Enhanced Structure-Based Prediction of Chiral Stationary Phases for Chromatographic Enantioseparation from 3D Molecular Conformations.

The accurate prediction of suitable chiral stationary phases (CSPs) for resolving the enantiomers of a given compound poses a significant challenge in chiral chromatography. Previous attempts at developing machine learning models for structure-based CSP prediction have primarily relied on 1D SMILES strings [the simplified molecular-input line-entry system (SMILES) is a specification in the form of a line notation for describing the structure of chemical species using short ASCII strings] or 2D graphical representations of molecular structures and have met with only limited success. In this study, we apply the recently developed 3D molecular conformation representation learning algorithm, which uses rapid conformational analysis and point clouds of atom positions in the 3D space, enabling efficient chemical structure-based machine learning. By harnessing the power of the rapid 3D molecular representation learning and a data set comprising over 300,000 chromatographic enantioseparation records sourced from the literature, our models afford notable improvements for the chemical structure-based choice of appropriate CSP for enantioseparation, paving the way for more efficient and informed decision-making in the field of chiral chromatography.

Investigation into the performance and stability of immobilized and coated polysaccharide columns in supercritical fluid chromatography.

In this study, the performance of the widely used "golden four" coated chiral stationary phases (Chiralpak AD-3, Chiralcel OD-3, Chiralpak AS-3, and Chiralcel OJ-3) was compared with their corresponding immobilized versions (Chiralpak IA-3, Chiralpak IB-3, Chiralpak IB N-3, Chiralpak IH-3, and Chiralpak IJ-3) under supercritical fluid chromatography (SFC) conditions with a set of 30 racemic compounds. Using the traditional modifiers, methanol and isopropanol, the immobilized columns (Chiralpak IB N-3 and Chiralpak IH-3) showed an improved general ability to successfully resolve the enantiomers of the target analytes relative to their coated versions (Chiralcel OD-3 and Chiralpak AS-3), while the coated columns (Chiralpak AD-3, Chiralcel OD-3, and Chiralcel OJ-3) performed better than their immobilized versions (Chiralpak IA-3, Chiralpak IB-3, and Chiralpak IJ-3). An investigation of the non-traditional modifiers, dichloromethane, ethyl acetate, and tetrahydrofuran with immobilized columns, revealed a generally decreased ability to successfully resolve the enantiomers of the target analytes, relative to the use of the traditional modifiers, methanol and isopropanol. The stability of the coated columns (Chiralpak AD-H and Chiralcel OD-H) was evaluated by injecting "forbidden" solvents, including dichloromethane, dimethyl sulfoxide, and tetrahydrofuran. After 200 injections of these solvents on coated columns, the retention factors and resolutions slightly decreased, and a significant increase in column backpressure was observed, indicating some degree of stationary phase degradation.

Quantitative Determination of Water in Organic Liquids by Ambient Mass Spectrometry.

This study uses a rapid tandem mass-spectrometry method to determine water content in complex organic solutions. Emphasis is placed on trace-water analysis by a fast and accurate alternative to the Karl-Fischer method. In this new method, water is captured by a charge-labeled molecular probe. Water binds strongly with high specificity to the strongly electrophilic aldehyde site in a charge-labelled molecule (N-methylpyridinium); competitive binding by other analytes is effectively discriminated against in the mass-measurement step. Quantitative determinations are made over a wide concentration range, 0.001 % (10 ppm) to 99 %, with better than 10 % relative standard deviation, along with short (1 min) analysis times using small sample volumes (several µL). Applications include water measurement in simple organic solvents, for example, deuterated solvents, as well as in complex mixtures, for example, organic reaction mixtures. Additionally, this method allows for water monitoring in levitated droplets. Mechanistic investigations into the impact of water on important chemical processes in organic synthesis and environmental science are reported.

3DMolMS: prediction of tandem mass spectra from 3D molecular conformations.

