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Chiral resolution

Chiral resolution, also known as enantiomeric resolution, is the process of separating a racemic mixture—a 50:50 mixture of two enantiomers—into its individual pure enantiomeric forms.^[1] Enantiomers are non-superimposable mirror-image stereoisomers that possess identical physical properties, such as melting point and solubility, but interact differently with other chiral entities, including biological systems.^[1] This separation is essential in pharmaceuticals, agrochemicals, and materials science, where enantiomers can exhibit profoundly different biological activities, potencies, and toxicities; for instance, one enantiomer of a drug may be therapeutic while the other is inactive or harmful.^[2] Regulatory agencies, including the FDA and EMA, require evaluation of enantiomeric purity and often prefer single-enantiomer formulations to minimize adverse effects and optimize efficacy, as exemplified by the thalidomide tragedy in the 1950s–1960s, where the racemic mixture led to severe birth defects due to the teratogenic (S)-enantiomer.^[1]^[2] Key methods for chiral resolution include classical approaches like diastereomer formation and crystallization, as well as modern techniques such as chromatography and enzymatic kinetic resolution.^[1] In the diastereomer method, a racemic compound is reacted with an enantiomerically pure chiral resolving agent—such as tartaric acid for amines or brucine for carboxylic acids—to form diastereomeric salts with differing solubilities, enabling separation via fractional crystallization, followed by regeneration of the pure enantiomers.^[1] Kinetic resolution exploits the selective reactivity of enzymes or chiral catalysts toward one enantiomer, leaving the other enriched in the mixture, and is particularly valuable for large-scale production.^[1] Chromatographic techniques, including high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) with chiral stationary phases (CSPs), have become dominant for both analytical and preparative separations due to their versatility and efficiency.^[2] Over 100 CSPs are available for HPLC, while advances in CSP design continue to improve resolution for complex molecules.^[2] Historically, the field traces back to Louis Pasteur's 1848 manual separation of tartrate enantiomers by crystallization, which demonstrated the existence of molecular chirality and laid the foundation for modern stereochemistry.^[1]

Fundamentals

Historical Development

The discovery of chiral resolution traces back to the mid-19th century, when Louis Pasteur achieved the first manual separation of enantiomers in 1848. While studying sodium ammonium tartrate crystals, Pasteur observed that they formed as mirror-image hemihedral crystals below 28°C, which he painstakingly sorted into two piles using tweezers under a microscope.^[3] He then dissolved each pile separately and confirmed their optical activity using a polarimeter, finding that one rotated plane-polarized light to the right (dextrorotatory) and the other to the left (levorotatory) with equal magnitude, thus demonstrating the existence of enantiomers and establishing the foundational principle of molecular dissymmetry.^[4] This serendipitous experiment, conducted at age 26, marked the birth of stereochemistry and chiral resolution as scientific pursuits.^[4] Building on this breakthrough, Pasteur advanced to chemical methods five years later in 1853, performing the first resolution of a racemic mixture through diastereomeric salt formation. He treated racemic tartaric acid with optically active cinchotoxine (a derivative of quinine obtained by treating quinine with sulfuric acid), yielding diastereomeric salts with differing solubilities that could be separated by fractional crystallization.^[5] The individual salts were then decomposed to isolate the pure enantiomers of tartaric acid, confirming their optical purity via polarimetry. This technique, leveraging the physical property differences of diastereomers, laid the groundwork for classical resolution strategies and highlighted the potential of chiral auxiliaries in separation processes. In the early 20th century, chiral resolution evolved with the adoption of naturally occurring alkaloids as resolving agents, expanding the scope beyond Pasteur's initial examples. By the 1900s, brucine and quinine—readily available chiral bases derived from plant sources—became widely used for resolving racemic acids through salt formation, owing to their ability to form sparingly soluble diastereomers with targeted enantiomers./06%3A_Stereochemistry_at_Tetrahedral_Centers/6.08%3A_Resolution_(Separation)of_Enantiomers) These agents facilitated resolutions of compounds like mandelic acid and enabled industrial-scale applications, as their natural chirality and commercial availability made them practical for routine laboratory use./06%3A_Stereochemistry_at_Tetrahedral_Centers/6.08%3A_Resolution(Separation)_of_Enantiomers) This period saw a shift toward systematic screening of resolving agents, solidifying diastereomeric crystallization as a cornerstone method. Post-World War II developments in the 1950s underscored the pharmaceutical relevance of chiral resolution, driven by growing awareness of enantiomer-specific biological effects. The thalidomide tragedy of the early 1960s, where the racemic sedative caused severe birth defects due to the teratogenic activity of one enantiomer despite the other being benign, dramatically highlighted the risks of administering unresolved racemates and spurred regulatory demands for enantiopure drugs.^[6] This event catalyzed advancements in resolution techniques for pharmaceutical synthesis, emphasizing the need for stereoselective purity in therapeutic agents.^[6] From the 1980s onward, chiral resolution integrated with the burgeoning field of asymmetric synthesis, providing complementary strategies for accessing enantiopure compounds when direct synthesis proved inefficient. Pioneering work in catalytic asymmetric hydrogenation and epoxidation during this decade, later recognized by the 2001 Nobel Prize in Chemistry to Knowles, Noyori, and Sharpless, reduced reliance on resolution for many targets but reinforced its role in resolving synthetic intermediates or natural product derivatives.^[7] Key milestones included the commercialization of chiral stationary phases for chromatography in the 1990s, enabling preparative-scale resolutions, and the rise of biocatalytic methods in the 2000s for eco-friendly separations.^[8] By the 2010s and into 2025, hybrid approaches combining resolution with computational screening of resolving agents have further streamlined processes, supporting the production of over half of modern small-molecule drugs as single enantiomers.^[9]

