In Situ Separation of the Chiral Target Compound (S)-2-Pentanol in

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In situ Separation of Chiral Target Compound (S)-2-Pentanol in Biocatalytic Reactive Distillation Steffen Kuehn, Gerrit Sluyter, Marc-Andreas Christlieb, Rene Heils, Anne Stoebener, Joscha Kleber, Irina Smirnova, and Andreas Liese Ind. Eng. Chem. Res., Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 20, 2017

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Industrial & Engineering Chemistry Research

In situ Separation of Chiral Target Compound (S)-2-Pentanol in Biocatalytic Reactive Distillation Steffen Kühna, Gerrit Sluytera, Marc-Andreas Christlieba, René Heilsb, Anne Stöbenera, Joscha Klebera, Irina Smirnovab, Andreas Liesea,*

a

Institute of Technical Biocatalysis, Hamburg University of Technology, Denickestr. 15, D-

21073 Hamburg, Germany b

Institute of Thermal Separation Processes, Hamburg University of Technology, Eissendorfer

Str. 38, D-21073 Hamburg, Germany

*e-mail: [email protected]; Fax: +49-40-42878-2127; Tel: +49-40-42878-3018

Keywords: Reactive distillation, kinetic resolution, integrative processes, in situ separation, lipases

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Abstract In situ separation of a chiral target compound is realized for the first time as distillate on top of an integrated biocatalytic batch reactive distillation column. The applied reaction system is the racemic resolution of (R/S)-2-pentanol in a transesterification with propyl butyrate, which is catalyzed by immobilized Candida antarctica lipase B (Novozym435®). Biocatalyst integration is realized in baskets of wire gauze catalytic packings as column internals. The equilibrium limited reaction is shifted to the product side by fractional distillation of low boiling propanol and the target compound (S)-2-pentanol. Increased molar fractions up to x(S)-2-PeOH = 65 ± 4% with an enantiomeric excess of 90 ± 4 % and 51 ± 3 % overall conversion are reached for (S)-2-pentanol. Feasibility of the reaction system is investigated in our preselection tool. This tool is developed to evaluate predefined operation conditions in the column setup for transesterification reactions based on criteria regarding biocatalyst stability and boiling point differences between the reactants. The subsequently selected reaction of (R/S)-2-pentanol with propyl butyrate is successfully carried out in a batch reactive distillation column with in situ separation of our target compound (S)-2-pentanol. In contrast to current examples in literature, this clearly demonstrates the possibility of one-pot target compound isolation within chiral synthesis applying biocatalytic reactive distillation.


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1 Introduction In reactive distillation, a chemical reaction is combined with thermal separation in one unit operation as an integrated process. More than 150 industrial applications are known for this well-established intensified process to achieve cost efficient product formation1. Main advantages are the reduction in the number of unit operations, to overcome existing azeotropes during reaction and a shift of the chemical equilibrium towards the product side1. The focus in industry is to synthesize bulk chemicals via esterification, hydrolysis and etherification reactions, which are up today catalyzed by chemical catalysts e.g. ion exchange resins2,3. Integrated chemical catalysts reveal a broad range in process conditions regarding column pressure (p = 0.1-30 bar) and column temperature (T = 27-296°C)2. In contrast to the production of bulk chemicals, the performance of asymmetric synthesis in a reactive distillation setup could allow formation of enantiomerically pure fine chemicals in future times. The specific feature of asymmetric synthesis is the selective production of an enantiomer in excess to existing stereoisomers of the chiral molecule. Especially for the preparation of pharmaceuticals, chiral precursors and chiral products are one of the key issues in process engineering4. A study by Okasinski et al. demonstrates the potential of reactive distillation for the synthesis of optically pure propylene glycol and propylene oxide catalyzed by Co(II)-Salen complex in a simulation based approach5. Nevertheless, chemical catalysts are often limited regarding stereo- or enantioselective synthesis. An alternative is the application of biocatalysts, which are able to differentiate between enantiomers in their active site yielding optically pure compounds. Known challenging aspects about biocatalysts are thermal deactivation even at moderate temperatures and high prices for their preparation. However, several industrial examples of biocatalysts with increased thermal stability are published mainly focusing on the class of hydrolases. The applications are ranging from lipase catalyzed esterification processes at 60°C to the production of high fructose corn syrup by α-Amylases at 105-115°C4,6,7. Additionally, lipases are characterized to be tolerant towards organic solvents8. Further increase in thermal stability can be induced by immobilization of the biocatalysts. Recently, Poojari et al investigated Lipase B from Candida antarctica (CalB) immobilized on polyacrylic resin (Novozym435® manufactured by Novozymes in Bagsvaerd, Denmark) and the data revealed no significant loss in residual activity at 80°C after 30 days of operation9. Beside thermal stability, integration of reaction and separation requires careful selection of process parameters (e.g. temperature and pressure) since both processes are taking place simultaneously. ACS Paragon Plus Environment