MOTIVATION: Tandem mass spectrometry is an essential technology for characterizing chemical compounds at high sensitivity and throughput, and is commonly adopted in many fields. However, computational methods for automated compound identification from their MS/MS spectra are still limited, especially for novel compounds that have not been previously characterized. In recent years, in silico methods were proposed to predict the MS/MS spectra of compounds, which can then be used to expand the reference spectral libraries for compound identification. However, these methods did not consider the compounds' 3D conformations, and thus neglected critical structural information. RESULTS: We present the 3D Molecular Network for Mass Spectra Prediction (3DMolMS), a deep neural network model to predict the MS/MS spectra of compounds from their 3D conformations. We evaluated the model on the experimental spectra collected in several spectral libraries. The results showed that 3DMolMS predicted the spectra with the average cosine similarity of 0.691 and 0.478 with the experimental MS/MS spectra acquired in positive and negative ion modes, respectively. Furthermore, 3DMolMS model can be generalized to the prediction of MS/MS spectra acquired by different labs on different instruments through minor fine-tuning on a small set of spectra. Finally, we demonstrate that the molecular representation learned by 3DMolMS from MS/MS spectra prediction can be adapted to enhance the prediction of chemical properties such as the elution time in the liquid chromatography and the collisional cross section measured by ion mobility spectrometry, both of which are often used to improve compound identification. AVAILABILITY AND IMPLEMENTATION: The codes of 3DMolMS are available at https://github.com/JosieHong/3DMolMS and the web service is at https://spectrumprediction.gnps2.org.

Rapid Quantitation of Various Therapeutic Monoclonal Antibodies Using Membranes with Fc-Specific Ligands.

Therapeutic monoclonal antibodies (mAbs) provide effective treatments for many diseases, including cancer, autoimmune disorders, and, lately, COVID-19. Monitoring the concentrations of mAbs is important during their production and subsequent processing. This work demonstrates a 5 min quantitation of most human immunoglobulin G (IgG) antibodies through capture of mAbs in membranes modified with ligands that bind to the fragment crystallizable (Fc) region. This enables binding and quantitation of most IgG mAbs. Layer-by-layer (LBL) adsorption of carboxylic acid-rich polyelectrolytes in glass-fiber membranes in 96-well plates allows functionalization of the membranes with Protein A or a peptide, oxidized Fc20 (oFc20), with high affinity for the Fc region of human IgG. mAb capture occurs in <1 min during the flow of solutions through modified membranes, and subsequent binding of a fluorophore-labeled secondary antibody enables quantitation of the captured mAbs using fluorescence. The intra- and inter-plate coefficients of variations (CV) are <10 and 15%, respectively, satisfying the acceptance criteria for many assays. The limit of detection (LOD) of 15 ng/mL is on the high end of commercial enzyme-linked immunosorbent assays (ELISAs) but certainly low enough for monitoring of manufacturing solutions. Importantly, the membrane-based method requires <5 minutes, whereas ELISAs typically take at least 90 min. Membranes functionalized with oFc20 show greater mAb binding and lower LODs than membranes with Protein A. Thus, the membrane-based 96-well-plate assay, which is effective in diluted fermentation broths and in mixtures with cell lysates, is suitable for near-real-time monitoring of the general class of human IgG mAbs during their production.

Microporous affinity membranes and their incorporation into microfluidic devices for monitoring of therapeutic antibodies.

Control of monoclonal antibody (mAb) concentrations in serum is important for maintaining the safety and efficacy of these lifesaving therapeutics. Point-of-care (POC) quantification of therapeutic mAbs could ensure that patients have effective mAb levels without compromising safety. This work uses mimotope-functionalized microporous alumina affinity membranes in vertical flow assays for detection and quantitation of therapeutic mAbs. Selective capture of bevacizumab from 1000:1 diluted serum or plasma and binding of a fluorescently labelled anti-human IgG secondary antibody enable fluorescence-based analysis of bevacizumab at its therapeutically relevant concentration range of ∼50-300 µg/mL. The assay results in a linear relationship between the fluorescence intensity of the antibody capture spot and the bevacizumab concentration. A simple prototype microfluidic device containing these membranes allows washing, reagent additions and visualization of signal within 15 min using a total of 5 mL of fluid. The prototype devices can monitor physiologically relevant bevacizumab levels in diluted serum, and future refinements might lead to a POC device for therapeutic drug monitoring.

Quantitation of Trastuzumab and an Antibody to SARS-CoV-2 in Minutes Using Affinity Membranes in 96-Well Plates.