Basic Concepts of Chirality

Chirality is a geometric property of a molecule that renders it non-superimposable on its mirror image, analogous to the relationship between a left hand and a right hand.^[10] This handedness arises in molecules lacking an improper axis of rotation, such as a plane of symmetry, and is exemplified by helical structures or tetrahedral carbons with four different substituents.^[11] The concept was first observed by Louis Pasteur in 1848 through the manual separation of mirror-image crystals of sodium ammonium tartrate.^[11] Molecules exhibiting chirality exist as enantiomers, which are pairs of stereoisomers that are nonsuperimposable mirror images of each other. Enantiomers possess identical physical properties, including melting points, boiling points, and solubilities, in achiral environments, but they differ in their interaction with plane-polarized light. Specifically, one enantiomer rotates the plane of polarized light clockwise (dextrorotatory, denoted as (+)), while its mirror image rotates it counterclockwise (levorotatory, denoted as (-)), by equal magnitudes.^[10] A racemic mixture, or racemate, consists of an equimolar (1:1) blend of two enantiomers, resulting in no net optical rotation due to the cancellation of their opposing effects.^[12] Such mixtures are optically inactive and commonly arise from chemical syntheses starting from achiral precursors.^[13] The degree of optical rotation is quantified using polarimetry, which measures the angle of rotation \alpha of plane-polarized light passing through a sample. The specific rotation [\alpha], a standardized measure independent of concentration and path length, is calculated by the formula

[\alpha] = \frac{\alpha}{c \cdot l}

where c is the concentration in g/mL and l is the path length in decimeters.^[14] The significance of chirality extends to biology, where homochirality predominates: proteins are composed almost exclusively of L-amino acids, while nucleic acids incorporate D-sugars.^[15] This selective use of one enantiomer underscores the importance of chiral resolution in producing enantiomerically pure compounds for biological and pharmaceutical applications.^[15]