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In different approaches immobilized lipases were already used as catalysts in integrated processes like reactive distillation. As model reaction in several studies the synthesis of butyl butyrate (BuBu) starting from ethyl butyrate (EtBu) and n-butanol is applied. Initial work on BuBu formation by Paiva et al (2003) demonstrates the applicability of lipase from Mucor miehei, immobilized on anion-exchange resin, in a batch separation column (h = 230 mm, d = 30 mm). The lipase is placed in 13 inverted pear bulbs over the length of the separation column under constant removal of the low boiling ethanol10. Further published investigations of Heils et al (2012) on the same reaction system presented an alternative integration of CalB by entrapment in a silica-gel coating applied on wire gauze packings in a batch reactive distillation setup (h = 960 mm, d = 45 mm). The column setup shows increased conversion by in situ product removal of low boiling ethanol (EtOH) in comparison to the same reaction in a stirred tank reactor (STR)11. Additional work on the formation of BuBu illustrates the theoretical feasibility in a continuous setup based on simulations in Aspen Custom Modeler with conversions up to 90% for n-butanol (simulated process conditions: T = 343 K, p = 0.2 bar)12. In the given examples, BuBu formation in distillation setups is successfully performed by implementation of immobilized CalB. In our work, the same biocatalyst is applied due to its high thermal stability and approved feasibility studies for various biocatalyst preparations. As the implementation of chiral synthesis was not addressed by forming BuBu, Au-Yeung et al (2013) described the potential of a continuous horizontal distillation apparatus for multicomponent chiral resolution including subsequent separation of produced enantiomers. The setup consists of multiple external side-reactors filled with immobilized lipase for generating increased residence times in the liquid phase and providing decreased pressure drop in the horizontal separation part13. Asymmetric synthesis in a fully integrated batch reactive distillation setup is introduced recently by Heils et al. (2015) for the racemic resolution of (R/S)-2-pentanol ((R/S)-2-PeOH) with ethyl butyrate (EtBu) catalyzed by CalB immobilized on a hydrophobic silica coating14. Predominantly, CalB catalyzes the reaction of (R)-2-PeOH with EtBu while (S)-2-PeOH is not converted by the biocatalyst and stays in the column setup as target compound of the asymmetric synthesis. This system achieves high enantiomeric excess of the target molecule accumulating in the bottom of the system (ee(S)-2-PeOH > 99%) at 69% conversion related to (R/S)-2-PeOH while removing low boiling EtOH at the top of the column under process conditions (T = 30-60°C, p = 60115 mbar)14. The technology even allows in situ coating to renew biocatalytic activity in the process after deactivation15. More recently, Wierschem et al (2016) published a simulative study ACS Paragon Plus Environment

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involving model based process analysis for the CalB catalyzed transesterification of (R/S)-1phenyl ethanol and isopropenyl acetate in a continuous column setup. Low boiling acetone and residual isopropenyl acetate are stripped at the top of the column in their model. Additional simulated integration of a zeolite-catalyzed racemization step into the reactive section revealed high productivity in the performed simulation16. The results in all given studies clearly demonstrate feasibility of biocatalysts with reasonable thermal stability (especially lipases) in reactive distillation setups. However, the target compounds are accumulated in the bottom of the column setups due to increased boiling points of the target compound in comparison to the starting materials. In classical chemically catalyzed reactive distillation processes, in situ product separation preferably takes place at the top of the column to minimize the number of downstream-processing steps. Especially for batch reactive distillation processes, in situ separation at the column top is necessary to prevent impurities with higher boiling compounds in the bottom. In this work, we wanted to investigate the potential of different substrate combinations for their application in biocatalytic batch reactive distillation (RD) aiming at in situ product separation and isolation of a chiral target compound at the top of the batch RD column. In current literature on biocatalytic reactive distillation, the in situ isolation of the target compound is not addressed so far. However, this will be one of the main targets to allow industrial application and illustrate the advantage of the integrated process approach. For the selection of a feasible substrate combination comprising the feature of in situ target compound isolation, a criteria-based theoretical tool is developed to enable substrate availability and separation. Therefore, predefinition of the boiling points and boiling point differences between the reactants as well as the applicability of the biocatalyst under process conditions are considered. 120 different CalB catalyzed transesterification reactions are implemented including the boiling points of the reactants at varied operating pressures. Operating at low pressures in RD decreases the boiling temperatures of the reactants and an operation at moderate temperatures for improved biocatalytic conditions can be guaranteed. This procedure allows a fast evaluation for substrate availability and separation based on the boiling points of the corresponding starting materials, because especially for chiral molecules availability of vapor-liquid equilibria data are limited. Moreover, the effect of a changed boiling point order by substituted ester compounds is addressed in the preselection tool as well as experimentally by the comparison of small scale flash distillation under vacuum (FD) and RD performance. Based on fulfilled criteria within the ACS Paragon Plus Environment

