Introduction
Nature is rich in biologically active and structurally complex compounds that have intrigued organic chemists for centuries A significant challenge in this field arises from stereochemistry issues To obtain enantiomerically pure compounds, there are three primary approaches based on the type of starting material utilized.
Enzymes, known as biocatalysts, are highly effective in synthesizing enantiopure compounds due to their exceptional chemo-, regio-, and enantioselectivity They facilitate asymmetric synthesis from prochiral compounds, achieving yields of up to 100%, or perform kinetic resolution of racemates, with a maximum yield of 50%.
Lipases, specifically triacylglycerol acyl hydrolases (EC 3.1.1.3), are recognized as effective catalysts for creating enantiomerically enriched compounds due to their stability, wide substrate range, and ability to function without co-factors under mild reaction conditions Among these, lipase B from Candida antarctica (CAL-B) stands out as a particularly valuable biocatalyst for the kinetic resolution of sec-alcohols and similar compounds CAL-B is available as a patented recombinant protein from Novo-Nordisk.
Scheme 1 Methods to obtain enantiomerically pure compounds Enzymes are used in two pathways (i) asymmetric synthesis from prochiral substrates or (ii) KR of racemates crystallization
(bio) catalysis (bio) catalysis asymmetric synthesis
Prochiral compounds can be effectively synthesized using Novozym 435®, an immobilized lipase that demonstrates excellent stereoselectivity for a variety of non-natural substrates Notably, CAL-B maintains high activity and stability in non-aqueous environments, making it suitable for esterification and transesterification reactions, alongside conventional hydrolysis processes.
The application of biotransformation in organic synthesis has expanded with the incorporation of enzymes in organic solvents and eco-friendly nonaqueous media, including ionic liquids and supercritical fluids Supercritical carbon dioxide (scCO2) has gained attention as a nonflammable, nontoxic, and abundant solvent for enzymatic reactions due to its advantageous properties such as low viscosity and ease of product recovery While carbon dioxide can also function as a liquid solvent below its critical temperature, there is a lack of research on its practical use with biocatalysts The distinct physical properties of liquid CO2 compared to its supercritical form suggest differing enzymatic behaviors, and the ability to maintain liquid CO2 at relatively low pressures can reduce equipment costs Additionally, using liquid CO2 at lower temperatures may enhance enantioselectivity in reactions.
This study pioneers the use of liquid CO2 as an alternative solvent for biotransformation, specifically investigating its feasibility in a batch reactor and two continuous-flow reactors through the kinetic resolution of sec-alcohols catalyzed by Candida antarctica lipase B Remarkably, the use of liquid CO2 expanded the substrate scope of the lipase to include bulky phenyl alkyl sec-alcohols The structure of the thesis is outlined in Scheme 2.
Scheme 2 Outline of this study
Liquid CO2 as a superior solvent for CAL-B catalyzed kinetic resolution of sec-alcohols
Kinetic resolution of rac-1-phenylethanol
CO 2 and organic solvents using a batch reactor
Liquid CO 2 in flow reactors for large-scale biosynthesis
Comparison of productivity between reactors
Candia antarctica lipase B catalyzed kinetic resolution of rac-1-phenylethanol in
CAL-B catalyzed KR of 1-phenylethanol in organic solvents and in liquid CO 2
The kinetic resolution (KR) of rac-1-phenylethanol catalyzed by CAL-B was conducted in liquid CO2 and various conventional organic solvents using a batch reactor Vinyl acetate served as the acylating agent for irreversible transesterification, as the resulting vinyl alcohol tautomerizes irreversibly to acetaldehyde To ensure accuracy, all reactions were carried out in identical reactors and vigorously stirred with a magnetic bar to minimize mass transfer effects.
Scheme 3 KR of rac-1-phenylethanol by CAL-B
The activity of lipase is closely linked to the hydrophobicity of solvents, indicated by their log P values, with more hydrophobic solvents typically yielding higher results Notably, CAL-B demonstrated the highest transesterification activity and exceptional enantioselectivity (ee p>99%) in liquid CO2, outperforming hexane and toluene.
