to withstand applied loads, but does not seem to reduce the applied low back loads relative to noncontextual expert groups.

Factors influencing the likelihood of females passing the Ottawa paramedic physical ability test.

The Ottawa Paramedic Physical Ability Test (OPPAT) was launched in Ontario as a physical employment standard for front-line paramedics. When considering pass rates based on sex, males had a higher likelihood of passing than females. To help understand how to improve pass rates among females we aimed to understand if factors such as participant demographics, college type, employment status and/or peak heart rate (a surrogate of fitness level) were related to OPPAT pass rates. Females who were employed, who were educated in a public paramedic college, and who had higher body mass were more likely to successfully complete the OPPAT. Those educated in a public paramedic college were more than twice as likely to pass relative to those educated in a private college program. This may underscore a need to further explore different modes of paramedic education to understand why public college program trained students are more likely to pass.

Predictors of milk cortisol in North American women.

OBJECTIVES: Human milk content varies across mother-child dyads, environments, and populations. Among the hormones in milk is cortisol, a glucocorticoid; its impact on the breastfeeding child is unknown. Milk cortisol may constitute a signal to the child's developing physiology which can shape characteristics (e.g., growth, temperament) to prevailing environmental conditions. This exploratory study evaluated the maternal, breastfeeding, and infant characteristics associated with milk cortisol. METHODS: We evaluated archived milk specimens for cortisol using enzyme immunoassay and employed an information-theoretic approach to assess associations between milk cortisol and participant characteristics with linear regression modeling. Because we employed secondary data, information for some variables likely to impact milk cortisol variation (e.g., time of day, socioeconomic status, maternal or infant body mass index, milk energy density) was unavailable. RESULTS: Participants were 48 lactating mothers from upstate New York, aged 21-40 years. Milk cortisol ranged from 0.098 to 1.007 µg/dL. Child age ranged from 1 to 26 months. In linear regression employing best fit modeling criteria, milk cortisol increased with child age (B: 0.069; p: .000; a 7.1% increase in milk cortisol for each month of child age), while child symptoms of illness (B: -0.398; p: .057; a 33% decrease) and consumption of complementary foods (B: -.525; p: .020; a 41% decrease) were associated with lower milk cortisol. CONCLUSIONS: We speculate that increasing milk cortisol with child age plays a role in signaling development (e.g., as increasing independence increases risk for injury and other negative health outcomes), independent of the maternal stressors we could capture.

Increased Ability to Perceive Relevant Sensory Information Minimizes Low Back Exposures in Lifting.

We have previously shown evidence that some individuals seem to consistently minimize low back loads when lifting, while others do not. However, it is unknown why. Individual differences in ability to perceive relevant sensory information may explain differences in minimization of low back loads during lifting, consistent with considering load reduction in one's movement objective in an optimal feedback control theory framework. The purpose of this study was to investigate whether individuals' ability to perceive proprioceptive information (both force- and posture-senses) at the low back was associated with peak low back loads when performing generic or occupation-specific lifts. Seventy-two participants were recruited to perform 10 barbell (generic) and backboard (occupation-specific) lifts, while whole-body kinematics and ground reaction forces were collected. Peak low back compression and anteroposterior shear forces normalized to body mass were calculated as dependent variables. Both posture matching ability and force matching ability at the heavier force targets were associated with lower means and variability of peak low-back loads in both lift types, albeit with small effect sizes (R2 ≤ .17). These findings support the utility of an optimal feedback control theory framework to explore factors explaining interindividual differences in low back loads during lifting. Further, this evidence suggests improving proprioceptive ability may be a useful strategy in lift training programs designed for workplace injury prevention.

Differential effects of sex on upper body kinematics and kinetics during fatiguing, Asymmetric lifting.

This study quantified sex-specific biomechanical adaptations to fatigue in asymmetric lifting. Twenty-one females and fifteen males performed a prolonged asymmetric lifting protocol while upper body, trunk and pelvis kinematics were collected. Features of movement identified with principal component analysis, and peak joint angular velocities and moments were calculated. Sex-specific kinematic adaptations to fatigue included females adopting a 'stoop-like' lifting strategy to a greater extent than males. Additionally, females exhibited higher vertical elbow positions during load rotation, moved their body toward the destination for load deposit, and did not reduce peak right shoulder flexion velocities, in contrast to male participants. Females also had greater low back and shoulder peak normalized joint moments. When fatigued, females adopted an asymmetric lifting strategy that minimized metabolic demand as supported by smaller decreases in maximum voluntary contractions. However, females' fatigue-related adaptations increased biomechanical exposures associated with injury risk.

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.

Quantifying how functional and structural personal factors influence biomechanical exposures in paramedic lifting tasks.

It is unknown how structural (sex, stature, body mass) and functional (strength, flexibility) personal factors influence lifting strategy in paramedic work. We explored whether variance in peak low back forces and kinematic coordination patterns could be explained by structural and functional personal factors in paramedic lifting tasks. Seventy-two participants performed backboard and stretcher lifts. Peak low back forces normalised to body mass, as well as kinematic coordination patterns, were calculated as dependent variables. Being female, stronger, shorter, having higher body mass, and/or having greater lower body range of motion (ROM) were all independently associated with lower normalised low back forces across backboard and stretcher lifting. Females and stronger individuals seemed to define a movement objective to consistently minimise compressive forces, while individuals with greater hip ROM consistently minimised anteroposterior shear forces. The efficacy of improving strength and hip ROM to reduce low back forces in paramedic lifting should be investigated.Practitioner summary: Females, stronger individuals, and individuals with greater hip range of motion consistently exhibited lower normalised low back forces in paramedic lifting. Improving strength and hip range of motion via training is a potential proactive ergonomics approach to reduce peak low back forces in paramedic lifting tasks.

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.

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.

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.

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.