Quantitation of therapeutic monoclonal antibodies (mAbs) in human serum could ensure that patients have adequate levels of mAbs for effective treatment. This research describes the use of affinity, glass-fiber membranes in a 96-well-plate format for rapid (<5 min) quantitation of the therapeutic mAb trastuzumab and a mAb against the SARS-CoV-2 spike protein. Adsorption of a poly(acrylic acid)-containing film in membrane pores and activation of the -COOH groups in the film enable covalent-linking of affinity peptides or proteins to the membrane. Passage of mAb-containing serum through the affinity membrane results in mAb capture within 1 min. Subsequent rinsing, binding of a secondary antibody conjugated to a fluorophore, and a second rinse yield mAb-concentration-dependent fluorescence intensities in the wells. Calibration curves established from analyses on different days have low variability and allow determination of mAb levels in separately prepared samples with an average error <10%, although errors in single-replicate measurements may reach 40%. The assays can occur in diluted serum with physiologically relevant mAb concentrations, as well as in undiluted serum. Thus, the combination of 96-well plates containing affinity membranes, a microplate reader, and a simple vacuum manifold affords convenient mAb quantitation in <5 min.

Fragmentation of Polyfunctional Compounds Recorded Using Automated High-Throughput Desorption Electrospray Ionization.

Using desorption electrospray ionization (DESI) as part of an automated high-throughput system, tandem mass spectra of the compounds in a pharmaceutical library were recorded in the positive mode under standardized conditions. Quality control filtering yielded an MS/MS library of 16 662 spectra. Fragmentation of subsets of the compounds in the library chosen to contain a single instance of a particular functional group (amide, piperazine, sulfonamide) was predicted by experts, and the results were compared with the experimental data. Expert performance was good to excellent for all the cases evaluated. Substituents on the functional groups were found to exert important secondary control over the fragmentation, with the main effect observed being product ion stabilization by aromatic substitution, which was consistent across the different groups evaluated. These substituent effects are generally explicable in terms of standard physical organic chemistry considerations of product ion stability as controlling fragmentation. A somewhat unexpected feature was the incidence of homolytic cleavages, driven by the stability of substituted amine radical cations. The findings of this study are intended to lay the groundwork for machine learning approaches to performing MS/MS spectrum → structure and structure → MS/MS spectrum operations on the same experimental data set. The effort involved and the success achieved in computer-aided interpretation, now underway, will be compared with the expert performance as described here.

High-Throughput Determination of Enantiopurity by Microplate Circular Dichroism.

Methods for the rapid determination of enantiomeric excess (ee) in asymmetric synthetic methodology development are increasingly in demand as high-throughput experimentation protocols in academia and industry are adopted. Optical approaches have been reported, many of which rely on the use of chemical derivatization or molecular assemblies, resulting in UV/vis, fluorescence, or circular dichroism (CD) signals that report the ee values. While UV/vis and fluorescence approaches benefit from readily available 96- and 384-well plate readers, until recently, no CD plate readers existed. Herein, we report the utility of using the EKKO CD plate reader to analyze a chlorocoumarin amine derivatization methodology for the ee determination of a diverse set of chiral amines with an error margin within ±7%. Linear calibration curves of ee versus CD responses for each amine were obtained, the minimum detectable and quantifiable ee values were calculated, the technique was applied to an asymmetric hydrogenation, and various interferents expected to be present in crude samples are explored. The technique described herein is found to be suitable for high-throughput experimentation that requires a parallel and rapid ee determination step.

Next-Generation TLC: A Quantitative Platform for Parallel Spotting and Imaging.

A high-throughput screening approach for simultaneous analysis and quantification of the percent conversion of up to 48 reactions has been developed using a thin-layer chromatography (TLC) imaging method. As a test-bed reaction, we monitored 48 thiol conjugate additions to a Meldrum's acid derivative (1) in parallel using TLC. The TLC elutions were imaged using a cell phone and a LEGO brick-constructed UV/vis light box. Further, a spotting device was constructed from LEGO bricks that allows simple transfer of the samples from a well-plate to the TLC plate. Using software that was developed to detect "blobs" and report their intensity, we were able to quantitatively determine the extent of completion of the 48 reactions with one analysis.

William H. Pirkle: Stereochemistry pioneer.