Figure 1 Effect of solvents on CAL-B catalyzed KR of 1-phenylethanol Reaction conditions:
Substrate 0.83 mmol, vinyl acetate 5.4 mmol, Novozym 435 ® 5 mg, solvent 10 ml, 20 o C, 2 h, pressure for liquid CO2 6.5 MPa Enantiomeric excess of the (R)-acetate was found to be excellent (ee p > 99%) in all media a No reaction occurred due to insufficient mixing of the enzyme and substrate since the density of the solvent greatly exceeds that of the immobilized enzyme N.a.: not available Dielectric constants of selected solvents: liquid CO2 (20 o C, 6 MPa) 1.48, hexane 1.88, isooctane 1.94, toluene 2.38, i-Pr2O 3.88, CHCl3 4.81, THF 7.58
Liquid CO2 exhibits behavior similar to hydrocarbon solvents with low polarizability, although its log P value remains unreported To compare polarity, the dielectric constants of selected solvents were utilized The high activity of lipase in liquid CO2 and in solvents with elevated log P values can be attributed to the inability of hydrophobic solvents to remove essential water molecules from the enzyme's outer layers Despite lipase being sensitive to water and often prepared in a dry form, it requires residual water to maintain catalytic activity Additionally, the enhanced activity of lipase in liquid CO2 may be associated with the pre-opening of the enzyme's lid and its interfacial activation in hydrophobic environments.
The kinetics of the kinetic resolution (KR) of rac-1-phenylethanol by CAL-B were evaluated in various medium systems, including hexane, liquid CO2, supercritical CO2 (scCO2), and a solvent-free environment at 20°C Hexane emerged as the optimal organic solvent for CAL-B-catalyzed KR Notably, the lipase exhibited enhanced activity in liquid CO2, achieving a 50% conversion after 12 hours, indicating its stability and effectiveness under high pressure Furthermore, the enzyme demonstrated strong performance in a solvent-free system, yielding approximately 30% and 35% after 6 hours, respectively, compared to hexane.
Figure 2 The time courses of the KR of 1-phenylethanol in different nonaqueous media
Reaction conditions: Substrate 0.83 mmol, vinyl acetate 5.4 mmol, Novozym 435 ® 5 mg, with or without 10 ml solvent, 20 o C, pressure for liquid CO2 6.5 MPa Enantiomeric excess of the
(R)-acetate was excellent (ee p > 99%) in all cases.
The impact of temperature on CAL-B catalyzed kinetic resolution of rac-1-phenylethanol was examined across various media, including hexane, liquid CO2, supercritical CO2 (scCO2), and a solvent-free system Results indicated that the reaction rate consistently increased with rising temperatures, achieving excellent enantioselectivity (ee p > 99%) Notably, transesterification in scCO2 at 40°C and 10 MPa demonstrated activity comparable to that in hexane, with approximately 35% conversion after 2 hours However, direct comparison of lipase activity between liquid CO2 and scCO2 was not feasible due to temperature differences.
The effect of temperature on the CAL-B catalyzed kinetic resolution (KR) of 1-phenylethanol was investigated under specific reaction conditions, including a substrate concentration of 0.83 mmol, vinyl acetate at 5.4 mmol, and 5 mg of Novozym 435® The reactions were conducted at 20°C for 2 hours, with a liquid CO2 pressure of 6.5 MPa, and included variations with or without 10 ml of solvent The study focused on measuring the enantiomeric excess achieved under these conditions.
(R)-acetate was excellent (ee p > 99%) in all cases.
Pressure has minimal impact on the reaction rate of lipase-catalyzed kinetic resolution of rac-1-phenylethanol, with enantioselectivity remaining exceptionally high (all ee p >99%) Csajagi et al also found that varying pressure from 0.1 to 12 MPa did not significantly affect the reaction outcomes.
