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67126dc812ff75c3a1cc63fb | 6 | Density of DSILs was determined using an Automatic Density Meter DDM2911 (Rudolph Research Analytical, Hackettstown, NJ) equipped with a Peltier module for precise temperature control, with the mechanical oscillator method. The measurement was acquired at 20 °C in 5 repetitions for each of the samples (approximately 1.0 cm 3 ). Before the series of measurements, the apparatus was subjected to a two-point calibration using deionized water and air as the references. After each series of measurements, the densimeter was washed with water and organic solvents (methanol and acetone) and dried with airflow. The uncertainty of the density measurement was estimated to be less than 5×10 -4 g cm -3 . Based on the experimental density values obtained at 20 °C (ρ20), and average molecular weights of ILs and DSILs (Mw), the following parameters were calculated: molar volume (Vm 20 ), excess molar volume (V E20 ), average volume of a single ionic pair (Vip 20 ), lattice energy (UPOT 20 ), and standard molar entropy (S°2 0 ), according to the equations below : |
67126dc812ff75c3a1cc63fb | 7 | The surface tension and contact angle measurements were carried out using a DSA 100E analyzer (Krüss, Germany) at 25 °C. According to the manufacturer, the measurement accuracy of this instrument amounts to ±0.001 mN m -1 . The surface tension was determined using the drop shape method. Basically, the principle of this method is to form an axisymmetric drop at the tip of a needle of known diameter. The image of the drop is taken with a CCD camera and digitized. The surface tension (γ, mN m -1 ) was calculated according to the results of the drop profile analysis according to the Laplace equation. The value of the surface tension and contact angle allowed for calculation of the CMC and surface tension at CMC (γCMC) based on the plot γ vs log C using a linear regression analysis method. The temperature was controlled using a Fisherbrand FBH604 thermostatic bath (Fisher, Germany, accuracy ±0.1 °C). The determination of the contact angle (CA) was based on the sessile drop method. The drops of solution are deposited on a solid hydrophobic surface (paraffin). The images of the drops were taken with a CCD camera, digitized and evaluated using Young-Laplace fitting. The CA was determined as the slope of the tangent line at the contact point between the 3 phases (solution, paraffin surface, and air). The measured error of the CA determination in this method is estimated to be less than 0.1° by the manufacturer. Where wdic is mass fraction of dicamba anion. |
67126dc812ff75c3a1cc63fb | 8 | where SEM is the standard error of the mean, s is the sample standard deviation, and n is the number of replications. Data were statistically analyzed using one-way ANOVA with a random series effect. Fisher's multiple post hoc test (α = 0.05) was used to compare treatments. Before tests, inoculum was diluted with pure medium until its absorbance of monochromatic light (688 nm) was in the range of 0.05-0.08 at 10 cm path. To obtain the desired range of concentrations, 19 cm 3 portions of diluted inoculum were mixed with 1 cm 3 of the prepared toxicant solution in OECD medium. For control, 1 cm 3 of pure OECD medium was added. In the range finding tests, the selected IL and DSIL systems were tested in triplicates at a series of geometrically decreasing concentrations: 100, 10, 1, and 0.1 mg dm -3 . The absorbance at 688 nm was tested for each sample using 10 cm-long glass cuvettes, and the measurements were conducted at the start of the experiment and after 72 hours. The contents of all test vessels were mixed and illuminated continuously during the test, and their position was randomized to achieve similar levels of illumination for each sample throughout the experiment. The tests were conducted at 22-23 °C. The rate of inhibition of algae growth was calculated according to the eq. 7: |
67126dc812ff75c3a1cc63fb | 9 | The EC50 range for each of the toxicants tested was determined based on the relationship between its concentration and the calculated 𝐼 𝑟 value., To determine the exact EC50 values, the experiments were replicated for each of the toxicants using the same methodology as described above, but different series of decreasing concentrations based on the EC50 ranges found for each compound: 2.84, 2.13, 1.60, 1.20, 0.90, and 0.625 mg dm -3 for ILs comprising a single anion, less compared to previous reports. The resulting bromide was isolated by filtration followed by washing with an apolar solvent, but originally used hexane was substituted by significantly safer heptane. These changes made it possible to reduce the negative environmental impact of the process. were obtained by mixing AB0.9 and B according to an analogous method. All of the obtained systems were colorless liquids at 25 °C. The formation of water during synthesis, combined with highly hygroscopic properties of ion exchange products, results in a high water content in the raw products (approximately 4-6%, Table ) and difficulties in its removal. Such a high water content would significantly affect and alter analysis of the physicochemical properties of the obtained DSILs (e.g., density, refractive index). Therefore, an effort was made to remove most of the water from the DSILs via a two-step azeotropic distillation. Thorough removal of solvents was facilitated by subsequent drying of the residue using a Schlenk line with a vacuum of approximately 2•10 -5 bar. |
