“Separating the inseparable: how chemists resolve mirror-image molecules.”
Introduction
Despite advances in asymmetric synthesis (Part 5), sometimes we still end up with a racemic mixture of enantiomers. When direct stereoselective routes are impractical, chemists must separate the enantiomers – a process known as resolution. This part examines classical and modern methods for resolving enantiomers, their pros and cons, and how they are applied in pharmaceutical contexts. We will cover:
- Chemical Resolution: Using a chiral resolving agent to form diastereomers, which can be separated (e.g., forming diastereomeric salts with a chiral acid/base).
- Enzymatic Resolution: Using enzymes to react with or consume one enantiomer preferentially (kinetic resolution).
- Chromatographic Resolution: Using chiral stationary phases in chromatography (HPLC, SFC, GC) to separate enantiomers on an analytical or preparative scale.
- Miscellaneous methods: Such as preferential crystallization (sometimes a racemate can crystallize into enantioenriched crystals under certain conditions) and newer techniques like membrane separations.
We’ll also discuss the racemic mixture vs single enantiomer decision from a regulatory and practical standpoint. Why were many old drugs racemic (hint: separation and asymmetric synthesis were hard), and how technological advances changed this equation. Regulatory considerations by FDA/EMA – for example, guidelines stating that if you choose to market a racemate, you should justify it and characterize both enantiomers fully. The concept of “racemic switches or chiral switches” (developing a single-enantiomer version of an existing racemic drug) will also be addressed.
💡 For a detailed journey into chiral resolution, visit: #Chiral_Resolution
Classical Chemical Resolution
Louis Pasteur performed the first resolution in 1848 by manual sorting of tartrate crystals, but for most molecules we rely on chemical means. The classic method is to use a chiral resolving agent that will react or associate with the enantiomers to form diastereomeric derivatives, which have different physical properties (solubility, etc.) and thus can be separated. For instance, alkaloid bases like strychnine or brucine, or tartaric acid and its derivatives, have been historically used.
- Diastereomeric Salt Formation: Perhaps the most common approach for acids and bases. Suppose we have a racemic acidic drug (like a carboxylic acid). We can add an enantiomerically pure chiral base (such as (+)-α-phenethylamine or quinine or brucine) to form salts. The salt of one enantiomer may crystallize out preferentially because diastereomeric salts often have different solubilities in a given solvent. By filtering the less soluble salt, we enrich one enantiomer in solid form. Then we free the acid from the salt and recover the pure enantiomer. This method was widely used: e.g., (S)-naproxen was originally resolved using cinchona alkaloids. Penicillin derivates (like early semi-synthetic penicillins) often utilized resolving bases. Another example: mandelic acid enantiomers can be resolved by forming diastereomeric amides with (R)-1-phenylethylamine. Key considerations are finding a good resolving agent that gives crystalline, easy-to-separate diastereomers and can be recovered. Sometimes a “double resolution” is needed for high purity (two successive crystallizations).
- Diastereomeric Derivative Formation: Beyond salts, one can attach a chiral auxiliary and then separate. For example, Mosher’s acid chloride (α-methoxy-α-trifluoromethylphenylacetyl chloride) reacts with racemic alcohols to form diastereomeric esters, which can be separated (Mosher’s method is often used analytically to determine enantiomeric composition by NMR, but in principle, could be preparative). Another classical reagent: 1,1’-binaphthyl-2,2’-diyl hydrogen phosphate (BNP, “Naphtyl phosphoric acid”) for resolving chiral amines, forming diastereomeric phosphates.
- Mechanical/Preferential Crystallization: A few racemates crystallize as conglomerates (physical mixture of enantiopure crystals rather than racemic combined crystals). If a saturated solution of such a racemate is seeded with one enantiomer’s crystal, often that enantiomer preferentially crystallizes out (a process called seeding or preferential crystallization). This is rarer and works only for certain compounds (sodium ammonium tartrate was one such case, as Pasteur discovered). It requires careful control (racemates might spontaneously crystallize as racemic compound or as separate enantiomers depending on conditions).
Chemical resolution by crystallization is efficient on large scale if the conditions are right – it’s not unusual to achieve kilograms of single enantiomer by a simple crystallization nowadays if you have the right resolving agent. However, yield is inherently limited: in a perfect scenario, you recover at most 50% of the desired enantiomer (the other 50% is the undesired one left in solution or another fraction). If the undesired can be racemized (turned back into racemic mixture) and recycled, the yield can approach 100%. This is racemization-recycle strategy – e.g., the undesired enantiomer of naproxen after one resolution can be racemized by heat or base and then subjected to resolution again, thus not wasting it. This approach (resolution + racemization) is used industrially for some cases (often called dynamic kinetic resolution if racemization happens concurrently with resolution).