In liquid CO₂ Solvent free
In scCO₂ the productivity or enantioselectivity of the CAL-B when it was used for KR of rac-1- phenylpropan-2-ol in a mixture of organic solvents (hexane-THF-vinyl acetate) 17
The effect of pressure on the CAL-B catalyzed kinetic resolution (KR) of 1-phenylethanol was investigated under specific reaction conditions: using 0.83 mmol of substrate, 5.4 mmol of vinyl acetate, 5 mg of Novozym 435®, and 10 ml of solvent at 20°C for 2 hours The results showed an excellent enantiomeric excess of the (R)-acetate, exceeding 99% in all cases.
The use of liquid CO 2 in continuous flow reactors for large-scale biosynthesis of
Continuous flow reactors offer significant advantages over batch processes, such as increased productivity, reduced operating costs, and enhanced safety These reactors can be smaller than their batch counterparts while producing comparable amounts of product Operating costs for batch processes tend to be higher due to the frequent need to empty and refill the reactors The two most commonly used types of continuous flow reactors are the ideal continuous stirred-tank reactor (CSTR), which ensures complete mixing of the reacting stream.
In hexane In liquid CO₂
In an ideal continuous packed-bed reactor (CPBR), the substrate stream flows uniformly at a constant velocity along the reactor axis, eliminating any back-mixing The reactor operates at a pressure of 12 MPa, ensuring that the product stream remains consistent with the liquid phase throughout the reactor and stable over time.
Selecting the appropriate reactor for specific applications relies on the physical states of substrates, enzymes, and products, as well as economic considerations The Continuous Packed Bed Reactor (CPBR) optimally utilizes immobilized enzymes, whereas the Continuous Stirred Tank Reactor (CSTR) is favored when pH control is critical or when the substrate stream's physical characteristics do not align with CPBR functionality.
Liquid CO2 is an ideal candidate for continuous flow processes due to its fluid nature, yet there have been no documented uses of liquid CO2 in biocatalyzed reactions within such systems This study explores the lipase-catalyzed kinetic resolution of rac-1-phenylethanol utilizing a continuous flow of liquid CO2 in both a stirred-tank reactor and a packed-bed reactor, aiming to assess the compatibility of liquid CO2 for continuous biocatalysis.
2.2.1 KR of 1-phenylethanol in a continuous-flow stirred-tank reactor (CSTR) using liquid CO 2
An ideal Continuous Stirred-Tank Reactor (CSTR) achieves complete back-mixing, which minimizes substrate concentration and maximizes product concentration Its well-mixed design allows for easy control of temperature and pH, as well as the efficient management of gas supply or removal Consequently, CSTRs are the preferred choice for processes that involve substrate inhibition or product activation.
The experimental setup, illustrated in Figure 5, involved sealing 0.2 g of immobilized CAL-B in a pressure-resistant stainless steel vessel with a capacity of 10 mL, which was fitted with a magnetic bar Temperature regulation was achieved using a water bath integrated with a circulation chiller.
CO2 was introduced into the vessel using a CO2 pump until the target pressure was reached, regulated by a back pressure regulator Simultaneously, the substrate solution was continuously pumped with an HPLC pump while the reaction mixture was stirred vigorously with a magnetic bar The outflow from the reactor was collected and analyzed by GC to assess the enantiomeric excess of both the substrate (ee s) and the product (ee p).
Figure 5 Experimental apparatus of a CSTR using Novozym 435 ®
The investigation began with a CO2 flow rate of 1.5 mL/min and a substrate flow rate of 0.02 mL/min, achieving a volume ratio of vinyl acetate to 1-phenylethanol of 4:1 and a residence time of 6.7 minutes Analysis of the outflow revealed that the continuous-flow reaction reached a stationary state within 30 minutes, resulting in a 39% conversion to (R)-acetate with an enantiomeric excess (ee) greater than 99%, and (S)-alcohol with an ee of 56% These findings indicate that liquid CO2 is highly effective for continuous kinetic resolution (KR) using lipase.