67126dc812ff75c3a1cc63fb | 10 | As a result of the above method, it was possible to significantly reduce the amount of water in the obtained DSILs: from 2.5 times (AB0.2) up to 6.5 times (AB0.9). The exact water contents after drying the systems are summarized in Table , they ranged from 0.6% (AB0.9) to 1.7% (AB0.2). It is likely that this is due to an increase in the strength of DSIL-water hydrogen bonding as the molar proportion of the MCPA anion increases. Drying process revealed that the remaining water must be therefore relatively highly bound with the ionic system. Further dehydration of such highly hygroscopic DSILs would require sophisticated methods, and conducting further analyses would have to take place in a dry inert gas atmosphere to achieve substantially reduced water contents (<2000 ppm) demonstrated in other reports. However, it should be borne in mind that the obtained ILs are intended for use in aqueous solutions, thus the increased water content in raw products does not constitute significant issue from a point of view of their industrial application. |
67126dc812ff75c3a1cc63fb | 11 | Detailed 1 H NMR spectra of each system and 13 (Supplementary Data). The occurrence on each of the analyzed spectra of a broad signal of high intensity in the range from 3650 to 3050 cm -1 originating from stretching vibrations in the O-H bonds is indicated by the formation of numerous hydrogen bonds, most likely both between the ions of the analyzed systems, and also between the hydroxyl group and water that remains in the ILs and DSILs after drying. |
67126dc812ff75c3a1cc63fb | 12 | The following basic physicochemical properties were studied for both single-ion ILs (A, B) and DSILs (AB0.1-AB0.9): density and refractive index at 20 °C, as well as the characteristics of phase transformations in the temperature range from -80 °C to 120 °C. The two single-anion ILs exhibited values of density equal to 1.0511 (A) and 1.0886 g cm -3 (B), while all the studied DSILs possessed densities between these two values. The density dependence of xdic is shown in Fig. (detailed data are summarized in Table .8, Supplementary Data). These results |
67126dc812ff75c3a1cc63fb | 13 | showed that there is a close correlation between the density at 20 °C and the molar proportion of the dicamba anion (xdic) in a given system; across the xdic range, a 0.1 increase in this parameter resulted in an increase in DSIL density of approximately 0.0038 g cm -3 . This relationship can be approximated to a linear function with very high accuracy (R² = 0.997), with exact regression parameters provided in Table .9 in Supplementary Data. The obtained results are consistent with previous studies on multi-ion ILs, where such systems have often been reported as having good correlation with a linear mixing law. Since the average molar mass of ILs and DSILs (Mw) increases proportionally with increasing xdic, it should be emphasized that a close linear relationship also exists between the average molar mass of systems A, AB0.1-AB0.9 and B and their measured density. This is confirmed by the fact that all analyzed systems are characterized by a very similar molar volume (Vm 20 ), defined as the quotient of the molar mass of a substance and its density at a given temperature. |
67126dc812ff75c3a1cc63fb | 14 | The average value of this parameter for all ILs and DSILs is approx. 531.4 cm 3 mol -1 , and the calculated extreme values (530.7 cm 3 mol -1 for AB0.9 and 532.0 cm 3 mol -1 for AB0.1) differ from the average value by about 0.1%. It should be noted here that DSILs containing a higher molar proportion of MCPA anion (AB0.1-AB0.5) have positive excess molar volume (Vm E20 ) values than the average, while at higher xdic values Vm E20 values fall below zero. This relationship is shown in Fig. . Positive values of Vm E20 indicate the presence of non-additive physical contributions in the analyzed system, including non-specific interactions between mixture components that alter the molar volume, while negative values are associated with chemical or structural interactions (e.g., changes in coulombic interactions or hydrogen bonds between mixture components, changes in free volume or ion packing). Thus, it can be predicted that an increase in the molar proportion of the dicamba anion above 0.5 plausibly causes a significant change in the nature of the interactions in DSIL. These changes may affect the chemical environment of MCPA anions, which explains the observed deshielding of hydrogen atoms in NMR spectra. |
67126dc812ff75c3a1cc63fb | 15 | The negligible differences between Vm 20 values also indicate that other parameters proportional to this physical quantity also have similar values. The average volume occupied by a single ion pair of a given system (Vip 20 ) is nearly identical (from 0.881 nm 3 for AB0.9 to 0.883 nm 3 for AB0.1) in all analyzed cases. Moreover, there are no significant differences between the studied ILs and DSILs systems in terms of lattice energy at 20 °C (UPOT 20 ) and standard molar entropy (S°2 0 ) values. |
67126dc812ff75c3a1cc63fb | 16 | However, the linear correlation of the refractive index with xdic is not as accurate as in the case of density; the R 2 value is equal to 0.888 (Fig. ). It should be noted here that the experimental values for DSILs containing similar or the same proportion of MCPA anions as dicamba (AB0.4-AB0.6) were close to the theoretical ones. Thus, the relationship between ΔnD 20 and xdic followed a similar pattern as the dependence of excess molar volume (Vm E20 ) on the molar fraction of dicamba. This corroborates with a known relationship between density and refractive index: with a higher density of a substance, the spatial packing of its chemical constituents increases, and refraction results from the interaction of light with molecules (or ions). |