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preselection tool, CalB catalyzed transesterification of propyl butyrate (PrBu) with (R/S)-2-PeOH is selected for in situ product separation of a chiral target compound at the top of the RD column. The necessity of stepwise temperature controlled reflux with changing boundary conditions is approved in experiments by the concentration profiles at different RD column heights. Biocatalyst integration is realized by placing CalB immobilized on polyacrylic resin (available as Novozym435®) in baskets of wire gauze elements to create catalytic packings.


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2. Materials and Methods 2.1 Chemicals (R/S)-2-pentanol (≥ 98%), ethyl butyrate (≥ 98%), propyl butyrate (≥ 98%), (R/S)-2-pentyl butyrate (≥ 98%), tetramethyl orthosilicate (98%), trimethoxymethyl silane (98%), polyethylene glycol (Mn = 400 g·mol-1) and p-nitrophenyl acetate (97%) were purchased from Sigma Aldrich; methanol (≥ 99.9%), ethanol (≥ 99.8%), acetonitrile (≥ 99.9%), ethyl acetate (≥ 99.5%), isoamyl alcohol (≥ 98%), Roti® Nanoquant solution, potassium dihydrogen phosphate (≥ 99%) and dipotassium hydrogen phosphate (≥ 99%) from Roth and sodium fluoride from Prolabo (East Grinstead, United Kingdom). All chemicals were used for experimental investigation without further purification. 2.2 Biocatalyst Immobilized Candida antarctica lipase B (CalB) on methacrylic carrier available as Novozym435® (CAS: 9001-62-1) was applied in stirred tank reactors as well as RD column experiments. CalB for Novozym435® is produced by submerged fermentation of a genetically modified Aspergillus niger microorganism and adsorbed on a macroporous resin with respect to the manufacturer. Activity is described to be ≥ 5000 U·g-1 according to propyl laurate assay. Free biocatalyst solution (Lipozyme CalB L) is used for preparation of catalytic xerogel, which was kindly

provided

by

Novozymes

measurements result in 52 ± 5 U·ml

(Bagsvaerd, -1

Denmark).

Spectrophotometric

activity

according to p-nitrophenyl acetate assay (50 mM p-

nitrophenyl acetate in phosphate buffer, 30°C, pH 7, λ = 400 nm). The protein content is 0.50 ± 0.01 mass%, obtained in Bradford assay17. Storage of Novozym435® and Lipozyme CalB L takes place at < 7°C in a refrigerator. 2.3 Experiments in Stirred Tank Reactor (STR) and Flash Distillation (FD) Biocatalytic activity of CalB preparations is obtained in jacketed STRs (60°C, 5 mL, x(R/S)2-PeOH

= 0.1, xEtBu or xPrBu = 0.9, 400 rpm) mixed by magnetic stir bars. Prior to the start of an

experiment, solvent-free stock solution of the substrates is heated to 60°C to avoid temperature differences in the beginning of the reaction. Novozym435® was used without further treatment, whereas silica xerogel formation was carried out with Lipozym CalBL solution to produce sol-gel particles. For xerogel composition, the existing protocol of Heils et al. is used14. In further treatment, the sol-gel is granulated with a cone mill and separated in different sieve fractions. Average particle sizes of 450-630 µm are used in the experimental procedures. After placing