Enzymatic Resolution
Enzymes often display high enantioselectivity. For example, lipases and esterases may hydrolyze one enantiomer of an ester faster than the other. If you have a racemic ester, an enzyme can convert, say, the R-ester to R-acid + alcohol, while leaving S-ester largely unreacted. By stopping at partial conversion, you can separate the acid (now enriched in R) and recover the remaining S-ester. This is a kinetic resolution – it doesn’t exceed 50% yield ideally, but you get both enantiomers isolated (one in product, one unreacted). Example: ibuprofen in the lab can be resolved by esterifying racemic ibuprofen and using Candida rugosa lipase, which might hydrolyze preferentially one enantiomer. Another common one: amino acid resolution by acylases – in early days, racemic amino acids were resolved by treating with enzymes that only act on L (or D) forms. Epoxide hydrolases can take a racemic epoxide and hydrolyze one enantiomer to a diol, leaving the other epoxide intact; separation is then easy (diol vs epoxide differ).
Enantioselective enzymatic transformations can also be designed as dynamic kinetic resolutions where the substrate enantiomers interconvert under reaction conditions (often with a chemical catalyst or conditions causing racemization) while the enzyme selectively removes one enant (driving equilibrium). For instance, amide racemization can be coupled with enzymatic hydrolysis of one enant to drive a near 100% yield of one acid. This has been done in manufacturing of some chiral acids.
Chromatographic Resolution
Chiral chromatography has become a powerful method, especially for analytical purposes but also preparative if needed. Chiral HPLC uses a column with a chiral stationary phase that interacts differently with each enantiomer, thus separating them as distinct peaks. There are many chiral stationary phases (CSPs): derivatized polysaccharides (like Chiralcel OJ with cellulose tribenzoate, etc., which are versatile for many drugs), protein-based phases, macrocyclic glycopeptide phases, etc. For small scale, this is routine: any medicinal chemistry group can purify 10-100 mg of one enantiomer from a racemate by chiral HPLC. For large scale (multi-kg), chiral prep HPLC is expensive but possible – simulated moving bed (SMB) chromatography is a method used in industry to continuously separate enantiomers in a more efficient way than batch HPLC. One example: some early production of prilosec (omeprazole) enantiomers was done by SMB chromatography before synthetic routes were improved.
Chiral GC can separate enantiomers if they are volatile enough and a suitable chiral phase (often cyclodextrin-based) is used, but it’s mainly analytical (e.g., separating D- vs L-limonene). In pharma, GC is used for chiral purity of small volatile compounds or as a method of analysis, but preparative GC is rare.
💡 Learn more about chiral chromatography in our dedicated series: #Chiral_Chromatography
Other Methods
– Diastereomeric Complexation: Certain metal complexes can preferentially bind one enant. e.g., [Co(III) complexes can form diastereomers with amino acids (saccharinate complex method). Not common nowadays. – Membrane-based separation: experimental enantioselective membranes that let one enantiomer permeate faster via chiral carriers – mostly in research, not mainstream industrial use yet. – Capillary electrophoresis with chiral additives (like adding cyclodextrin to separate enantioomers by forming transient diastereomeric inclusion complexes) – this is analytical, not for production.
Pros and Cons of Resolution:
– Pros: Straightforward if a good method is known; uses often simple equipment (crystallizer, filter) or existing chromatography setups. Some racemic compounds might be easier to make than designing an asymmetric route. For early drug development (when you need a small sample of each enantiomer to test toxicity or efficacy), resolution is the fastest way to get material. Indeed, a common practice in drug discovery is to do an initial resolution to see if the enantiomers differ, then if one is clearly better, invest in asymmetric synthesis of that one. – Cons: Wastage of material (the undesired enantiomer, unless you racemize and recycle, which adds steps/energy). Additional steps increase cost and time. Also, some resolutions are tricky: need multiple crystallizations to reach high e.e., etc. Moreover, for very complex molecules with many stereocenters, resolution is not practical (e.g., you can’t easily resolve something with 3 chiral centers by typical means – though you could by chiral HPLC perhaps, but yield would be low).