Principles of Separation

Enantiomers and Diastereomers

Enantiomers are nonsuperimposable mirror-image stereoisomers that possess identical physical properties, including melting points, boiling points, solubilities, and reactivities toward achiral reagents.^[16] This similarity arises because enantiomers have the same molecular formula, connectivity, and conformational possibilities, making them indistinguishable in achiral environments.^[17] However, enantiomers exhibit different behaviors in chiral settings, such as interactions with other chiral molecules or polarized light, which forms the basis for their resolution but complicates direct separation due to the lack of exploitable physical differences.^[14] In contrast, diastereomers are stereoisomers that are not mirror images of each other, often arising in molecules with multiple chiral centers where configurations differ at one or more sites.^[17] Unlike enantiomers, diastereomers are constitutionally distinct compounds with differing physical properties, such as varying melting points, solubilities, and chromatographic behaviors, which allow for straightforward separation by conventional methods like crystallization or distillation. For instance, the unequal solubilities of diastereomers enable selective precipitation from solution, a key principle in chiral resolution strategies.^[18] A common approach to resolving enantiomers exploits their conversion into diastereomers by reacting the racemic mixture with a chiral auxiliary, creating separable diastereomeric derivatives whose property differences can be leveraged.^[16] This temporary modification allows the enantiomers to behave as diastereomers during separation, after which the auxiliary is removed to recover the pure enantiomers. A representative example is the resolution of racemic 1-phenylethylamine by forming diastereomeric salts with enantiopure tartaric acid; the (S)-amine-(R,R)-tartrate salt is less soluble and crystallizes preferentially from methanol solution, enabling isolation of the enantiomers.^[19] The thermodynamic foundation for these separations lies in the distinct free energies of diastereomers in solution and solid states, stemming from their non-mirror-image structures that lead to different intermolecular interactions and lattice energies.^[18] This results in unequal solubilities, as the dissolution free energy (ΔG = ΔH - TΔS) varies between diastereomers due to differences in solvation and crystal packing, facilitating selective crystallization without requiring chiral environments.^[20]

Core Strategies for Resolution

Chiral resolution strategies are broadly classified into direct and indirect methods. Direct methods achieve enantiomeric separation without chemical modification of the racemate, typically employing chiral environments such as stationary phases or selectors that interact differently with each enantiomer based on their stereochemical properties.^[21] In contrast, indirect methods involve temporary derivatization of the enantiomers with a chiral auxiliary to form diastereomers, which exhibit distinct physical properties and can be separated using achiral techniques before regenerating the original enantiomers.^[21]^[22] These strategies further divide into equilibrium and kinetic approaches. Equilibrium resolution exploits reversible thermodynamic differences between enantiomers or diastereomers, such as variations in solubility or stability, to drive separation under conditions where the system reaches a balanced state.^[21] Kinetic resolution, however, relies on irreversible differences in reaction rates between enantiomers when interacting with a chiral reagent or catalyst, allowing selective processing of one enantiomer over the other.^[21] In kinetic processes, the selectivity factor s quantifies the efficiency of enantiomer discrimination, defined as s = \frac{k_1}{k_2}, where k_1 and k_2 are the rate constants for the two enantiomers. Higher values of s (ideally >20) enable greater enantiomeric enrichment at lower conversions. Key efficiency metrics include enantiomeric excess (ee), calculated as ee = \frac{|R - S|}{R + S} \times 100\%, where R and S represent the concentrations of the respective enantiomers, providing a measure of optical purity.^[21] In chromatographic resolutions, the resolution factor R_s = \frac{2(t_2 - t_1)}{w_1 + w_2}, with t_1 and t_2 as retention times and w_1 and w_2 as peak widths, assesses baseline separation, where R_s > 1.5 indicates complete resolution. Despite their efficacy, core strategies face challenges including scalability for industrial production, high costs associated with chiral agents or media, and the need for efficient recovery of auxiliaries or selectors to minimize waste.^[21]^[22]