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immobilized CalB in the thermostated reactors, addition of solvent-free substrate mixture starts the reaction. Independent from the applied biocatalyst preparation, experiments were carried out at concentrations of cNovozym435 = cSol-Gel = 35 mg·mL-1 (EtBu) and cNovozym435 = cSol-Gel = 7 mg·mL1

(PrBu). Comparison between Novozym435® and gel coating is carried out related to the applied

amount of the catalyst preparation. Sample volumes of 100 µL were withdrawn in certain time intervals and subsequently analyzed by gas chromatography. Calculation of initial activity is performed with non-linear regression of formed (R)-2-PeBu at low conversions (Conv. 5°C. At 10 mbar, no effect on the number of reactions is observed in a temperature range of 60 - 80°C. In the same temperature range at 100 mbar, a rising number of feasible reactions is detected at increased column temperatures. Hence, at reduced pressures the corresponding boiling temperatures of the reactants are lowered as well and an increased number of reactions can theoretically be implemented to the batch RD setup. As RD column temperature is fixed by the thermal stability of the biocatalyst, reducing the pressure in the setup is necessary to increase the number of applicable reactions.

Fig. 3: Feasible number of transesterification reactions in the preselection tool; biocatalyst application is considered by defined temperatures in RD (60-80°C); RD operation constraints are: ∆TS < 15°C, ∆TLB = ∆THB > 5°C; TC,max=60, 70, 80°C; RD pressure: 10 & 100 mbar

Based on the preselection tool, solvent free CalB catalyzed transesterification of (R/S)-2-pentanol ((R/S)-2-PeOH) with either ethyl butyrate (EtBu) or propyl butyrate (PrBu) were chosen as reaction system in this study (Fig. 4). Residual (S)-2-PeOH is the target compound in both reaction schemes.


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Fig. 4: Selected transesterification reactions catalyzed by Candida antarctica lipase B (CalB). Residual (S)-2-PeOH is the target compound and (R)-2-pentyl butyrate ((R)-2-PeBu) the reaction product Both selected reactions fulfill the temperature criteria and can theoretically be implemented in the batch RD column. The substrates evaporate at temperatures of 55 - 70°C at 100 mbar and 80 mbar to allow application in the biocatalytic RD setup, respectively. The temperature differences and boiling points of the reactants for the chosen reactions are reported in Tab. 3. Formed low boiling primary alcohol is ethanol (EtOH) or 1-propanol (1-PrOH) depending on the selected starting ester. Due to their low boiling temperatures, the primary alcohols can be separated at the top of the RD column. High boiling 2-PeBu is the formed reaction product in both reactions and can easily be accumulated in the column bottom. The major difference between the reactions is the boiling point order of the substrate compounds. This effect is demonstrated for (R/S)-2-PeOH19, EtBu20 and PrBu21,22 by comparing calculated boiling temperatures by Eq. 1 at varied operating pressure in (Fig. 5). Calculated values are in accordance with experimental data from literature. Hence, the combination of (R/S)-2-PeOH with PrBu involves a higher boiling temperature for the racemic alcohol whereas the opposite behavior is evident by using EtBu. Tab. 3: Calculated boiling temperatures and temperature differences for the transesterifications of (R/S)-2-PeOH either with EtBu or PrBu Parameter

pRD [mbar]

(R/S)-2PeOH & EtBu 100

(R/S)-2PeOH & PrBu 80


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Tb,(R/S)-2-PeOH [°C] Tb,(R/S)-2-PeBu [°C] Tb,EtBu [°C] Tb,PrBu [°C] Tb,EtOH [°C] Tb,PrOH [°C] ∆TLB [°C] ∆THB [°C] ∆TS [°C]

65 95 55 29 26 30 10

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61 89 70 42 18 19 10

Fig. 5: Boiling point of reactants at varied RD column pressures; the dotted line is the implemented constraint for the maximum RD column temperature using CalB (Tmax,RD); lines represent calculated boiling temperatures by Eq. 1; data points account for experimental data from literature:

= (R/S)-2-PeOH19,

= EtBu20,

= PrBu21,22

Beside boiling point calculation for pure compounds, vapor liquid equilibria (VLE) are considered for the chosen reaction system. In literature, experimental data on binary mixtures are only described at ambient pressure for EtBu and PrBu with isopropanol and (R/S)-2-BuOH as well as EtBu and PrBu with EtOH and 1-PrOH23,24. Because experimental VLE data is not available for all substrate combinations, an estimation for non-ideal behavior is performed in Aspen properties V8.0 (Aspen Technology, Bedford, Massachusetts, USA) using the universal quasi-chemical UNIQUAC equation as a gE model25. The UNIQUAC parameters are estimated based on UNIFAC Dortmund parameters (UNIFAC DMD), which allow the calculation of ACS Paragon Plus Environment