Figure 6 The time course of KR of rac-1-phenylethanol in a CSTR filled with Novozym
435 ® Experimental points are shown at the time after pumping the substrate mixture
Reaction conditions: immobilized enzyme 0.2 g, CO2 flow rate 1.5 mL/min (residence time 6.7 min), substrate flow rate 0.02 mL/min (4 volume ratio of vinyl acetate to 1- phenylethanol), 20 o C, 7 MPa
The study examined how the conversion rate of 1-phenylethanol in a Continuous Stirred-Tank Reactor (CSTR) is influenced by residence time With a low substrate flow rate of 0.02 mL/min in comparison to the CO2 flow rate, the residence time in the system could be adjusted by altering the flow rates.
The study examined the impact of varying CO2 flow rates on reaction rates while maintaining a constant substrate flow rate CO2 flow rates were set at 0.5, 1, 1.5, 2, and 3 mL/min, leading to residence times of 20, 10, 6.7, 5, and 3.3 minutes, respectively Results indicated that the reaction rate was significantly influenced by residence time, with the highest conversion rate of 29% occurring at a residence time of 6.7 minutes Longer residence times enhanced conversions due to increased contact time between the substrate and the enzyme However, excessively low CO2 flow rates resulted in decreased conversions, likely due to inadequate solubilization of the substrate in liquid CO2.
Figure 7 Effect of residence time on the KR of rac-1-phenylethanol in a CSTR filled with
Novozym 435 ® Reaction conditions: immobilized enzyme 0.2 g, substrate flow rate 0.02 mL/min (0.5 volume ratio of vinyl acetate to 1-phenylethanol), 20 o C, 7 MPa Enantiomeric excess of the (R)-acetate was excellent (ee p > 99%) in all cases
2.2.2 KR of 1-phenylethanol in a continuous packed – bed reactor (CPBR) using liquid
An ideal Continuous Packed Bed Reactor (CPBR) operates without back-mixing, allowing enzymes to interact with a decreasing substrate concentration gradient and an increasing product concentration gradient These reactors are particularly effective for processes characterized by product inhibition, substrate activation, and reaction reversibility Additionally, an ideal CPBR of appropriate length can achieve any desired level of reaction efficiency.
To optimize the KR reaction using liquid CO2 in a CPBR, an experimental setup was established utilizing Novozym 435 ® The immobilized CAL-B (1.4 g) was contained within a pressure-resistant stainless steel column measuring 3.94 mL and 28.5 cm in length A CO2 pump introduced gas into the vessel, achieving the desired pressure, which was regulated by a back pressure regulator, followed by the pumping of the substrate solution.
The residence time, measured in minutes, is continuously regulated by an HPLC pump as the solution flows into a turbulent mixer, which consists of a 0.5 mL tube filled with cotton After mixing, the outflow is collected at the reactor's exit for analysis.
GC to determine ee of substrate (ee s) and product (ee p)
Figure 8 Experimental apparatus of a CPBR using Novozym 435 ®
The system operated for three cycles, each lasting 24 hours, successfully producing enantiopure acetate and alcohol products In line with green chemistry principles aimed at waste reduction, Sheldon's E-factor of 20 serves as a key metric for evaluating the environmental impact of manufacturing processes An ideal E-factor is zero, indicating no waste generation; however, many chemical processes, particularly in fine chemicals and pharmaceuticals, often exceed an E-factor of 25 In contrast, our system achieved a remarkably low E-factor of less than 0.3, effectively minimizing waste without the use of organic solvents and utilizing an adequate amount of vinyl acetate.
Table 1 Novozym 435 ® catalyzed KR of rac-1-phenylethanol with continuous flow of liquid
In a packed-column reactor, the reaction conditions involved using 1.6 g of immobilized enzyme with a CO2 flow rate of 1.0 mL min⁻¹ and a substrate flow rate of 0.02 mL min⁻¹, maintaining a volume ratio of rac-1-phenylethanol to vinyl acetate at 2:1, at a temperature of 20°C and a pressure of 6.5 MPa The process consisted of four steps: pressurization and stabilization for 1 hour, sampling for 8 hours, washing with liquid CO2 for 1 hour, and depressurization to 0.1 MPa with a 14-hour storage at ambient conditions The influx was monitored using a precision balance to measure the substrate mixture over time, with the recovered outflow collected at a stationary state for 8 hours Enantiomeric excess (ee) of the substrate (alcohol) and product (acetate) was determined through chiral GC analysis, while conversion (c) was calculated from ee values and verified by independent calculations using 1H NMR spectrum ratios Enantiomer selectivity (E-value) was derived from the ee values, and the E-factor was calculated as the kg of waste (acetaldehyde and excess vinyl acetate) divided by kg of products (enantiopure acetate and alcohol), excluding recyclable factors like CO2 and reused immobilized enzyme.