67126dc812ff75c3a1cc63fb | 17 | The phase transitions of the studied systems were also determined using the differential scanning calorimetry technique. For both parent ILs A and B, as well as all systems containing MCPA and dicamba (AB0.1-AB0.9), melting (Tm) and crystallization (Tc) temperatures were observed during heating and cooling cycles, respectively. The thermograms are available on Figs. A.32-A.42 (Supplementary Data), while values of the phase transition temperatures are summarized in Table .10 (Supplementary Data). The presence of a long hexadecyl substituent in the cation of ILs and DSILs significantly reduced lattice energy. This is evidenced by the fact that analogous MCPA and dicamba salts with shorter substituents in the cation underwent only glass transition at temperatures lower than -30 °C. As shown in Fig. , the observed Tm values ranged from -28 °C (IL A) and rose with increasing xdic value up to -12 °C for B. |
67126dc812ff75c3a1cc63fb | 18 | Thus, the melting point reduction typical of eutectic mixtures was not observed for DSILs. The crystallization temperatures (Tc), on the other hand, for each of the studied systems were 1-3 °C lower than the corresponding Tm values. The dependence of both phase transformation temperatures on xdic can be approximated to a linear function with high accuracy (R 2 > 0.98). |
67126dc812ff75c3a1cc63fb | 19 | In this study, the critical micelle concentration (CMC) and surface tension at CMC for ILs A and B and DSILs at various molar ratios (AB0.1-AB0.9) were determined. The obtained data is depicted in Fig. , and the exact values are provided in Table .11 (Supplementary Data). to the Clint's equation (Fig. ; the deviation is visualized as a blue area). The most notable discrepancy was observed for the DSIL AB0.4, where the experimental CMC value was as much as 45% lower compared to the theoretical one. Similar deviations from the "ideal" binary mixture behaviour were also observed previously for mixtures of anionic/cationic and ionic/non-ionic surfactants. These non-additive effects in surfactant mixtures predominantly stem from the entropy of the diffuse counter-ion layer outside the micelle interfacial boundaries, as well as the length of the tails and the charges in the cation and anion. The occurrence of the above-described synergistic effect in DSILs makes it a legitimate strategy to combine two active surfactants in well-defined ratios to reduce the amount of surfactants introduced into the environment. Synthetic auxins show higher efficiency when applied in the form of aqueous solutions with the addition of a surface-active adjuvant, therefore, such DSILs with a surface-active cation and pesticidal anions can guarantee excellent efficacy without necessity of use any other additives. Furthermore, the analysis of the surface tension of the aqueous solution for the DSILs AB0.1-AB0.9 and ILs A and B at their respective CMCs indicated a linear dependence on the xdic value, as illustrated in Fig. . No significant deviation from the linear relationship (R > 0.98) indicates lack on any unexpected interactions in the analyzed systems with water and air. |
67126dc812ff75c3a1cc63fb | 20 | To determine the ability of the DSILs obtained from two popular herbicides, greenhouse experiments were performed on common lambsquartersa plant susceptible to both, MCPA and dicamba, accordingly. It should be noted that dicamba is characterized by higher activity towards dicotyledonous weeds compared to MCPA, so the dose of both active agents differed. |
67126dc812ff75c3a1cc63fb | 21 | The method of determining Dtotal value for systems with different proportions of the dicamba anion is visualized in Fig. The applied ILs or DSILs were capable of reducing the fresh weight of lambsquarters plants in the range from 61% (AB0.6) to 86% (AB0.2) compared to the fresh weight of control objects, while the commercially available herbicides or their tank mixes were characterized by lower activity in the range from 11% to 57%. These findings indicate that not only ILs but also DSILs were characterized by higher biological activity compared to the reference formulations. In the case of AB0. means that the addition of dicamba allows the reduction of the MCPA dose in DSILs with higher xdic values to be fully compensated, and also confirms that the proposed method of selecting the total dose of both herbicides (Dtotal) proved to be correct. |
67126dc812ff75c3a1cc63fb | 22 | Noteworthy, DSIL AB0.2 exhibited the highest biological activity of all studied systems, and its application resulted in an 85% reduction in fresh weight of the test plants compared to the control. This result, indicating the possibility of effective control of common lambsquarters growth in the crop, was clearly superior to other tested active forms. For larger xdic values, the addition of dicamba is able to compensate for the significant loss of MCPA, but it does not result in a clear improvement in DSIL activity. Moreover, the compensation effect facilitates the use of DSILs at lower doses of the active ingredient. AB0.2 applied at a reduced total dose of 305 g of a.i. per ha enables slightly better results compared to the application of the IL A (with MCPA anion only) at a higher dose (400 g ha -1 ). These results confirm our previous findings, in which it was revealed that the DSIL containing the same cation and anions of MCPA and its less biologically active analog, mecoprop-P, along with a small addition of dicamba. Effectiveness at the level of 85% for AB0.2, with a 30% reduction in the dose of active ingredients compared to the recommended doses of commercial formulations indicates the possibility of common lambsquarters control in a crop production, while also reducing the amount of pesticides released into the environment. |