Classical Methods

Diastereomeric Salt Crystallization

Diastereomeric salt crystallization is a widely used classical technique for chiral resolution, particularly suited for racemic mixtures containing acidic or basic functional groups. The process involves reacting the racemate with an enantiomerically pure chiral resolving agent of complementary ionic character, such as a chiral base (e.g., an alkaloid) for racemic acids or a chiral acid (e.g., tartaric acid) for racemic bases, to form a pair of diastereomeric salts. These salts, unlike the original enantiomers, possess different physical properties, including solubility, due to their diastereomeric nature, enabling their separation.^[18]^[23] The mixture is dissolved in a suitable solvent, often polar media like water or alcohols (e.g., methanol), selected to exploit the solubility disparities between the diastereomers while ensuring complete dissolution at elevated temperatures. The solution is then cooled gradually—typically at rates of 0.04–0.10°C/min—to promote the selective crystallization of the less soluble diastereomer. To enhance control and yield, the process may include seeding with a small amount (e.g., 2 m/m%) of the target diastereomer crystals at an appropriate temperature, followed by agitation to facilitate nucleation and growth.^[18]^[24] Following crystallization, the solid diastereomer is isolated via filtration, often without additional washing to preserve yield. The bound enantiomer is then regenerated by pH adjustment: acidification for salts formed with basic resolving agents to liberate acidic enantiomers, or basification for the reverse, allowing simultaneous recovery of the resolving agent for potential reuse. Yields typically range from 40–50%, depending on the system.^[18]^[23] This method is advantageous for its simplicity, low operational costs, and scalability to industrial levels, making it a preferred choice for producing enantiopure compounds in bulk. However, it demands stoichiometric quantities of the chiral resolving agent, which can elevate expenses if the agent is costly or poorly recyclable, and it carries risks of partial racemization for enantiomer-sensitive substrates under acidic or basic conditions. It is also restricted to ionizable compounds capable of forming stable salts.^[23]^[25] A classic example illustrates the procedure: racemic lactic acid is mixed with (S)-1-phenylethylamine, a chiral base, to generate diastereomeric salts that are dissolved in acetone and cooled to selectively crystallize the salt of (S)-lactic acid, which is then filtered and the enantiomer regenerated by acidification.^[26]

Resolving Agents and Case Studies

Common resolving agents for diastereomeric salt formation include natural products such as (R,R)-tartaric acid, which is widely used for resolving chiral amines due to its availability and ability to form selectively crystallizable salts, and cinchonidine, an alkaloid effective for resolving carboxylic acids by exploiting differences in salt solubility.^[25] Other classical agents include brucine for acids and ephedrine for bases. Synthetic agents like (S)-mandelic acid are also employed, particularly for amino alcohols, offering versatility in industrial applications through tunable interactions with racemic substrates.^[27] Effective resolving agents must achieve high enantiomeric excess (ee) in the resolved product, typically exceeding 95%, while being recyclable to minimize costs and waste. Additionally, low toxicity and compatibility with green solvents are critical criteria, ensuring scalability without environmental harm. A prominent case study is the industrial resolution of a key intermediate for duloxetine, an antidepressant, using (S)-mandelic acid to form diastereomeric salts with 3-(methylamino)-1-(2-thienyl)propan-1-ol; this process, optimized in 2-butanol, achieves >99% ee on a multi-ton scale and has supported commercial production since duloxetine's approval in 2007.^[27] Another example involves the resolution of ibuprofen enantiomers with S-α-methylbenzylamine, forming diastereomeric salts that preferentially crystallize the active (S)-ibuprofen, enabling enantioselective pharmaceutical manufacturing with ee values up to 99% and highlighting the agent's role in targeting the bioactive isomer responsible for anti-inflammatory activity.^[28]