Professor William H. Pirkle (1934-2018) made a profound impact on modern chemistry by inventing and popularizing widely used techniques for the analysis and purification of enantiomers, contributions that paved the way for the subsequent advances in the discovery, development, and manufacture of enantiopure pharmaceuticals, agrochemicals, and specialty chemicals. Pirkle's pioneering 1966 demonstration of the use of chiral solvating agents for the nuclear magnetic resonance determination of enantiopurity led to a lifelong interest in understanding the supramolecular interactions responsible for enantiodifferentiation. Ongoing research into the chromatographic resolution of stereoisomers throughout the 1970s led in 1981 to the very first commercialization of a chiral stationary phase for the high-performance liquid chromatography (HPLC) separation of enantiomers. The availability of this and subsequent "Pirkle columns" had a deep and lasting impact, becoming widely embraced by the chemical sciences research community worldwide and spearheading the wholesale changeover to HPLC as the preferred technique for measuring enantiopurity. Doc Pirkle was a highly creative, independent, and fun-loving collaborator whose circle of friends extends around the globe. His research group at the University of Illinois, often referred to as The Pirkle Zoo, became a refuge for an interesting assembly of characters who flourished under his mentorship and guidance.

Mass Activated Droplet Sorting (MADS) Enables High-Throughput Screening of Enzymatic Reactions at Nanoliter Scale.

Microfluidic droplet sorting enables the high-throughput screening and selection of water-in-oil microreactors at speeds and volumes unparalleled by traditional well-plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high-throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI-MS). Droplets are split, one portion is analyzed by ESI-MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s-1 is achieved with 98 % accuracy using a self-correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label-free ESI-MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.

Optical Chirality Sensing with a Stereodynamic Aluminum Biphenolate Probe.

The determination of the enantiopurity and the concentration of chiral compounds by chiroptical sensing with molecular probes is increasingly attractive for high-throughput screening applications including streamlined asymmetric reaction development. In this study, we use stereodynamic aluminum biphenolate complexes for quantitative ee and concentration analysis of amino alcohols and α-hydroxy acids. An important feature of the tropos biphenolate ligand used is the presence of a phenylacetylene antenna for optimal chirality recognition and CD/UV responses at high wavelengths. The complexation-driven chirality amplification yields strong CD signals, which allows quantitative chiroptical sensing with good accuracy. We show that aluminate biphenolate sensors can exhibit linear and nonlinear correlations between the induced CD signals and the enantiomeric composition or concentration of the chiral substrate.

Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry.

Directed Evolution is a key technology driving the utility of biocatalysis in pharmaceutical synthesis. Conventional approaches to Directed Evolution are conducted using bacterial cells expressing enzymes in microplates, with catalyzed reactions measured by HPLC, high-performance liquid chromatography-mass spectrometry (HPLC-MS), or optical detectors, which require either long cycle times or tailor-made substrates. To better fit modern, fast-paced process chemistry development where solutions are rapidly needed for new substrates, droplet microfluidics interfaced with electrospray ionization (ESI)-MS provides a label-free high-throughput screening platform. To apply this method to industrial enzyme screening and to explore potential approaches that may further improve the overall throughput, we optimized the existing droplet-MS methods. Carryover between droplets, traditionally a significant issue, was reduced to undetectable level by replacing the stainless steel ESI needle with a Teflon needle within a capillary electrophoresis (CE)-MS source. Throughput was improved to 3 Hz with a wide range of droplet sizes (10-50 nL) by tuning the sheath flow within the CE-MS source. The optimized method was demonstrated by screening reactions using two different transaminase libraries. Good correlations (r2 ∼ 0.95) were found between the droplet-MS and LC-MS methods, with 100% match on hit variants. We further explored the capability of the system by performing in vitro transcription-translation inside the droplets and directly analyzing the intact reaction mixture droplets by MS. The synthesized protein attained comparable activity to the protein standard, and the complex samples appeared well tolerated by the MS. The success of the above applications indicates that the MS analysis of the microfluidic droplets is an available option for considerably accelerating the screening of enzyme evolution libraries.

Mapping the dark space of chemical reactions with extended nanomole synthesis and MALDI-TOF MS.