67126dc812ff75c3a1cc63fb | 23 | Since the application of AB0.2 resulted in the greatest efficacy towards the tested dicotyledonous plant, it was also necessary to analyse its potential threat to non-target organisms. It is assumed that only 5% of active substances of herbicide formulations interact with target organisms, while the remaining 95% affects non-target organisms or penetrates the environment, either accumulating in the soil or leaching into groundwater. In addition, it is well known that cationic surfactants exhibit very high toxicity to aquatic organisms. For example, benzalkonium chloride, popularly used as an active ingredient in disinfectant formulations, exhibits very high acute toxicity to freshwater crustaceans (Daphnia magna, EC50 = 0.016 mg dm -3 ), algae (Chlorella vulgaris, EC50 up to 0.0576 mg dm -3 ) or fish (Oncorhynchus mykiss, gill cell line-W1, EC50 = 0.31 mg dm -3 ). To provide a preliminary evaluation of the risks associated with obtaining ILs and DSILs with a surface-active amphiphilic cation, an acute toxicity analysis was performed using a model organism from the freshwater algae group, C. vulgaris. The study was performed for both parent ILs, A and B, as well as DSIL AB0.2. Based on the analysis of the obtained dose-response (BAC16, EC50: 0.16 mg dm -3 ). This type of activity also indicates additional potential for the use of AB0.2 as an algicide (e.g., to counteract algal growth in pools or ponds) at welldefined concentrations. Algicides are also used to counteract the eutrophication in natural water bodies. One of the most commonly used substances to counteract algal growth is copper(II) sulfate, however, this compound exhibits considerable toxicity to other aquatic organisms. |
67126dc812ff75c3a1cc63fb | 24 | In the course of the present study, a series of new double salt ionic liquids (DSILs The greatest reduction in CMC value (lower by 45% compared to assumptions) was observed for DSIL containing MCPA:dicamba in a molar ratio amounting to 6:4. Thus, the strategy involving formation of these surface-active DSILs may allow for effective reduction of the amount of surfactants or active agent used for treatment. |
67126dc812ff75c3a1cc63fb | 25 | All DSILs evaluated for herbicidal efficacy against common lambsquarters showed similar efficacy compared to parent ILs with a single anion (MCPA or dicamba). These results indicate that there were no noticeable synergistic effects for the analyzed systems comprising large amounts of dicamba anion. Interestingly, the system containing a smaller amount of dicamba (MCPA:dicamba in a molar ratio amounting to 8:2) exhibited the highest biological activity within all the active substances tested and achieved 85% of fresh weight reduction compared to control. It can be concluded that a small addition of dicamba can more than compensate for the loss of MCPA dose. Therefore, the total dose of pesticide can be effectively reduced up to 25% |
67126dc812ff75c3a1cc63fb | 26 | (from 400 to 305 g ha -1 ) without any loss of efficacy. Most likely, the occurring synergistic effects cause a noticeable increase in the toxicity of this system towards C. vulgaris compared to parent ILs. This result confirms that a more thorough evaluation of the ecotoxicity of new compounds is crucial in the process of development of novel herbicidal formulations that are not only effective in use but also safe for non-target species. |
6661b56b91aefa6ce1eaad8b | 0 | The specific case of the synthesis of chiral amines, which are essential building blocks for the pharmaceutical industry , is a prominent illustration of this issue. Industrially, the synthesis of chiral amines is operated via multi-step batch processes which usually feature low overall yield, produce large amounts of waste, and are energy-intensive. They are typically catalysed by organometallic homogeneous catalysts based on toxic and depleted heavy metals (Ru, Rh, Pd) which usually operate at relatively high temperature, are not 100% enantioselective, and are difficult to recover . In this context, it is of particular interest to develop more sustainable chiral amines synthesis methods . |
6661b56b91aefa6ce1eaad8b | 1 | Biocatalytic routes have gained considerable attention in the last decades as potentially effective and sustainable alternatives. Remarkably, amine transaminases (TAs) catalyse the direct synthesis of chiral amines from pro-chiral ketones, using cheap and readily available amino donors (e.g. amino-acids) through transamination, with excellent enantioselectivity and in mild conditions. TAs are catching the eye as tremendous achievements have been made recently, both at the fundamental and applied levels . Industrial applications of biocatalytic transamination, however, remain scarce for TAs are usually employed as free enzymes in solution, which display limited stability. Batch processes utilizing such free enzymes do not allow easy catalyst separation, recovery, and reuse . Thus, immobilization strategies are often proposed . Additionally, thermodynamic limitations and substrate/product inhibitions tend to limit the applicability of transaminases in asymmetric synthesis of enantiopure amines . |
6661b56b91aefa6ce1eaad8b | 2 | To overcome these limitations, scientists aim at enhancing the TA robustness and at developing equilibrium shifting strategies. The first point can be achieved through enzyme immobilization, as the resulting heterogeneous biocatalysts are often more versatile and amenable to more productive flow processes. The second point usually relies on using large amino donor excess or on consuming/removing the (co)product during reaction . Besides the widely reported multi-enzymatic cascade reactions or non-catalytic consecutive reactions that can be used to push the equilibrium of the transamination reaction towards the production of the target amine, one alternative possibility is the physical separation of one of the transamination products towards another phase in the system. For example, in-situ (co)product removal (ISPR) strategies were recently employed in batch with free transaminases to drive the reaction towards the formation of valuable chiral molecules . In these examples, the acetophenone co-product was removed from the aqueous phase reaction medium by liquidliquid extraction (using an organic co-solvent), or the targeted chiral amine was selectively crystallized by salt formation. |