Chromatographic Techniques

Chiral Stationary Phases

Chiral stationary phases (CSPs) are specialized chromatographic media designed to achieve enantioseparation by selectively interacting with enantiomers through stereospecific binding sites. These phases are typically immobilized or coated onto a solid support, such as silica particles, enabling the formation of transient diastereomeric complexes that differ in retention times based on the chiral selector's structure. The development of CSPs has revolutionized chiral resolution, particularly in high-performance liquid chromatography (HPLC), by providing reproducible separations for a broad range of pharmaceuticals and natural products.^[29] The primary types of CSPs include polysaccharide-based, cyclodextrin derivatives, protein-based, and Pirkle-type phases, each exploiting distinct molecular recognition principles. Polysaccharide-based CSPs, derived from cellulose or amylose, are among the most versatile and widely used; for instance, cellulose tris(3,5-dimethylphenylcarbamate), commercially known as Chiralcel OD, facilitates separations through helical polymer structures that accommodate diverse analytes via multiple interaction sites. Cyclodextrin derivatives, such as β-cyclodextrin bonded to silica, form inclusion complexes with hydrophobic portions of enantiomers, enhancing selectivity for compounds with aromatic or alkyl groups. Protein-based CSPs, exemplified by ovomucoid (a glycoprotein from egg white), mimic biomolecular interactions like those in enzymatic binding, offering high specificity for amino acids and peptides but with narrower applicability. Pirkle-type CSPs, or brush-type phases, rely on π-π interactions between aromatic moieties on the selector and analyte, often combined with hydrogen bonding and steric effects, making them suitable for non-polar chiral molecules.^[30]^[31] Chiral recognition in CSPs is fundamentally described by the three-point attachment model, which posits that effective enantioseparation requires at least three simultaneous, stereochemically dependent interactions between the analyte and selector, such as hydrogen bonding, dipole-dipole forces, and steric repulsion or attraction. This model ensures that one enantiomer forms a more stable complex than the other, leading to differential adsorption energies and chromatographic retention. For example, in polysaccharide CSPs, the carbamate groups provide hydrogen-bonding sites, while the polymer backbone contributes steric differentiation.^[32]^[33] Immobilization techniques for CSPs are critical for enhancing durability, particularly in preparative applications. Coated phases involve physical adsorption of the chiral selector onto the support, offering simplicity and high initial performance but limited solvent compatibility and risk of leaching under harsh conditions. In contrast, covalently bonded or immobilized phases, achieved through radical polymerization or silane coupling, provide superior chemical and mechanical stability, allowing use with aggressive solvents and higher pressures in scaled-up separations; for instance, immobilized polysaccharide CSPs retain over 80% of the chiral recognition ability of their coated counterparts while enabling preparative loading up to grams per run.^[34] Selection of a CSP depends on the analyte's chemical class and structural features, with empirical screening often guided by databases correlating molecular descriptors to phase performance. Amylose-based CSPs, such as Chiralpak AD, excel for alcohols and amines due to their open helical cavities that favor polar interactions, while cyclodextrin phases are preferred for compounds with cyclic structures. As of 2021, more than 150 commercial CSPs were available from manufacturers like Daicel and Regis Technologies, facilitating rapid method development through standardized kits.^[35] Despite their efficacy, CSPs face limitations including high production costs—often exceeding $1,000 per analytical column due to complex synthesis—and restricted loading capacities in preparative modes, typically limited to 1-10% of column weight for optimal resolution, which can increase separation times and expenses for large-scale resolutions. Protein-based CSPs are particularly constrained by low capacity and sensitivity to pH extremes, while even robust polysaccharide phases may require mobile phase optimization to mitigate peak broadening.^[30]^[36]

High-Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) serves as a cornerstone technique for the enantiomeric resolution of chiral compounds, enabling the separation of enantiomers based on their differential interactions with chiral stationary phases. In chiral HPLC, the setup typically involves either normal-phase (NP) or reversed-phase (RP) modes, with polar organic mode (POM) often preferred for LC-MS compatibility. Mobile phases commonly consist of hexane/isopropanol mixtures for NP separations or acetonitrile/alcohol combinations for POM, while RP uses organic-aqueous mixtures; detection is achieved via UV absorbance at wavelengths such as 214 nm or 220 nm, or mass spectrometry (MS) for enhanced sensitivity.^[37] Optimization of chiral HPLC parameters is crucial for achieving efficient separations. Flow rates typically range from 0.5 to 2 mL/min, with lower rates (e.g., 0.2–1 mL/min) used to balance resolution and analysis time. Temperature control, often set around 35–45°C, influences selectivity by altering analyte-stationary phase interactions, thereby enhancing peak separation. For instance, in the separation of beta-blockers like bisoprolol using a Chirobiotic V column (5 μm, 250×4.6 mm) with a methanol/acetic acid/triethylamine mobile phase (100/0.20/0.15 v/v/v) at 0.5 mL/min and UV detection at 230 nm, retention times of 10.85 min and 11.43 min were observed for the enantiomers, yielding a resolution (Rs) of 1.30.^[38] The chromatogram displayed two distinct peaks with baseline separation, though Rs values around 1.3–1.4 indicate near-baseline resolution, highlighting the need for further optimization to reach the ideal Rs > 1.5 for complete baseline separation. Resolution is calculated using the formula:

R_s = \frac{2(t_2 - t_1)}{w_1 + w_2}

where t_1 and t_2 are the retention times of the two peaks, and w_1 and w_2 are their baseline widths; this metric ensures quantifiable separation efficiency.^[39] Scale-up from analytical (microgram) to preparative (kilogram) levels is a key advantage of chiral HPLC, facilitating production for pharmaceutical applications. Analytical methods handling micrograms transition to preparative columns for gram-to-kilogram yields, often employing simulated moving bed (SMB) chromatography for continuous operation and improved productivity, as demonstrated in the enantioseparation of metalaxyl racemate. Supercritical fluid chromatography (SFC), using CO₂ as a mobile phase, emerges as a greener alternative to traditional HPLC by reducing organic solvent consumption, achieving up to 98% enantiomeric purity in processes like the isolation of levosimendan intermediates.^[40] In pharmaceutical applications, chiral HPLC ensures high enantiomeric purity (>98% ee) for active pharmaceutical ingredients (APIs), critical for regulatory compliance and therapeutic efficacy. For example, it is routinely used for purity analysis of beta-blockers, where enantiomeric excess directly impacts pharmacological activity. Recent advances in the 2020s, particularly in ultra-high-performance liquid chromatography (UHPLC) with sub-2 μm particles, have enabled faster separations (reduced run times by 2–5 fold) while maintaining resolution, enhancing throughput in high-volume screening.^[40]^[38]^[37]

Specialized Techniques

Spontaneous and Preferential Crystallization

Spontaneous resolution refers to the rare phenomenon in which a racemic mixture of enantiomers crystallizes into a mechanical mixture of separate crystals, each containing a single enantiomer, without the need for external chiral influences.^[41] This process occurs only for compounds that form conglomerates, where the homochiral crystal lattices are thermodynamically stable and do not incorporate the opposite enantiomer.^[42] Such spontaneous resolution is observed in approximately 5-10% of chiral organic compounds, based on analyses of crystallographic databases and thermodynamic studies.^[42] A classic example is sodium ammonium tartrate, famously resolved by Louis Pasteur in 1848 through manual separation of the conglomerate crystals under a microscope.^[41] Another instance involves guaifenesin, a chiral drug that forms conglomerate crystals suitable for spontaneous resolution, as identified through structural analyses.^[43] Detection of conglomerate-forming racemates is crucial for identifying candidates for spontaneous resolution and typically involves X-ray crystallography to confirm the presence of homochiral space groups (Sohncke groups) in the crystal structure.^[41] Solubility measurements provide an additional diagnostic tool, as conglomerates exhibit a solubility for the racemate that is roughly twice that of the pure enantiomer, following the "double solubility rule" under ideal conditions. These methods ensure accurate identification, though challenges arise from the lack of explicit metadata in databases like the Cambridge Structural Database, requiring manual verification of synthetic routes and stereochemistry.^[41] Preferential crystallization extends the utility of conglomerate systems by selectively inducing the crystallization of one enantiomer from a near-saturated racemic solution, often initiated by seeding with a small amount of the desired enantiomer.^[44] This method leverages differences in nucleation and growth rates between enantiomers under controlled conditions, such as temperature cycling, where the solution is alternately cooled to promote crystallization and heated to redissolve impurities or small crystals.^[44] Ultrasound can further enhance the process by facilitating nucleation and reducing induction times, improving efficiency in chiral separations.^[45] A notable advancement is Viedma ripening, a deracemization technique involving grinding of racemic conglomerate crystals in a saturated solution to promote Ostwald ripening and attrition, leading to complete conversion to a single enantiomer with enantiomeric excess exceeding 99%.^[46] This process relies on nonlinear autocatalysis and solution-phase racemization for amenable compounds, achieving high purity without initial seeding in some setups.^[47] Recent developments as of 2025 include mechanochemical deracemization, which applies grinding without solvent to achieve enantiopure compounds from racemates, offering a sustainable alternative for scalable production.^[48] Preferential crystallization, including Viedma ripening, is particularly scalable for amino acids such as glutamic acid, where yields of up to 80% enantiopure product have been reported in laboratory-scale operations up to 320 mL.^[46] The method's key advantages include the absence of chiral additives or waste products, enabling a straightforward recycling of the mother liquor and high atom economy.^[44] However, its application is inherently limited to the small fraction of compounds (5-10%) that form stable conglomerates, and success depends on precise control of thermodynamic and kinetic parameters to avoid nucleation of the undesired enantiomer.^[42]