Regulatory Considerations
Historically, many drugs were developed as racemates because resolution or asymmetric synthesis was too difficult. The thalidomide disaster prompted regulatory bodies to pay attention to chirality. The FDA’s 1992 policy statement advocated that sponsors determine absolute configuration and understand the pharmacology of each enantiomer. If a racemate is developed, they should justify that choice and ensure that the manufacturing produces a consistent racemic composition. In fact, FDA said if stereoisomers are “biologically distinguishable, they might be considered different drugs” yet historically racemates were developed due to difficulty separating. Now that it’s easier to produce single enantiomers (via chiral separation or synthesis), the FDA expects critical examination: is there a reason to keep it racemic, or should it be single? They even advise that if one enantiomer is inactive, it’s just as well to remove it (unless it’s benign and too hard to remove). However, if a racemate has some synergistic benefit or the other enantiomer is innocuous and racemate is easier, they allow racemate with justification. They discourage developing mixtures of diastereomers at all unless absolutely necessary.
Single Enantiomer Drugs vs Racemates
By the 2000s, a large fraction of new small-molecule drugs were single enantiomers. For instance, one analysis found >80% of chiral small-molecule new drugs in the 2010s were single isomers. This reflects both technological capability and perhaps also regulatory caution. Some racemic drugs (like Prilosec to Nexium, Celexa to Lexapro) got “upgraded” to single enantiomer for patent advantages and sometimes slight clinical improvements. The question arises: are single enantiomers always better? Not necessarily – sometimes the other enantiomer is harmless or even beneficial (e.g., perhaps smoothing pharmacokinetics). A cited analysis suggests single enantiomers rarely offer dramatic efficacy/safety improvements; often the benefit is marginal despite higher cost. For example, levocetirizine (Xyzal) vs racemic cetirizine – both treat allergies effectively; levocetirizine’s advantage is subtle (maybe slightly less sedation or lower dose). Esomeprazole vs omeprazole gave more consistent acid control but efficacy was similar if dosing adjusted. Nonetheless, in drug development, if one enantiomer is clearly superior, it’s ethically and scientifically logical to develop that one alone to avoid unnecessary exposure to a possibly inactive or harmful isomer.
Racemate Considerations
Sometimes developing a racemate is acceptable or even necessary. For example, racemic bupivacaine was standard as an anesthetic until levobupivacaine (S-enantiomer) was introduced to reduce cardiotoxicity associated with the R form. If both enantiomers have similar profile (like ibuprofen’s both are anti-inflammatory, and R converts to S in vivo), a racemate might be fine. The FDA does not force single enantiomers – it requires understanding. In some cases, the non-eutomer’s presence could even be beneficial (like providing a longer action via slower metabolism, or counteracting some side effect of the eutomer). The FDA guidance notes there are racemates with “few recognized adverse consequences” and that developing racemates “may continue to be appropriate” in cases, but stresses appropriate controls and studies per enantiomer.
You might find this blog article an interesting read in this context. <https://chiralpedia.com/blog/racemates-%e2%89%a0-less-safe-rethink-chirality/>.
Racemic Switches
Later (often as the old patent expires) – a way to extend patent life (so-called “evergreening”). Examples: esomeprazole (from omeprazole), escitalopram (from citalopram), R-albuterol (levalbuterol from albuterol), dexmethylphenidate (Focalin from methylphenidate), etc. Regulatory authorities require that the single enantiomer shows some advantage or at least equivalence. These single enantiomer versions often gain traction if they show reduced side effects or better dosing, but sometimes it’s primarily a patent strategy. Read more @ <https://chiralpedia.com/blog/chiral-switch-unlocking-the-potential-of-single-enantiomers/>.
A cautionary note: As mentioned, thalidomide’s enantiomers interconvert in vivo with a half-life of ~8 hours. This means giving a single enantiomer might not prevent the issue, since it will partially turn into the harmful form inside the body. For any racemic drug considered for switching to a single enantiomer, one must check if interconversion happens (some chiral drugs can racemize under physiological conditions, particularly those with acidic chiral centers like α-carbonyl compounds). Thalidomide is developed now as a racemate for cancer therapy (with severe controls in place), because separating enantiomers doesn’t help due to racemization.
Summary (Part 6)
– Resolution = separation of enantiomers. Methods:
– Chemical: Form diastereomers using chiral reagents (salts or covalent derivatives) and separate by crystallization. Traditional, still widely used for large-scale separations if asymmetric synthesis not available. E.g., resolving agents like tartaric acid, alkaloids (brucine, cinchonine).