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activity coefficients based on group contribution methods26. For (R/S)-2-PeOH and PrBu, estimated azeotropic behavior is present at 80 mbar, 60.8°C and y(R/S)-2-PeOH > 0.79 mol·mol-1. The azeotrope is estimated based on a comparison of literature data and UNIFAC DMD parameters for PrBu and 1-PrOH at ambient conditions (Fig. 6)24. However, the azeotropic behavior in this case can be overcome by the reaction. Comparing the boiling point of pure reactants to estimated VLE-data revealed a lower boiling temperature of the azeotropic mixture (temperature minimum azeotrope). Hence, a preselection based on pure compounds is presenting suitable substrate combinations even though consideration of boiling points from mixtures can lead to an increased number of feasible reactions. Within the preselection process, a criteria-based platform is established to get an overview on the application of different substrate combinations in biocatalytic reactive distillation without necessity of VLE data in the stage of deciding, if a system is in principle applicable. However, VLE data have to be considered after selection of a reaction system for the detection of present azeotropes. Overall, Antoine parameters from NIST allowed fast initial comparison of solvent-free CalB catalyzed kinetic resolution reactions at varied RD column pressures18. Conservative evaluation of the boiling point order and theoretical applicability in the RD setup is performed considering biocatalyst application to select the transesterifications of (R/S)-2-PeOH with EtBu and PrBu.


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Fig. 6: Estimated VLE data with UNIFAC DMD parameters in Aspen properties for: PrBu and 1PrOH at 1013 mbar (A) and 80 mbar (B); (R/S)-2-PeOH and PrBu at 1013 mbar (C) and 80 mbar (D); experimental data for the liquid (x1-PrOH) and vapor molar fraction (y1-PrOH) of mixtures containing PrBu and 1-PrOH at 1013 mbar are taken from Ortega et al24

3.2 STR Experiments: Biocatalytic Activity & Enantioselectivity Catalytic activity and enantioselectivity of the applied reaction system as well as thermal stability of the catalyst were investigated for the selection of a biocatalyst preparation and its integration in reactive distillation. To be flexible in the position of the reactive zone and guaranteeing substrate availability for product formation, increased catalytic activities within the RD column internals are of interest. Different possibilities for placing the biocatalyst in the RD


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setup are suitable to allow fixation as well as increased thermal stability in the RD column. Two catalyst preparations for CalB were selected in this work including either entrapment of the biocatalyst in a silica matrix (gel coating)11 or adsorption on poly-acrylic resin (Novozym435®). For the chosen reaction systems, a comparison of both preparations is given in Tab. 4. In our STR experiments, Novozym435® revealed 12-13 times increased activity compared to the gel coating independent from the performed reaction. The activities for Novozym435® and gel coating are compared based on the amount of catalyst preparation (U·gNovozym435-1 and U·ggel coating1

). Moreover, the theoretically applicable total amount of both catalyst preparations is considered

assuming similar conditions to Heils 201514 for the gel coating material. Within our column packing height of hpacking = 1200 mm at maximum 34.3 ggel coating in 12 gel coated Montz-A3-500 elements and 60.4 gNovozym435 applying 8 Katapak SP-like catalytic packing elements with 7.5 g Novozym435® can be placed in the setup. Hence, in addition to increased catalytic activity of Novozym435® a higher total amount of biocatalyst preparation can be integrated into the RD setup applying Novozym435® and an increased flexibility in the position of the reactive zone is possible. An increased activity of the biocatalyst becomes more important at lower RD column sections and increased boiling points of the substrates as well as increased temperature differences between the substrates. Substitution of the ester component from EtBu to PrBu resulted in reduced catalytic activity by 46 - 49 % within the gel coating as well as Novozym435®. In literature, thermal stability tests with respect to residual activity of the used catalyst preparations revealed applicability at elevated temperatures in the range of 60° C for the gel coating11 and 80°C for Novozym435® 9 for operation times of 30 d. At least, the gel coating experiments were already obtained in reactive distillation experiments and both preparations were tested to allow high residual activities for different reaction systems. As the presented experiments in this study are in the range of up to 7 h, deactivation of both catalyst preparations does not have a significant impact on the results. With respect to the presented preselection tool, increased thermal stability of Novozym435® allows a broadened process window regarding the number of feasible reactions. Calculations by comparing the initial reaction rates of (R)-2-PeOH and (S)-2-PeOH according to Eq. 2 revealed high enantioselectivities (E) at our operating conditions for the solvent free transesterification reactions. =