6661b56b91aefa6ce1eaad8b | 3 | When aiming to perform such reactions in continuous flow, possibly coupled with product separation, membrane technologies can be of particular interest . Membrane contactors are known to offer operational flexibility, large and tunable interfacial area, modular linear scaleup which allows easy concatenation with other operations, compactness, and low energy consumption. Therefore, researchers have implemented membrane contactors at the outlet of the transamination flow reactor to separate their outputs . In these processes, membranes are solely employed as separation unit for downstream processing and the transaminases are immobilized separately (onto classical supports) and packed into distinct fixed-bed reactors. |
6661b56b91aefa6ce1eaad8b | 4 | Taking this to the next level, it would be of particular interest to immobilize enzymes directly onto active membrane supports, and hence to develop bifunctional membranes allowing to simultaneously host the immobilized enzymes and perform the product separation to intensify the transamination process. The immobilization of enzymes onto polymeric membranes has already been reported with lipases, carbonic anhydrase, and glucose oxidases . Recently, Howdle et al. developed an electrospun polycarvone acrylate diepoxide/polyvinylidene fluoride (PCADE/PVDF) membrane and exploited it for the immobilization of the TA from Halomonas Elongata (HeWT) . This epoxy-functionalized membrane allowed 61.0 % immobilization yield and 43.6 % of specific activity recovery (no TA leaching), paving the way for potential application in combined reaction-separation processes. |
6661b56b91aefa6ce1eaad8b | 5 | In the perspective of designing effective hybrid chemical processes (i.e. combining reaction on immobilized enzymes and in situ separation through a membrane), it is essential to first master the step of enzyme immobilization on conventional polymeric membranes that are routinely employed industrially. Such supports differ from usual enzyme carriers such as porous silica, or resins beads (i.e. typically 100 µm particles, with average pore size of 20-60 nm 45 ), in the sense that polymeric membranes tend to display lower specific surface area available for immobilization , resulting in potentially lower enzyme loadings . Also, their surface is usually not directly amenable to enzyme grafting, so that chemical functionalization is needed. Thus, it is of prime importance to develop robust enzyme immobilization strategies on these membranes, with the aim to optimize enzyme loading, preserve specific activity of immobilized enzymes, and avoid leaching. |
6661b56b91aefa6ce1eaad8b | 6 | Polyacrylonitrile membranes (PAN) and polypropylene (PP) were selected as commercially available and industrially relevant membranes showing good mechanical resistance and featuring respectively hydrophilic and hydrophobic surface chemistry. We leverage electrostatic interactions and covalent grafting strategies to avoid leaching. The membrane carriers are characterized at different stages of the preparation. After TA immobilization, using a model kinetic resolution, we show that these functional materials exhibit high catalytic performance (specific activity), minor leaching and excellent reusability. This paves the way to a future use in flow mode hybrid processes, possibly concatenated with purification strategies. |
6661b56b91aefa6ce1eaad8b | 7 | Figure describes the protocoladapted from Shi et al used to immobilize transaminases onto PAN membranes. The PAN surface was partially hydrolyzed by dipping a 5 cm 2 disc of the PAN membrane in 50 mL of a 1.5 M NaOH solution for 2 hours at 50 °C under gentle stirring (as recommended by Pérez-Álvarez et al. ). The resulting hydrolyzed PAN membrane (HPAN) was then washed with 100 mL distilled water for 1 hour, and this washing step was repeated 3 times, before being dipped into 50 mL of a 1 wt. % (unless stated otherwise) aqueous solution of branched polyethyleneimine (PEI) for 18 hours at 37 °C under gentle stirring. The resulting membrane was washed again 4 times, stored in distilled water and is denoted HPAN_PEI. |
6661b56b91aefa6ce1eaad8b | 8 | The functionalized membrane was transferred into round bottom glass flasks containing 5 mL of buffered solution (MES or HEPES 0.1 M buffer, PLP 1 mM, sodium pyruvate 10 mM) for enzyme immobilization. The latter contained the desired TA concentration (C0) and was set either at pH 8 with the HEPES buffer or at pH 5.5 with the MES buffer. Incubation was done for 18 hours at 35 °C under gentle stirring. The resulting membrane-immobilized transaminase was either directly rinsed or post-treated and then rinsed. Post-treatment was done with glutaraldehyde (GA, 1 wt.%) or sodium alginate (SA, 0.2 wt.%) aqueous solutions for 1 hour (unless stated otherwise) at 25 °C in an attempt to prevent TA leaching . Rinsing was done by suspending the membrane in 5 mL of rinsing solution (containing HEPES 0.1 M buffer, PLP 1 mM, sodium pyruvate 10 mM) for 30 minutes (repeated two times), to eliminate the loosely attached enzymes. |
6661b56b91aefa6ce1eaad8b | 9 | This TA immobilization was performed on each PAN membrane support employed in this study (i.e. PAN, HPAN, HPAN_PEI), in order to evaluate the impact of the different steps of functionalization on the catalytic performance of the resulting immobilized TAs. Depending on the immobilization pH, TAs immobilized on pristine PAN were labelled as TA_PANa (if pH was 8) or TA_PANb (if pH was 5.5). Similarly, TAs immobilized on HPAN were labeled as TA_HPANxa or TA_HPANxb, where x = / stand for TAs immobilized on HPAN, x = 1 for HPAN_PEI (without post-treatment), x = 2 for HPAN_PEI (with SA post-treatment) and x = 3 HPAN_PEI (with GA post-treatment), respectively. TA_HPAN_xa or TA_HPAN_xb. In the case of TA_HPAN_2a, TA_HPAN_2b, TA_HPAN_3a and TA_HPAN3b, an additional post-treatment was applied (with SA or GA, respectively) before the rinsing step. |