Kinetic and Enzymatic Resolution

Kinetic resolution is a method for separating enantiomers in a racemic mixture by exploiting differences in their reaction rates with an achiral reagent, where the selectivity factor s (or enantiomeric ratio E) quantifies the preference, with s > 1 indicating viable resolution. The enantiomeric ratio E is calculated using the formula

E = \frac{\ln[1 - C(1 + ee_p)]}{\ln[1 - C(1 - ee_p)]},

where C is the conversion of the substrate and ee_p is the enantiomeric excess of the product; this metric, introduced by Chen et al., remains independent of conversion and is widely used to assess selectivity in both chemical and enzymatic processes. In practice, maximum enantiomeric excess for both product and remaining substrate is achieved at approximately 50% conversion, theoretically yielding up to 50% of each enantiomer in high purity, though actual yields are often lower without additional steps. Enzymatic kinetic resolution represents a prominent subset, leveraging biocatalysts like lipases for high selectivity under mild conditions, often via ester hydrolysis or acylation of alcohols or amines. Lipase B from Candida antarctica (CALB) is particularly effective for resolving secondary alcohols, such as in the acetylation of racemic 1-phenylethanol using vinyl acetate as the acyl donor, where CALB exhibits an E value exceeding 50, enabling isolation of the (R)-acetate and (S)-alcohol in >98% enantiomeric excess at 50% conversion. This approach benefits from the enzyme's stability in organic solvents and broad substrate tolerance, making it suitable for preparative-scale separations. To overcome the inherent 50% yield limitation of classical kinetic resolution, dynamic kinetic resolution (DKR) integrates in situ racemization of the slower-reacting enantiomer, often using metal catalysts like ruthenium complexes alongside lipases, allowing theoretical yields approaching 100% for the favored enantiomer. For instance, combining CALB with a ruthenium-based racemization agent enables efficient DKR of secondary alcohols like 1-phenylethanol, producing the (R)-ester in high yield and enantiopurity. Post-2010 advances in directed evolution have enhanced enzymatic performance, with variants of CALB engineered for improved enantioselectivity and stability in kinetic resolutions of pharmaceutical intermediates like profens, achieving E values up to 200 through targeted mutations in the active site. These engineered lipases have found industrial application, such as in the kinetic resolution of 4-arylmethoxy-3-hydroxybutanenitriles, a key step in synthesizing statin intermediates for cholesterol-lowering drugs. Recent progress as of 2024 includes bienzymatic DKR systems that couple two enzymes for deracemization of secondary alcohols, achieving near-complete conversion with high enantiopurity without metal catalysts, advancing green chemistry applications.^[49] Despite these successes, kinetic and enzymatic resolutions face challenges, including inherently low yields without racemization in DKR and issues with enzyme stability under non-aqueous or high-temperature conditions, which can limit scalability without immobilization or engineering.