Eq. 2

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Hence, both catalyst preparations revealed appropriate behavior for racemic resolution of (R/S)-2PeOH either with EtBu or PrBu. In terms of increased overall catalytic activity as well as increased catalytic activity per single column parts (e.g. in the bottom of the setup), Novozym435® is selected for further investigations.

Tab. 4: Comparison of CalB preparations: gel coating and Novozym435®. Activity experiments were performed at 60°C in 5 mL reactors. Determined standard deviations belong to the number of performed experiments: EtBu and PrBu with gel coating (n = 2), EtBu with Novozym435® (n = 5), PrBu with Novozym435® (n = 3). Enantioselectivity was calculated from experimental data according to Eq. 2. Data for residual activity were taken from literature9,11. Parameter Catalyst preparation vinitial [U·gpreparation-1] E [-] Residual activity after 30 d operation [%]

(R/S)-2-PeOH & EtBu gel coating Novozym435® 151 ± 7 2003 ± 172 >100 ≈ 80 (at 60°C)11 -

(R/S)-2-PeOH & PrBu gel coating Novozym435® 83 ± 7 1020 ± 112 >100 ≈ 100 (at 80°C)9

3.3 Biocatalytic Reactive Distillation 3.3.1 Validation of batch RD column experiments with flash distillation (FD) at reduced pressure and calculated data In order to validate our constructed batch RD column, the transesterification reaction of (R/S)-2-PeOH with EtBu was performed applying Novozym435® in catalytic packings. Obtained data in RD (section 2.5) were compared to FD under vacuum conditions (section 2.3) and calculated behavior of the reaction, based on the method introduced by Chen et al 198227. Within this method, the course of enantiomeric excess (ee) can be determined at varied conversions (X) for constant E-values (E) (Eq. 3 and Eq. 4). In the investigated transesterification reaction, a differentiation between the reaction product (eeP reaction) and the residual substrate demonstrating the target compound (eetarget compound) needs to be done. !"∙$ %&&,()*+,-. /

= =

!"∙$ !&&,()*+,-. /

!"∙ !&& +)(0+ *-12-3.4 !"∙ %&&+)(0+ *-12-3.4


Eq. 3 Eq. 4 20

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While the ee relates to either (S)-2-PeOH (= target compound) or (R)-2-PeBu (= reaction product) (Eq. 5 and Eq. 6), X confers to the sum of chiral substrate material (R/S)-2-PeOH including both enantiomers (Eq. 7). ? !?

556789&6 :;?@ = ?

%?

?C3 !?C3

55A,8&7:6B;? = ? D=

C3 %?C3

?,E %?,E !? %? ?,E %?,E

Eq. 5 Eq. 6 Eq. 7

Independent from the setup, similar operation conditions were selected (x(R/S)-2-PeOH = 0.1, xEtBu = 0.9, 100 mbar, 60°C). The main difference between both setups was the placement of the immobilized biocatalyst. In the RD setup 4 catalytic packing elements were placed in the height of the column, whereas the biocatalyst was introduced in the liquid phase performing FD. Calculation of ee and X in both setups considered present concentrations in the bottom. In the beginning, the RD column was operated at total reflux ratio (rr = ∞). Within the time range between 135 and 255 min, a reflux ratio of 20 was adjusted (rr = 20). By this procedure, in situ product removal of low boiling EtOH (Tboil = 29°C at 100 mbar) was performed. After successful EtOH removal, the temperature in the RD column top rose due to increased concentrations of higher boiling materials in the vapor flow. During the course of reaction with an applied reflux ratio, rising temperature in the top of the RD column was observed. Simultaneously, accumulation of the target compound ((S)-2-PeOH) and the reaction product ((R)-2-PeBu) in the bottom of the RD column setup took place (Fig. S1 and Fig. S2 - Supporting information). In similar FD, variation of the operating pressure revealed differences in time-dependent behavior for a shift of thermodynamic equilibrium to the product side. Increased conversion was observed at reduced pressures of 100 mbar compared to ambient pressure (Fig. S3 – Supporting information). The results for comparison of enantioselective product formation in FD and RD column operation are given in Fig. 7. The dotted line presents calculated values for the course of eetarget compound and the filled line calculated values of eeP,reaction with increasing X at E = 100. Nonfilled data points belong to the results of RD. They are in good agreement with the calculated values and obtained data in FD at 100 mbar. The standard deviations for ee and X in the presented RD column experiment were determined by maximum error estimation. For FD, standard deviations account for two experiments performed at similar conditions (n = 2). In the