– Enzymatic: Kinetic resolution by selective reaction of one enantiomer (common with esters, amides). Yields max 50% per batch, unless combined with racemization. Often mild conditions, high selectivity. Many examples in pharma for producing chiral alcohols or acids (e.g., lipase resolution of a secondary alcohol intermediate in making an HIV protease inhibitor, yielding one enantiopure).
– Chromatographic: Chiral column separation – routine in labs for analysis and small prep; scaling up is costly but possible with SMB systems. Useful when no easy crystallization method exists, or for high-value low-volume products (e.g., separating enantiomers of an early clinical candidate for testing).
– Crystallization (preferential): If a racemate forms a conglomerate, can preferentially crystallize one enantiomer. Rare but elegant when it works (requires initial enantioenriched seed or external influence like stirring with a small amount of one crystal type).
– Efficiency: Resolutions inherently waste at least half the material (unless the other half can be racemized and reused). This makes them less ideal than asymmetric synthesis which (in principle) converts all material to desired enantiomer. However, sometimes resolution is simpler to implement (fewer steps to develop) and can be acceptable if the other enantiomer can be recycled or if raw materials are cheap.
– Regulatory/Industry trends: There is pressure to avoid wasteful resolution unless necessary. But many existing drugs (pre-1990s) were launched as racemates simply because resolution was the only viable route back then. Some of those have since been “switched” to single enantiomer products (with varying degrees of success).
– Racemic vs Single Enantiomer development: If one enantiomer is clearly superior pharmacologically, modern practice is to develop that enantiomer alone whenever feasible. Racemic drug development still occurs if both enantiomers contribute or separation is too difficult or the product of racemate is acceptable (the FDA acknowledges cases where a racemate is fine, e.g., if enantiomers interconvert or both active). But thorough evaluation of each enantiomer’s profile is required.
– Chiral switches: Many were done in the 2000s for incremental improvements or patent extension (e.g., esomeprazole, dexmethylphenidate, levofloxacin from ofloxacin, etc.). Regulatory agencies ensure that a chiral switch drug shows some clinical benefit or at least not worse than the racemate. The success of these in the market varies (some replaced the racemate almost entirely, like esomeprazole largely replaced omeprazole brand usage; some remain niche, like R-albuterol is used but racemic albuterol is still widely used because generic and cheaper).
– Case by case: Instances like thalidomide remind us that even if one enantiomer seems “safe”, resolution doesn’t help if in vivo racemization occurs. Or cases like sotalol where racemate intentionally combines two actions (the enantiomers have complementary effects), developing a single might lose half the spectrum of activity. So it’s not always a straightforward better/worse decision – it depends on pharmacology and chemistry.
Suggested Reading
“Stereochemical Issues in Chiral Drug Development” – Health Canada Guidance (2000). <https://www.canada.ca/en/health-canada/services/drugs-health-products/drug-products/applications-submissions/guidance-documents/chemical-entity-products-quality/guidance-industry-stereochemical-issues-chiral-drug-development.html>. (Covers resolution expectations; also an attachment in it discusses comments on racemates vs single enantiomers).
J. Agranat, et al. (2002). “Medicinal Chemistry and the chirality problem.” Reviews in Computational Chemistry, which includes a section on chiral switches and lists racemic vs single enant drugs and their differences.
Topics in Current Chemistry, Vol. 269 (Chirality in Drug Design and Development) – chapter on separation techniques for enantiomers (describes methods like crystallization, chromatography).
American Pharmaceutical Review article <https://www.americanpharmaceuticalreview.com/Featured-Articles/619871-Chiral-Separation-and-Enantiomeric-Analysis-Critical-Importance-in-Pharmaceutical-Development/#:~:text=Interestingly%2C%20only%20two%20chiral%20switches,%E2%80%9D%E2%81%B7> – mentions meta-analysis of single vs racemic and that pure enantiomers seldom drastically outperform racemates in safety/efficacy. Useful to temper the assumption that single is always better.
Zhou, P. & Xie, Z. (2012). “Chiral Drugs: Development and Patent Term Extension.” Journal of Pharmaceutical Sciences, 101(9). (Discusses racemic switches and regulatory aspects, with examples of drugs that got patent extension via enantiomers.)
Silvestri IP, Colbon PJJ. The Growing Importance of Chirality in 3D Chemical Space Exploration and Modern Drug Discovery Approaches for Hit-ID: Topical Innovations. ACS Med Chem Lett. 2021 Jul 16;12(8):1220-1229. doi: 10.1021/acsmedchemlett.1c00251.