Comparison of the productivity of continuous-flow and batch reactors using liquid
To compare the productivity of continuous-flow and batch reactors, the specific reaction rate (r) is utilized, which measures the product formed per minute by 1 g of enzyme For continuous-flow systems, the specific reaction rate (r flow) can be determined using the product concentration ([P] in mmol mL⁻¹), the flow rate (f in mL min⁻¹), and the mass of the applied enzyme (m e in g), as outlined in Equation 1.
Cycle No b In-flux rate c
(mg/min) Recovered d (g) ee s e (%) ee p e (%) c f (%) E-value g E- factor h
A stirred batch reaction is defined by its specific reaction rate (r batch), which is determined using the amount of product generated (n p in mmol), the duration of the reaction (t in minutes), and the mass of the enzyme used (m e in grams), as outlined in Equation 2.
To ensure accurate comparisons between the productivity of continuous-flow reactions and batch mode reactions, it is essential to calculate the specific reaction rates (r values) at the same conversion levels (c).
The productivity of reactions can be assessed using the space-time yield (Y), which measures the product amount generated per unit time per reactor volume This metric is crucial when reactor installation costs exceed enzyme expenses For continuous-flow systems, the space-time yield (Y flow) is determined by the product concentration ([P] in mmol mL -1), flow rate (f in mL min -1), and reactor volume (V r in mL), as outlined in Equation 3.
A space-time yield value of a batch reactor (Y batch) can be calculated from the amount of the product (n p (mmol)), the reaction time (t (min)), and the reactor volume (V r (mL)) according to
Table 2 presents the productivity metrics, including specific reaction rate (r) and space-time yield (Y), for Continuous Stirred Tank Reactors (CSTR), Continuous Packed Bed Reactors (CPBR), and Batch Stirred Tank Reactors (BSTR) utilizing immobilized lipase and liquid CO2 as the reaction medium for the kinetic resolution of 1-phenylethanol.
The BSTR demonstrated the ability to achieve high specific reaction rates and varying degrees of conversion, although it exhibited low space-time yields In contrast, the CSTR showed that lowering substrate concentration could enhance conversion rates, but this adjustment would lead to a decrease in overall productivity.
The CPBR achieved an impressive 50% conversion rate on its first attempt, generating 36 μmol of product per minute per gram of enzyme, while the CSTR only managed to increase its conversion from 29% to 38% despite a significant concentration reduction of 3.7 times This increase in the CSTR was accompanied by a nearly threefold drop in productivity for both reaction rate and yield In contrast, the CPBR demonstrated a remarkable yield value of 12.5 μmol min⁻¹ mL⁻¹, significantly outperforming other reactor types.
Table 2 Comparison of the productivity of continuous-flow and batch reactions a
Enzyme loading (mg) (Reaction type)
Residence time (min) c b (%) r c (mol min -1 g -1 )
In a flow reaction conducted at a stationary state with a liquid CO2 flow rate of 1.5 mL min^-1 and a substrate mixture of 1-phenylethanol and vinyl acetate at 20°C and 7 MPa, samples were taken 60 minutes after initiation The conversion rate was assessed using gas chromatography (GC), revealing an impressive enantiomeric excess of (R)-acetate exceeding 99% across all trials The specific reaction rate for the flow system was calculated using Equation 1, while the batch system's rate was determined via Equation 2 Additionally, the space-time yield for the flow system was computed according to Equation 3, with Equation 4 applied for the batch system.