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experiments, an ee > 99 % was reached for the target compound (S)-2-PeOH at X = 48 ± 3 % in the RD column and X = 50 ± 1 % in FD. Simultaneously, ee of the reaction product (R)-2-PeBu stayed > 95% until X = 50 ± 1 % and was decreasing at increased conversions. Decreased ee of the reaction product at X > 50% can be explained by the absence of the preferentially reacting enantiomer (R)-2-PeOH. Therefore, presented data clearly demonstrate the successful implementation of Novozym435® in catalytic packing elements in a batch RD column for the performance of racemic resolution reactions. The course of the reaction is in good agreement with previously published data of Heils et al (2015), in which gel coatings were applied to integrate immobilized CalB14.

Fig. 7: Selectivity of the transesterification reaction with (R/S)-2-PeOH and EtBu. Operation conditions: xEtBu = 0.9 [mol·mol-1], x(R/S)-2-PeOH = 0.1 [mol·mol-1], p = 100 mbar: flash distillation (FD, filled data points, n = 2): T = 60°C, V = 7 mL, cNovozym435 = 57.4 mg·g-1; RD experiment (non-filled data points, maximum error determination): mBottom,0 = 800 g, reflux ratio = 20 (between t = 155 – 255 min), TBottom = 58 – 64°C, cNovozym435 = 8.1 mg·g-1

3.3.2 In situ Separation of Chiral Target Compound For experimental investigation of the racemic resolution with (R/S)-2-PeOH and PrBu, batch RD was performed with catalytic packings similar to our proof of concept study. Due to


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changed order of boiling points with (R/S)-2-PeOH as lower boiling substrate compared to PrBu, in situ separation of the target compound (S)-2-PeOH is possible. RD column pressure was maintained at 80 mbar to have the same temperature difference between the substrates compared to (R/S)-2-PeOH and EtBu at 100 mbar (∆TS = 10°C). The course of temperature was monitored over the RD column height at 4 different positions, which was always below 75°C independent from the examined position (Fig. 8). This RD column temperatures prevent fast deactivation of the biocatalyst, respectively. The process strategy consisted of a stepwise in situ separation of accumulated compounds in the top of the RD column. At the RD column top, automated temperature controlled reflux ratio of 15:2 was applied at fixed temperature constraints. As soon as the temperature dropped below the adjusted temperature limit, fixed reflux ratio took place to withdraw low boiling reactants. After 45 min of operation, limiting temperature was set to 43°C to get rid of low boiling 1-PrOH followed by 46°C between 105 and 225 min. Finally, the separation of accumulated target compound (S)-2-PeOH was addressed at an adjusted temperature of 60°C between 225 and 345 min. Selection of the temperature limits was carried out with respect to the boiling temperature of the corresponding boiling points at 80 mbar for 1PrOH (Tboil = 42°C) and (R/S)-2-PeOH (Tboil = 61°C). Monitored temperatures in the upper part of the RD column (1.14 m and 1.5 m) showed the effect of successful in situ product removal during the experiment (Fig. 8), because rising temperatures were observed over the course of reaction.


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Fig.

8:

Temperature

profile

in

RD

over

column

height.

