Periodic Reporting for period 1 - CRMS (Microfluidic Diffusion-based Chiral Resolution)
Reporting period: 2023-05-01 to 2025-05-31
Context: Homochirality is a fundamental feature of animate matter, and nearly all biological processes occurring in nature are stereospecific, i.e. responsive to only one type of handedness of the chiral molecule. Most drugs are chiral and must be administered as a single enantiomer because their enantiomeric form may have detrimental effects. The thalidomide story is a tragic reminder of the importance of chirality for humanity. Indeed, left-handed thalidomide is pharmacologically active as a powerful tranquilizer, while the right-handed enantiomer proved to disrupt fatal development and cause phocomelia and other congenital malformations. There is no doubt that chiral resolution will get even more interest and importance in the future. For instance, to deal with the COVID-19 pandemic and inevitable future threats, there is an urgent need for rapid research on new drugs. However, identical chemical and physical properties of enantiomers require state-of-the-art analytical techniques for the isolation and quantification to determine the purity of synthesized drugs. Therefore, my MSCA project “Chiral Resolution using Microfluidics and helical Supramolecular polymers (HSPs)” (CRMS) aimed to develop a novel diffusion-based strategy by using HSPs and microfluidics for cost-effective and continuous chiral resolution under sustainable conditions.
Overall Objective: The primary goal of this research is to utilize the helicity and low diffusion rates of self-assembled supramolecular polymers (HSPs) to achieve enantioselective trapping and separation of chiral molecules within microfluidic channels. Specific Objectives are: Synthesize a series of HSPs capable of selectively interacting with one enantiomer of a given chiral compound. To achieve this, the HSPs must meet two key criteria: a) They must possess specific interaction sites for chiral auxiliaries and b) They must be sufficiently rigid to maintain their helical structure upon interaction. 2) Develop two distinct microfluidic channel designs for enantiomer separation: a) An H-type microfluidic channel and b) A Y-type microfluidic channel. 3) Optimize the flow conditions for both the supramolecular polymers and the chiral molecules within the microfluidic devices to ensure efficient enantioseparation. 4) Apply the developed technique to the chiral separation of pharmaceutical drug molecules, specifically alprenolol and ethambutol. Most chiral drugs are synthesized as racemic mixtures in the laboratory, although typically only one enantiomer is pharmacologically active. Delivering drugs in enantiopure form can enhance therapeutic efficacy and minimize side effects. Furthermore, from a green chemistry perspective, enantioseparation via microfluidic systems offers a cost-effective and environmentally friendly alternative to traditional methods.
Overall Objective: The primary goal of this research is to utilize the helicity and low diffusion rates of self-assembled supramolecular polymers (HSPs) to achieve enantioselective trapping and separation of chiral molecules within microfluidic channels. Specific Objectives are: Synthesize a series of HSPs capable of selectively interacting with one enantiomer of a given chiral compound. To achieve this, the HSPs must meet two key criteria: a) They must possess specific interaction sites for chiral auxiliaries and b) They must be sufficiently rigid to maintain their helical structure upon interaction. 2) Develop two distinct microfluidic channel designs for enantiomer separation: a) An H-type microfluidic channel and b) A Y-type microfluidic channel. 3) Optimize the flow conditions for both the supramolecular polymers and the chiral molecules within the microfluidic devices to ensure efficient enantioseparation. 4) Apply the developed technique to the chiral separation of pharmaceutical drug molecules, specifically alprenolol and ethambutol. Most chiral drugs are synthesized as racemic mixtures in the laboratory, although typically only one enantiomer is pharmacologically active. Delivering drugs in enantiopure form can enhance therapeutic efficacy and minimize side effects. Furthermore, from a green chemistry perspective, enantioseparation via microfluidic systems offers a cost-effective and environmentally friendly alternative to traditional methods.
My MSCA postdoctoral tenure was a highly rewarding experience that allowed me to work across multiple disciplines including supramolecular polymers, flow chemistry, and chiral recognition and separation. During this time, I also acquired hands-on expertise in a range of analytical techniques such as chiral HPLC, SEM, and DLS. The project began with the fabrication of H-type and Y-type microfluidic channels for flow experiments (Figure 1A). I systematically explored various flow parameters within these channels and investigated the diffusion behavior of small dye molecules, such as Rhodamine B and dansyl chloride, under different flow rates (Figure 1B). COMSOL simulations were employed to model the flow profiles under laminar conditions, and experimental results confirmed the expected diffusion trends (Figure 1C).