The batch system demonstrated significantly higher specific reaction rates—two to four times greater than continuous modes—across all conversion degrees This advantage is likely due to the inefficient mixing in continuous reactors, which increases diffusional resistance For optimal mixing in a Continuous Stirred Tank Reactor (CSTR), the volume of immobilized enzyme should ideally be ten times less than the reactor's volume, which can restrict the CSTR's capacity when reactor size is limited Although the specific reaction rate in the Continuous Packed Bed Reactor (CPBR) is lower, it shows promise for higher transformation rates with increased substrate loading Thus, further operational optimization could enhance its productivity.
Expanding substrate scope of Candida antarctica lipase B using liquid carbon
Substrate specificity of CAL-B catalyzed transesterification of alcohols
To explore the impact of liquid CO2 on CAL-B's activity, transesterification reactions were performed using a range of aromatic, aliphatic, and allylic primary and secondary alcohols, comparing the outcomes in both liquid CO2 and hexane (Table 3).
Lipase exhibits remarkable activity towards primary and secondary alcohols, particularly those with a methyl substituent at the hydroxymethine center, demonstrating excellent enantioselectivity in both liquid CO2 and hexane In its catalytically active conformation, the alcohol's large substituent positions itself at the entrance of CAL-B's active site, while the medium substituent is oriented into the stereoselectivity pocket However, substituting the methyl group with an allylic or ethyl group results in a significant decrease in activity, although enantioselectivity remains high (ee p >99%) in both solvents Notably, the enzyme facilitates the transesterification of 1-phenylpropanol more efficiently in liquid CO2, achieving a 25% conversion compared to just 4% in hexane.
The lipase exhibited a significant decrease in both activity and selectivity when reacting with the unique substrate 1-phenyl-2-propanol (4a), in contrast to other methyl alkyl sec-alcohols (3a-5a) This reduction can be attributed to the unfavorable spatial orientation of the benzyl group at the enzyme's active site Similar findings were reported in another study, where Novozym 435® showed diminished activity and selectivity for 1-phenylbut-3-en-2-ol compared to 1-phenylprop-2-en-1-ol Furthermore, the reaction for resolving 1-phenyl-2-propanol was notably faster in liquid CO2 than in hexane, achieving higher selectivity at lower temperatures.
Expanding substrate scope of CAL-B using liquid CO 2
From Table 3, a remarkably acceleration effect of liquid CO2 on the lipase catalyzed the transesterification could be observed with 1-phenyl-1-propanol (8a), but not with other
Table 3 Novozym 435 ® catalyzed transesterification of various alcohols in hexane or liquid
Liquid CO2 25 >99 (R) >200 a Reaction conditions: alcohol (0.40 mmol) with vinyl acetate (2.2 mmol) and Novozym 435 ®
The study involved using 10 mg of a substance dissolved in 10 ml of hexane or liquid CO2 at a pressure of 6.5 MPa and a temperature of 20°C The conversions and enantiomeric excess (ee) values reported are averages from a minimum of three reaction runs, analyzed via gas chromatography as outlined in Section 5.7 Additionally, Novozym 435® was utilized at a concentration of 5 mg, with the reaction conducted at a temperature of 5°C.
N.d.: not determined due to low conversion observed substrates containing smaller medium substituents, when it is compared with that of hexane Therefore, it is questioned that whether this effect could be extended to other substrates having bulky side chains, which are the limitations of this robust enzyme To answer this question, the transesterification of a series of phenyl sec-alcohols containing different size chains by CAL-B were examined in liquid CO2 medium and hexane as a control (Table 4)
The enzyme demonstrated remarkable activity for bulky 1-phenylalkanols (9-15a) in liquid CO2, while showing minimal activity in hexane, where only α-cyclopropylbenzyl alcohol (14a) was converted Notably, the lipase's performance appeared unaffected by the alkyl chain length, yielding consistently high results despite variations in alkyl substituents; however, (R)-selectivity significantly declined for substrates 9-13a Previous research by Hult et al sought to enhance the steroselectivity pocket of the robust wild-type CAL-B through the W104A mutation, which allowed activity for bulky alcohols like 12a, favoring (S)-enantiomers Nonetheless, this (S)-selective variant exhibited low enantioselectivity for 3b, suggesting that the modified variant's cavity can accommodate the unsubstituted phenyl group, leading to a shift in enantiopreference.