6661b56b91aefa6ce1eaad8b | 10 | PP membranes were functionalized in three steps. First they were coated with PDA with the aim to provide reactive amine functions for further functionalization of the PP support . A 5 cm 2 disc of PP membrane was immersed into 10 mL of ethanol in order to wet its surface and pores. Simultaneously, a dopamine (i.e. 3-hydroxytyramine) hydrochloride solution was prepared in a 10 mM Tris buffer (pH 8.5) at a concentration of 2 mg/mL, and left to stir. After about 15 min, dopamine started to self-polymerize and the colorless solution turned pale brown. |
6661b56b91aefa6ce1eaad8b | 11 | At this stage, the wetted PP membrane was immersed in the dopamine solution and kept for 20 hours (unless stated otherwise) at room temperature, under gentle stirring (Figure S1 ). The obtained PP_PDA membrane (dark brown to black) was then rinsed with 100 mL distilled water for 1 hour, and this washing step was repeated three times. |
6661b56b91aefa6ce1eaad8b | 12 | Second, the obtained PP_PDA membrane was modified with a bisepoxide coupling agent (glycerol diglycidyl ether; GDE) to confer an appropriate linker arm for the subsequent covalent grafting of the enzyme 56 (Figure , step 1). The PP_PDA was immersed in 50 mL of a 100 mg/mL GDE solution (in ethanol) and stirred for 18 hours (unless stated otherwise) at room temperature. The resulting PP_PDA_GDE membrane was then rinsed with 50 mL of ethanol for 1 hour first, then with 100 mL distilled water for 1 hour (repeated three times). |
6661b56b91aefa6ce1eaad8b | 13 | Third, in order to drive the covalent grafting of the transaminase on the epoxy linker arm, the PP_PDA_GDE membrane was partially functionalized with polyethyleneimine (prior to enzyme immobilization; Figure , step 2). Thus, the PP_PDA_GDE was immersed into 50 mL of a 5 mg/mL PEI solution in carbonate/bicarbonate 0.1 M buffer at pH 9.5 and stirred for 90 minutes (unless stated otherwise) at room temperature. |
6661b56b91aefa6ce1eaad8b | 14 | The functionalized PP membrane was transferred into 5 mL of buffered solution (HEPES 0.1 M buffer, PLP 1 mM, sodium pyruvate 10 mM) containing the desired TA concentration (C0) at pH 8, and incubated for 18 hours at 35 °C under gentle stirring (Figure , step 3). After immobilization, the resulting membrane-immobilized transaminase was rinsed with 5 mL of rinsing solution (containing PLP 1 mM, sodium pyruvate 10 mM in HEPES 0.1 M buffer pH 8) for 30 minutes (repeated two times) to eliminate the loosely attached TAs. For comparison, this TA immobilization was also performed on each PP membrane support employed in this study (i.e. PP, PP_PDA, PP_PDA_GDE and PP_PDA_GDE_PEI). The resulting catalysts were denoted TA_PPy, where y = / stand for TAs immobilized on pristine PP, y = 1 on PP_PDA, y = 2 on PP_PDA_GDE and y = 3 on PP_PDA_GDE_PEI. |
6661b56b91aefa6ce1eaad8b | 15 | Inspired López-Gallego et al. , we immobilized the enzyme onto PP_PDA_GDE_PEI (with either 0.1 mM or 1 mM PLP, sodium pyruvate 10 mM in HEPES 0.1 M buffer pH 8) and then rinsed the resulting membrane three time (5 mL sodium pyruvate 10 mM in HEPES 0.1 M buffer pH 8), and directly incubated it with PLP (1 mM in HEPES 10 mM pH 8 for 90 minutes at room temperature under gentle stirring). Additional rinsing was applied again (four times 5 mL sodium pyruvate 10 mM in HEPES 0.1 M buffer pH 8, 30 minutes). The obtained membranes were denoted TA_PP3_SSz, where z is the concentration of PLP (in mM) used during the TA immobilization step. The amount of PLP effectively loaded onto the membrane was evaluated by UV absorption (see ESI). |
6661b56b91aefa6ce1eaad8b | 16 | Enzyme loadings on the membranes were evaluated by mass balance. Typically, a functionalized membrane was incubated in a 5 mL (V0) TA solution of known concentration, C0 [mg.mL ]. After immobilization, the membrane was removed from the reactor and the TA concentration in the remaining solution (C1) was measured by Bradford titration (see ESI). The membrane was then washed with 5 mL (V0) of rinsing solution, and the enzyme concentration in the resulting solution was measured (C2). The same procedure was applied to the next rinsing solutions, leading to measure C3 and C4. The immobilized enzyme loading (L) was determined by Eq. 1. The immobilization yield (%) is defined as the ratio between immobilized TA (L) and the total TA mass introduced during the immobilization (5 x C0). |
6661b56b91aefa6ce1eaad8b | 17 | The kinetic resolution of BMBA was used as a model reaction to assess the catalytic activity of free and membrane-immobilized transaminases. Racemic BMBA was reacted with pyruvate, to produce BAP and either L-or D-alanine leaving unreacted R-or S-BMBA when using an S-or a R-selective TA, respectively (Figure ). When catalytic tests were run with immobilized transaminases, one disk of 5 cm 2 of membrane support was employed. |
6661b56b91aefa6ce1eaad8b | 18 | The yield is defined as the proportion of rac-BMBA converted into BAP (%). The maximum theoretical yield for the kinetic resolution is thus 50 %. The specific activity is defined as the number of µmol of 4'-bromacetophenone formed per minute per mg of immobilized enzymes and evaluated by Eq. 2, where L is the immobilized enzyme loading (determined by mass balance via the Bradford method (mg)) and, t is the reaction time (min). |