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Resolution of 3-(methylamino)-1-(2-thienyl)propan-1-ol, a new key ...
The conditions were optimized for an industrial-scale resolution of 3 by using (S)-mandelic acid 4 as a resolving agent and 2-butanol containing 2 equimolar ...
[27]
Recycling of resolving agent and solvent by water-based reactions ...
Chiral resolution of (R,S)-(±)-ibuprofen using (S)-(-)-1-phenylethylamine as a resolving agent was used as the model system. Ibuprofen (Scheme 1(a)) is common ...
[28]
Strategies for chiral separation: from racemate to enantiomer
Sep 27, 2023 · This review comprehensively summaries the latest developments in the main enantioseparation methods, including preparative-scale chromatography, ...
[29]
US5621140A - Resolution of ibuprofen - Google Patents
The use of (S)-α-methylbenzylamine as the resolving agent has been described ... The individual enantiomers are specifically designated as (S)-ibuprofen or (R)- ...
[30]
Turning Renewable Feedstocks into a Valuable and Efficient Chiral ...
Oct 12, 2021 · Known chiral 6 member cyclic PAs (chlocyphos, anycyphos, phencyphos, etc.) have only been employed as resolution agents for chiral amines.Missing: resolving post-
[31]
Chiral Stationary Phases for Liquid Chromatography: Recent ... - MDPI
This review gathered the most recent developments associated to the different types of CSPs providing an overview of the relevant advances that are arising on ...
[32]
Comprehensive Review on Chiral Stationary Phases in Single ... - NIH
Mar 18, 2024 · The study encompasses four crucial types of CSPs: cyclodextrin (CD)-, glycopeptide antibiotic-, protein-, and polysaccharide-based columns, with ...
[33]
[PDF] A Review on Chiral Stationary Phases for Separation of Chiral Drugs
Jun 4, 2020 · Chiral chromatography is a preferred method for the resolution of chiral drugs. Today, innovation in the chiral stationary phases has made ...
[34]
Chiral Recognition Mechanisms - ACS Publications
Apr 1, 2006 · The key points in the three-point interaction model are that at least three simultaneous interactions are required and that they should ...
[35]
The nature of chiral recognition: Is it a three‐point interaction?
Evolution of the initial “three-point attachment model” resulted in the understanding that an interaction in at least three configuration-dependent points ...
[36]
Comparison of Coated and Immobilized Chiral Stationary Phases ...
Mar 20, 2021 · In the present work, the direct and baseline enantioresolution of both chiral acids by HPLC on two amylose-derived chiral stationary phases is presented.Missing: techniques bonded
[37]
Chiral Separations | LCGC International
Breitbach and Armstrong: With more than 100 commercially available CSPs on the market today, choosing the right column and the proper mobile phase for new ...<|separator|>
[38]
Chiral chromatography method screening strategies: Past, present ...
Feb 8, 2021 · There are more than one hundred and fifty commercially available CSPs and more than fifteen-hundred CSPs reported in the literature [44], [45], ...
[39]
Recent progress in the development of chiral stationary phases for ...
Nov 3, 2021 · Generally, the amounts of polysaccharide derivative immobilized in a CSP is less than 20 wt%, whether coated- or bonded-type, implying that ...2 Chiral Small Organic... · 2.1 Cd-Based Csps · 4 Chiral Porous...
[40]
Recent developments in the high-throughput separation of ...
Jan 20, 2024 · This review focuses on the latest advancements in chiral SPs for LC. There is an urgent need for chiral screenings especially in the pharma industry.2. Fundamentals · 3. Stationary Phases: Chiral... · 3.1. Polysaccharide-Based...
[41]
Chiral Separation of Beta-blockers by High-performance Liquid ...
Here we present a chromatographic method we developed for chiral separation of five beta-blockers (acebutolol, atenolol, bisoprolol, labetalol, and metoprolol) ...
[42]
Modified resolution factor for asymmetrical peaks in ...
Citation Excerpt : The resolution (Rs) was calculated according to Eq. (3) [52]:Rs = 2 (t2 − t1)/(ω2 + ω1),where t1 and t2 are the retention times of two ...
[43]
Chiral Separations in Preparative Scale: A Medicinal Chemistry ...
Apr 21, 2020 · Despite the high E factor values, preparative SFC can be considered a greener process when compared to LC because a large proportion of the ...
[44]
https://pubs.rsc.org/en/content/articlelanding/2010/ce/c001895c
[45]
Identifying a Hidden Conglomerate Chiral Pool in the CSD | JACS Au
Here, a racemic material can undergo spontaneous resolution with each crystal containing a single enantiomer in a process referred to as a conglomerate ...
[46]
Reevaluating the Stability and Prevalence of Conglomerates: Implications for Preferential Crystallization
### Summary on Prevalence of Conglomerates and Implications for Preferential Crystallization
[47]
Pitfalls and rewards of preferential crystallization - RSC Publishing
When applicable, preferential crystallization is a productive and cost effective process which consists of alternate crystallizations of both enantiomers ...
[48]
Applications of ultrasound to chiral crystallization, resolution and ...
Ultrasound has been applied to several chiral crystallization processes. In diastereomeric crystallization, ultrasound can improve both yield and purity.
[49]
Viedma ripening: a reliable crystallisation method to reach single ...
Jul 13, 2015 · Viedma ripening in particular enables access to enantiopure products in a reliable way, simply through grinding of crystals in a solution.Missing: seminal | Show results with:seminal
[50]
https://doi.org/10.1103/PhysRevLett.94.065504