Following this, I synthesized several benzenetricarboxamide (BTA)-based helical supramolecular polymers (HSPs) and studied their polymerization behavior and enantioselectivity toward chiral guest molecules. A significant challenge encountered during this phase was that BTA derivatives bearing terminal hydroxyl or amino groups failed to form well-defined HSPs, instead aggregating randomly due to strong interactions with the BTA core’s carboxamide groups. The first successful HSP-forming molecule was ethyl 3-(3,5-bis(((S)-2-octyl)carbamoyl)benzamido)benzoate (BTAOCE) (Figure 2). Its synthesis and polymerization was confirmed via circular dichroism (CD), UV-Vis spectroscopy, and DLS. Flow experiments using BTAOCE in both H- and Y-type channels showed significantly reduced diffusion compared to small molecules, indicating its potential for chiral separation. However, when chiral guest molecules were introduced into the BTAOCE solution in methylcyclohexane (MCH), the polymerization was disrupted. This was likely due to the interference of guest molecules with the intermolecular hydrogen bonding of the carboxamide groups.
To address this, I designed and synthesized three new BTA-based monomers with enhanced binding sites while minimizing interference with carboxamide hydrogen bonding: (3-((3,5-dimethylphenyl)carbamoyl)phenyl)-S,S-dioctyl BTA (BTAAMI), (3-((3-chloro-5-methylphenyl)carbamoyl)phenyl)-S,S-dioctyl BTA (BTAAMICl) (4-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)-S,S-dioctyl BTA (BTAPEG). All three monomers formed supramolecular polymers in non-polar MCH solvent. NMR studies showed that chiral guest molecules interacted with these polymers, and CD spectra confirmed that polymerization remained intact after guest addition. Subsequently, I tested the enantioseparation capabilities of these polymers with chiral amino alcohols, diamines, and diols using microfluidic channels, with the flow outputs analyzed by chiral HPLC. Unfortunately, no significant enantioseparation was observed. However, reduced diffusion was noted for certain chiral molecules, suggesting some interaction with the supramolecular polymers during flow, albeit non-enantioselective.
To introduce enantioselectivity, I next focused on incorporating a known chiral selector, β-cyclodextrin (β-CD), into the BTA framework to create a supramolecular polymer. β-CD is well-documented for its enantioselective encapsulation abilities and its incorporation also imparts water solubility, thereby broadening the range of compatible chiral substrates. Before proceeding with polymer synthesis, I conducted preliminary separation experiments using β-CD alone with mandelic acid and tryptophan as guest molecules. The binding affinities of β-CD for R- and S-mandelic acid were 811 M-1 and 178 M-1, respectively; for D- and L-tryptophan, 447 ± 83 M-1 and 88 ± 17 M-1. Flow experiments with β-CD at 100 µL/min in water yielded enantiomeric excess (ee) values of 3% for mandelic acid and 1.8% for tryptophan. Encouraged by these results, I am currently synthesizing a β-CD-incorporated BTA-based supramolecular polymer (BTACD), which is expected to yield significantly higher enantioseparation due to the lower diffusion coefficient of the polymer compared to β-CD alone.
Following this, I synthesized several benzenetricarboxamide (BTA)-based helical supramolecular polymers (HSPs) and studied their polymerization behavior and enantioselectivity toward chiral guest molecules. A significant challenge encountered during this phase was that BTA derivatives bearing terminal hydroxyl or amino groups failed to form well-defined HSPs, instead aggregating randomly due to strong interactions with the BTA core’s carboxamide groups. The first successful HSP-forming molecule was ethyl 3-(3,5-bis(((S)-2-octyl)carbamoyl)benzamido)benzoate (BTAOCE) (Figure 2). Its synthesis and polymerization was confirmed via circular dichroism (CD), UV-Vis spectroscopy, and DLS. Flow experiments using BTAOCE in both H- and Y-type channels showed significantly reduced diffusion compared to small molecules, indicating its potential for chiral separation. However, when chiral guest molecules were introduced into the BTAOCE solution in methylcyclohexane (MCH), the polymerization was disrupted. This was likely due to the interference of guest molecules with the intermolecular hydrogen bonding of the carboxamide groups.