Understanding the practical limitations of chemical reactions is critically important for efficiently planning the synthesis of compounds in pharmaceutical, agrochemical, and specialty chemical research and development. However, literature reports of the scope of new reactions are often cursory and biased toward successful results, severely limiting the ability to predict reaction outcomes for untested substrates. We herein illustrate strategies for carrying out large-scale surveys of chemical reactivity by using a material-sparing nanomole-scale automated synthesis platform with greatly expanded synthetic scope combined with ultrahigh-throughput matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS).

Accelerated Forced Degradation of Pharmaceuticals in Levitated Microdroplet Reactors.

Forced degradation is a method of studying the stability of pharmaceuticals in order to design stable formulations and predict drug product shelf life. Traditional methods of reaction and analysis usually take multiple days, and include LC-UV and LC-MS product analysis. In this study, the reaction/analysis sequence was accelerated to be completed within minutes using Leidenfrost droplets as reactors (acceleration factor: 23-188) and nanoelectrospray ionization MS analysis. The Leidenfrost droplets underwent the same reactions as seen in traditional bulk solution experiments for three chemical degradations studied. This combined method of accelerated reaction and analysis has the potential to be extended to forced degradation of other pharmaceuticals and to drug formulations. Control of reaction rate and yield is achieved by manipulating droplet size, levitation time and whether or not make-up solvent is added. Evidence is provided that interfacial effects contribute to rate acceleration.

Modeling and predicting chiral stationary phase enantioselectivity: An efficient random forest classifier using an optimally balanced training dataset and an aggregation strategy.

Predicting whether a chiral column will be effective is a daily task for many analysts. Moreover, finding the best chiral column for separating a particular racemic compound is mostly a matter of trial and error that may take up to a week in some cases. In this study we have developed a novel prediction approach based on combining a random forest classifier and an optimized discretization method for dealing with enantioselectivity as a continuous variable. Using the optimization results, models were trained on data sets divided into four enantioselectivity classes. The best model performances were achieved by over-sampling the minority classes (α ≤ 1.10 and α ≥ 2.00), down-sampling the majority class (1.2 ≤ α < 2.0), and aggregating multicategory predictions into binary classifications. We tested our method on 41 chiral stationary phases using layered fingerprints as descriptors. Experimental results show that this learning methodology was successful in terms of average area under the Receiver Operating Characteristic curve, Kappa indices and F-measure for structure-based prediction of the enantioselective behavior of 34 chiral columns.

Are We Approaching a Speed Limit for the Chromatographic Separation of Enantiomers?

As the speed of chromatographic enantioseparations advances to the point where the enantiomers of most chiral compounds can be resolved in less than a minute, some in less than a second, we pose the question of how this field is likely to develop over the next few years. Are we approaching a fundamental speed limit, or will further technological advances continue to deliver faster and faster separations? Are faster separations even needed for chemical research, and if so, how will they help? We herein examine this trend, investigating the barriers that currently limit speed and offering some insights into the continued evolution of fast chromatographic enantioseparations.

An Asymmetric, Catalytic (4 + 3) Cycloaddition Reaction of Cyclopentenyl Oxyallylic Cations.

Treatment of 2-tosyloxycyclopentanone with substituted furans in the presence of a chiral amino alcohol catalyst and K2HPO4 results in the formation of (4 + 3) cycloaddition products with enantioselectivities that exceed 90% in certain cases.

Palladium-Catalyzed Enantioselective Arylation of Aryl Sulfenate Anions: A Combined Experimental and Computational Study.

A novel approach to produce chiral diaryl sulfoxides from aryl benzyl sulfoxides and aryl bromides via an enantioselective arylation of aryl sulfenate anions is reported. A (JosiPhos)Pd-based catalyst successfully promotes the asymmetric arylation reaction with good functional group compatibility. A wide range of enantioenriched diaryl, aryl heteroaryl, and even diheteroaryl sulfoxides were generated. Many of the sulfoxides prepared herein would be difficult to prepare via classic enantioselective oxidation of sulfides, including Ph(Ph-d5)SO (90% ee, 95% yield). A DFT-based computational study suggested that chiral induction originates from two primary factors: (i) both a kinetic and a thermodynamic preference for oxidative addition that places the bromide trans to the JosiPhos-diarylphosphine moiety and (ii) Curtin-Hammett-type control over the interconversion between O- and S-bound isomers of palladium sulfenate species following rapid interconversion between re- and si-bound transmetalation products, re/si-Pd-OSPh (re/si-PdO-trans).