The bulky phenyl alkyl substrates exhibited significant differences in reactivity between hexane and liquid CO2, with a preference for all (R)-enantiomers This suggests that the alcohols position the phenyl group towards the enzyme's active site entrance while directing the alkyl substituent into the stereoselective pocket of the lipase Notably, the lipase maintained consistently high conversion rates, irrespective of the alkyl chain length This phenomenon may be attributed to increased enzyme flexibility in liquid CO2, allowing for better accommodation of longer alkyl substrates Consequently, this flexibility could explain the unexpected (S)-selectivity and a marked decrease in substrate specificity Molecular dynamics simulations revealed that CAL-B displayed more flexible residues and reduced compactness when exposed to liquid CO2.
Table 4 Novozym 435 ® catalyzed KR of bulky phenyl alkyl sec-alcohols in liquid CO2 a
Liquid CO2 10 98 127 a Reaction conditions: alcohol (0.2 mmol) with vinyl acetate (1.1 mmol) and Novozym 435 ®
The study involved dissolving 100 mg of the compound in 10 ml of hexane or liquid CO2 at a pressure of 6.5 MPa and a temperature of 20°C Conversions and enantiomeric excess (ee) values were assessed through gas chromatography (GC) analysis The absolute configurations were established by comparing the optical rotation values to existing literature data Novozym 435® was used in a quantity of 20 mg, and the molar ratios were calculated based on GC results.
N.d.: not determined due to low conversion observed in CO2 medium 26 The study also pointed out that the enzyme exposed more hydrophobic surface and decreased the hydrophobic intra-molecular interactions where there are more than half of buried atoms of dissolved enzyme in water come to surface in supercritical CO2
The reaction of 10a in hexane at high pressure demonstrated a negative impact on the substrate scope expansion of CAL-B, a trend also noted in supercritical CO2 (scCO2) To further understand this acceleration effect, the lipase underwent pre-treatment in a liquid medium.
At 20°C and 7 MPa, CO2 was utilized for 3 hours prior to the resolution of 10a The lipase exhibited no residual activity in hexane; however, it maintained its activity and enantioselectivity in liquid CO2, achieving a 32% conversion rate and an E-value of 8, comparable to the untreated lipase This indicates that the observed phenomenon is not associated with an irreversible conformational change of the enzyme caused by the liquid CO2 medium.
Figure 10 Effect of pressure, liquid CO2 pretreatment, and scCO2 on the KR of 10a
Reaction conditions: substrate (10a) 0.2 mmol, vinyl acetate 1 mmol, Novozym 435 ® 100 mg,
10 ml solvent, 120 h Enantiomeric ratio (E-value) was given in numbers *Residual activity after 3 h pretreatment with liquid CO2 at 20 o C, 7 MPa.
20ᵒC, 7 MPa 20ᵒC, 7 MPa* 40ᵒC,12 MPa
Conclusion and perspective
This thesis highlights the advantages of using liquid CO2 as a solvent for biotransformations, showcasing its superior enzyme activity compared to traditional organic solvents like hexane, particularly for reactions involving bulky substrates Furthermore, the practical applications of liquid CO2 were explored through two types of continuous flow bioreactors Notably, a large-scale kinetic resolution of the model secondary alcohol rac-3a, facilitated by immobilized lipase in a continuous packed-column reactor, successfully produced enantiopure products while minimizing waste.
Liquid CO2, a byproduct of fossil fuel combustion, serves as an efficient solvent for biocatalysis in the synthesis of optically active compounds This innovative approach highlights the advantages of using a novel, non-flammable solvent in chemical processes.
CO2 can be employed with lower pressure and temperature than supercritical CO2
Future research will focus on enhancing enantioselectivity through low-temperature control and investigating the mechanisms by which CO2 accelerates enzymatic reactions This study aims to unlock new opportunities for exploring the benefits and potential industrial applications of CO2 as an alternative solvent.