6661b56b91aefa6ce1eaad8b | 19 | After 24 hours of reaction, the solid membrane was removed and the concentration of leached enzyme was evaluated via the Bradford method (ESI). The leached TA fraction (%) is defined as the ratio between the mass of leached TA after one catalytic test and the initial immobilized TA loading (L). 50 mM 3,3-diphenylpropionic acid (3-DPPA) was added to the reaction medium in order to crystallize with the remaining BMBA (in the form of a BMBA:DPPA salt). After 20 hours of crystallization, crystals were filtered, washed twice with 5 mL distilled water, then once with 5 mL tert-butyl methyl ether (MTBE) to remove residual BAP, and then dried at room temperature overnight. Semi-quantification of BMBA enantiomers was then determined using Chiral High-Performance Liquid Chromatography (Chiral-HPLC), by dissolving the obtained crystals in the mobile phase (95% isohexane/5% 2propanol/0.1% diethylamine) (see ESI). |
6661b56b91aefa6ce1eaad8b | 20 | The spectra of the pristine PAN membrane (Figure ) featured the main characteristic peaks of nitriles (at 2240 cm -1 ) and of alkanes (2925 and 1450 cm -1 ). However, it also features a broad band in the 3200-3500 cm -1 region as well as additional peaks at 1730 and 1230 cm -1 , which suggest the presence of some impurities (such as hydroxyl or carbonyls functions) at the surface of the PAN membrane. |
6661b56b91aefa6ce1eaad8b | 21 | Based on previous reports , we applied a mild hydrolysis treatment (120 minutes with NaOH 1.5 M at 50 °C) in order to favor the formation of COO -surface groups while preserving the HPAN membrane mechanical properties. Expectedly, new IR peaks highlighted the presence of carboxylic acid/carboxylate moieties at 1560, 1400 and around 3300 cm -1 51, , along with amides groups (characteristic peak at 1670 cm -1 ) coming from the partial surface hydrolysis of nitriles. |
6661b56b91aefa6ce1eaad8b | 22 | Additional surface-sensitive in-situ infrared experiments (DRIFTS; see Figure ) were performed on HPAN_PEI1 at 120 °C (to get rid of the broad O-H stretching band from 2800 to 3600 cm -1 due to surface hydration). It revealed characteristic peaks of amine (3420 and 2850 cm -1 ) as well as alkane (2925 and 1450 cm -1 ) and nitrile (2240 cm -1 ) moieties, which confirmed the results obtained from ATR-FTIR. |
6661b56b91aefa6ce1eaad8b | 23 | Consequently, the N/C ratio obtained at the PAN surface is lower (0.25) than the theoretical one (0.33). As expected, the basic hydrolysis of PAN (Figure ) resulted in an increase of the O/C ratio and in a decrease of the N/C due to the conversion of nitrile moieties into amides and carboxylates moieties (Table , entry 2). Addition of PEI by electrostatic adsorption at the HPAN surface (Figure ) logically led back to an increase of the surface N content. |
6661b56b91aefa6ce1eaad8b | 24 | ATR-FTIR analysis on the pristine PP membranes showed only the expected signals of alkyl groups (Figure ) . After dopamine polymerization (PP_PDA) the IR spectra showed an additional broad signal at 3200-3500 cm -1 , which can be attributed to the presence of hydroxyl (catechol) groups of PDA . The mechanism of polydopamine adhesion on hydrophobic surfaces such as PP is not clearly understood, but it is believed to involve strong non-covalent (e.g. hydrogen bondings, hydrophobic) interactions . The peak at around 1600 cm -1 may indicate the appearance of N-H (indole) groups generated by the PDA deposition , even though superposed with the O-H bending vibration mode of adsorbed water . |
6661b56b91aefa6ce1eaad8b | 25 | In the next steps, the PP_PDA membrane was functionalized with GDE and then with PEI. The signature peak of the epoxy groups (i.e. symmetric ring stretching, expected at 1250 cm -1 ) was not clearly observed on the PP_PDA_GDE spectrum, which might suggest an opening of the epoxy rings prior to the grafting of GDE, resulting in the presence of diol groups. |
6661b56b91aefa6ce1eaad8b | 26 | Consistently, the small shoulder observed at 1090 cm -1 may correspond to the C-C-O symmetric stretch of secondary alcohols present in the diols. However, upon functionalisation the membrane was turned hydrophilic (see water contact angle (WCA) analyses, Figure ) which creates large bands in the 3400 and 1630 cm -1 regions, hampering the observation of signature bands for amines, epoxides, or diols. a : These elements were detected in significant amounts (in some samples), but their presence is exclusively due to contaminations (either present on the original commercial membranes, or generated during the experiments). b : below detection limit. c : The value of the obtained ratio was < 0.01. |
6661b56b91aefa6ce1eaad8b | 27 | In XPS, pristine PP membranes (Figure ) showed nearly exclusively aliphatic C-(C,H) signal (Figure ), no nitrogen, and only traces of oxygen. Upon addition of PDA (Figure ) signals for oxygen and nitrogen logically appeared. Notably, the N/C ratio of the PP_PDA reaches a similar value to that of the theoretical value of the polydopamine polymer (N/C PDA = 0.125), suggesting the formation of a PDA coating of at least 10 nm thickness at the PP surface . As expected, the grafting of GDE (Figure ) on the amine residues present at the PP_PDA surface increased the oxygen surface concentration at the expense of nitrogen (Table , line 6), and the addition of PEI on PP_PDA_GDE (Figure ) resulted in a marked increase in the nitrogen content (and N/C ratio) (Table , line 7). |
6661b56b91aefa6ce1eaad8b | 28 | Scanning electron microscopy (SEM) allowed to verify that the morphology of the PP membrane was preserved after functionalization: the surface of pristine PP and PP_PDA_GDE_PEI showed similar porosity (Figure and), which confirmed that the membrane remains porous after functionalization. No change was pictured on cross-sections images either (Figure and), which indicates that the membrane porosity was intact. |