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Operation

conditions:

xPrBu = 0.9 [mol·mol-1], x(R/S)-2-PeOH = 0.1 [mol·mol-1], p = 80 mbar, mBottom,0 = 800 g, T = 70-74°C (in bottom), cNovozym435 = 7.7 mg·g-1. The time ranges and adjusted temperatures of applied controlled reflux ratio (rr = 15:2) is visualized by black boxes Moreover, the molar fraction of the reactants were analyzed over the RD column height at similar positions of the temperature probes. Presented horizontal standard deviations for the molar fraction at all column heights were calculated by the sum of propagated errors for all reactants. The propagated errors consist of the error in gas chromatography (n = 2) and the error of the slope in calibration lines (n = 12). A depletion of (R)- and (S)-2-PeOH as well as formation of the transesterification products (R)- and (S)-2-PeBu was observed in the bottom of the RD column (Fig. 9). High enantioselectivity of the biocatalyst towards (R)-2-PeOH was evident due to its faster depletion and simultaneous faster formation of (R)-2-PeBu (see Fig. 9, B). A molar excess of 9:1 for non-chiral PrBu in comparison to chiral (R/S)-2-PeOH was applied. At the end of the reaction, (R)-2-PeOH was completely converted to the corresponding (R)-2-PeBu.

Fig. 9: Molar fractions in the bottom of the RD column. A: all reactants; B: zoom in molar fractions with xi < 0.1. Operation conditions: see caption of Fig. 8. The molar fraction of the target compound (S)-2-PeOH constantly increased with column height and the course of reaction, caused by the change in adjusted temperature limits for the reflux ratio ACS Paragon Plus Environment

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at the RD column top (Fig. S4 and Fig. S5 – Supporting information). The highest boiling compound (R)-2-PeBu was only present in the lower parts, which indicated efficient separation of the reaction product. Looking at the molar fractions in the top of the RD column, accumulation of low boiling 1-PrOH took place in the beginning of the reaction (Fig. 10). The first step of in situ separation at T < 43°C decreased the molar fraction of 1-PrOH. Hence, the equilibrium was shifted to the product side and accumulation of the target compound in the top of RD column was observed in the second part of the in situ separation. With increasing reaction time and stepwise adjustment of the temperature limit up to T < 60°C, rising molar fractions up to x(S)-2PeOH

= 0.65 ± 0.04 mol·mol-1 were realized. At a temperature limit of T < 60°C, accumulated

target compound is decreasing after complete separation of low boiling 1-PrOH due to its stripping out of the RD column. Horizontal error bars in Fig. 10 account for the time period of collecting samples automatically. The samples were collected at the end of the sample period represented by the data points. The mean concentration of the corresponding time interval is analyzed in gas chromatography and the amount of withdrawn sample was dependent on the temperature at RD column top, respectively.

Fig. 10: Molar fractions in the top of the RD column. Stepwise adjustment of the temperature limit for automatically performed two-step in situ separation of low boiling 1-PrOH (T < 43°C & < 46°C) and (S)-2-PeOH (T < 60°C). Operation conditions: see caption of Fig. 8.


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The presented results demonstrate successful fractional distillation of two-step in situ removal of low boiling 1-PrOH and (S)-2-PeOH in dependency of the position in the RD column setup (Fig. 11). For (S)-2-PeOH, ee values and conversions of up to 90 ± 4 % and 51 ± 3% were obtained in the top of the column, which were determined considering the overall mass balance with deviations of 4.8 %. The mass balance includes the amount of applied substrate mixture in the beginning of the experiment, residual amount of reactants in the column setup at the end of the experiment (bottom and column height) as well as withdrawn amount of samples at all sampling points over the course of experiment. Compared to obtained data with a different boiling point order of the substrates including EtBu and (R/S)-2-PeOH, accumulation of (S)-2PeOH was 13 times higher for the application of PrBu and (R/S)-2-PeOH. The highest molar fraction of (S)-2-PeOH in the top of the RD setup with EtBu was 0.05 mol·mol-1. In comparison to the results of EtBu with (R/S)-2-PeOH, accumulation and in situ separation of the target compound was achieved by changing the substrate ester and thereby the order of boiling points based on our introduced preselection tool (section 3.1).

Fig. 11: Comparison of in situ separation of target compound ((S)-2-PeOH) at different substrate boiling point orders in the top of RD column by varied ester compound (EtBu or PrBu). ISPR = in situ product removal

Within the withdrawn sample fraction between t = 225 - 255 min shown in Fig. 11, the molar fraction of x(S)-2-PeOH taken at the column top accounts for 14.1 g of accumulated (S)-2-PeOH with ACS Paragon Plus Environment

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an ee = 90 ± 4 %. This refers to a volumetric productivity per batch of 1.04 g(S)-2-PeOH·L-1·h-1 determined by Eq. 8. FGH5IJK JLGKIMIN 5J OIKℎ = S

In Situ Separation of the Chiral Target Compound (S)-2-Pentanol in

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