To address this, I designed and synthesized three new BTA-based monomers with enhanced binding sites while minimizing interference with carboxamide hydrogen bonding: (3-((3,5-dimethylphenyl)carbamoyl)phenyl)-S,S-dioctyl BTA (BTAAMI), (3-((3-chloro-5-methylphenyl)carbamoyl)phenyl)-S,S-dioctyl BTA (BTAAMICl) (4-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)-S,S-dioctyl BTA (BTAPEG). All three monomers formed supramolecular polymers in non-polar MCH solvent. NMR studies showed that chiral guest molecules interacted with these polymers, and CD spectra confirmed that polymerization remained intact after guest addition. Subsequently, I tested the enantioseparation capabilities of these polymers with chiral amino alcohols, diamines, and diols using microfluidic channels, with the flow outputs analyzed by chiral HPLC. Unfortunately, no significant enantioseparation was observed. However, reduced diffusion was noted for certain chiral molecules, suggesting some interaction with the supramolecular polymers during flow, albeit non-enantioselective.
To introduce enantioselectivity, I next focused on incorporating a known chiral selector, β-cyclodextrin (β-CD), into the BTA framework to create a supramolecular polymer. β-CD is well-documented for its enantioselective encapsulation abilities and its incorporation also imparts water solubility, thereby broadening the range of compatible chiral substrates. Before proceeding with polymer synthesis, I conducted preliminary separation experiments using β-CD alone with mandelic acid and tryptophan as guest molecules. The binding affinities of β-CD for R- and S-mandelic acid were 811 M-1 and 178 M-1, respectively; for D- and L-tryptophan, 447 ± 83 M-1 and 88 ± 17 M-1. Flow experiments with β-CD at 100 µL/min in water yielded enantiomeric excess (ee) values of 3% for mandelic acid and 1.8% for tryptophan. Encouraged by these results, I am currently synthesizing a β-CD-incorporated BTA-based supramolecular polymer (BTACD), which is expected to yield significantly higher enantioseparation due to the lower diffusion coefficient of the polymer compared to β-CD alone.
In this project, I successfully developed a novel series of non-C₃ symmetric benzenetricarboxamide (BTA) supramolecular polymers featuring specific interaction sites for guest molecules. Their ability to form supramolecular polymers was thoroughly investigated using a range of spectroscopic techniques, including NMR, UV-Vis, circular dichroism (CD), and dynamic light scattering (DLS). These newly designed polymers not only offer promising features for chiral separation but may also find applications in other areas of supramolecular chemistry and materials science.
I further studied the diffusion behavior of these polymers within microfluidic channels under laminar flow conditions, systematically analyzing how the diffusion coefficients change with varying flow parameters. Importantly, I demonstrated that supramolecular interactions can influence the diffusion profiles of small molecules in flow, indicating a potential mechanism for selective separation. The feasibility of using microfluidics for chiral separation was validated through experiments involving β-cyclodextrin (β-CD) and chiral guest molecules. These results confirmed that the CRMS project holds promise for enantioselective separation. The final step underway involves the synthesis of a β-CD-incorporated supramolecular polymer and testing its separation performance with a range of chiral substrates in flow.
Beyond enantioseparation, this platform shows potential for regioisomer separation by replacing the β-CD moiety with alternative macrocyclic hosts such as cucurbiturils. Additionally, the diffusion-based separation approach could be extended to chiral nanotube resolution in the presence of surfactants like SDS, sodium cholate, or sodium deoxycholate. I recently presented this work as a poster at the Gordon Research Conference on Self-Assembly and Supramolecular Chemistry, where it received positive feedback and interest from the research community.
I further studied the diffusion behavior of these polymers within microfluidic channels under laminar flow conditions, systematically analyzing how the diffusion coefficients change with varying flow parameters. Importantly, I demonstrated that supramolecular interactions can influence the diffusion profiles of small molecules in flow, indicating a potential mechanism for selective separation. The feasibility of using microfluidics for chiral separation was validated through experiments involving β-cyclodextrin (β-CD) and chiral guest molecules. These results confirmed that the CRMS project holds promise for enantioselective separation. The final step underway involves the synthesis of a β-CD-incorporated supramolecular polymer and testing its separation performance with a range of chiral substrates in flow.
Beyond enantioseparation, this platform shows potential for regioisomer separation by replacing the β-CD moiety with alternative macrocyclic hosts such as cucurbiturils. Additionally, the diffusion-based separation approach could be extended to chiral nanotube resolution in the presence of surfactants like SDS, sodium cholate, or sodium deoxycholate. I recently presented this work as a poster at the Gordon Research Conference on Self-Assembly and Supramolecular Chemistry, where it received positive feedback and interest from the research community.