6661b56b91aefa6ce1eaad8b | 29 | TAs were immobilized on the membranes described above and tested in the kinetic resolution of BMBA. Figure shows the activity (in terms of BAP yield) displayed by the different heterogeneous biocatalysts obtained by the immobilization of TsRTA on the different membrane supports. When using the pristine PP or PAN membranes as supports for the enzyme, the activity was virtually nil. However, upon functionalizationand depending on the parameters of functionalization and immobilization (vide infra)significant biocatalytic activity was observed. In general, PP-immobilized TAs exhibited superior performance as compared to the PAN-immobilized TAs. Similar activity trends were obtained when employing HeWT as immobilized transaminase on these supports (Figure ). To interpret the raw yields obtained with the different enzyme-loaded membranes, complementary indicators must be considered. Table gathers the immobilization yield, specific activity recovery, and leaching fraction displayed by the obtained membraneimmobilized TAs, for both immobilization strategies. Regarding the HPAN_PEI immobilized biocatalysts, it can be observed that the immobilization yield is boosted when the TA immobilization was performed at pH 5.5 (HeWT_HPAN1b and TsRTA_HPAN1b) rather than 8 (HeWT_HPAN1a and TsRTA_HPAN1a). This can be explained by the fact the PEI is more positively charged at low pH and favors the electrostatic adsorption of a larger amount of TA at the membrane surface. Accordingly, the observed activity is higher. Importantly, the specific activity (activity normalized by the amount of immobilized TA on the membrane) was the same, which indicates that, on average, the intrinsic activity of each additional immobilized transaminases was maintained. However, leaching after catalytic test was important, highlighting the need of post-treatment strategies to improve the anchoring of the immobilized TAs at the membrane surface. |
6661b56b91aefa6ce1eaad8b | 30 | Regarding the HPAN_PEI immobilized biocatalysts, it can be observed that the immobilization yield is boosted when the TA immobilization was performed at pH 5.5 (HeWT_HPAN1b and TsRTA_HPAN1b) rather than 8 (HeWT_HPAN1a and TsRTA_HPAN1a). This can be explained by the fact the PEI is more positively charged at low pH and is thus able to electrostatically attract a larger amount of TA at its surface. Accordingly, the observed activity is higher. Importantly, the specific activity (activity normalized by the amount of immobilized TA on the membrane) was the same, which indicates that, on average, the intrinsic activity of each additional immobilized transaminases was maintained. However, leaching after catalytic test was important, highlighting the need of post-treatment strategies to improve the anchoring of the immobilized TAs at the membrane surface. Inspired by Shi et al. , we attempted to entrap the immobilized TA into a polymeric matrix formed by sodium alginate (SA, see Figure ; bottom). This biopolymer is able to electrostatically interact with the PEI layer, bringing additional negative charges that can in principle help stabilizing the enzyme. This post-treatment was found to preserve the enzyme loading and the specific activity, and concomitantly to reduce enzyme leaching (Table , compare HeWT_HPAN2b and TsRTA_HPAN2b to HeWT_HPAN1b and TsRTA_HPAN1b, respectively). |
6661b56b91aefa6ce1eaad8b | 31 | Such post-treatment strategy was found to (i) boost the enzyme loading (by securing the fixation of otherwise loosely attached TAs to the membrane surface), (ii) enhance the specific activity, and (iii) drastically curb the extent of enzyme leaching (Table , compare entry HeWT_HPAN3b and TsRTA_HPAN3b to HeWT_HPAN1b and TsRTA_HPAN1b, respectively). The surge in specific activity after treating with GA seems surprising, for crosslinking is known to rigidify the enzymes structure, and it is often argued to be the cause of partial deactivation (e.g. in cross-linked enzyme aggregates) . Yet the measurements were repeated (immobilization, activity assays, and Bradford tests to determine the loading) and the improvement was verified to be statistically significant (see the standard deviations in Table ). |
6661b56b91aefa6ce1eaad8b | 32 | Similar beneficial effect of such GA cross-linking of PEI-immobilized enzymes have been previously documented, with positive effects on specific activity (with lipases ), or on stability and reusability (with TAs 70 ). In fact, here, TA enzymes are not only cross-linked together but also bound to PEI via GA. We surmise that bonding occurs preferentially with PEI (rather than cross-linking). Hence, one hypothesis is that the higher specific activity obtained for TAs_HPAN3b is linked to a more favorable (more hydrated, less constrained) chemical microenvironment conferred by the PEI layer to the immobilized TAs . |
6661b56b91aefa6ce1eaad8b | 33 | Simple adsorption of TA on PP_PDA membranes led to important leaching (entry 9 and 12). However, using the covalent immobilization approach with GDE, TA leaching was significantly reduced (Table , compare HeWT_PP2 and TsRTA_PP2 with HeWT_PP1 and TsRTA_PP1, respectively). This highlights the beneficial role of the epoxy functions, able to immobilize the TA via covalent coupling . Interestingly, TA_PP2 also showed greater immobilization yield and specific activity with respect to TA_PP1 biocatalyst. Such enhanced specific activity obtained with GDE-immobilized TAs has already been observed in literature, and it was attributed to the hydrophilic and appropriate length of the epoxy linker-arm . |
6661b56b91aefa6ce1eaad8b | 34 | Further functionalization with PEI resulted in a lower immobilization yield, but a higher specific activity (Table , compare HeWT_PP3 and TsRTA_PP3 with HeWT_PP2 and TsRTA_PP2 respectively). The enhanced specific activity recovery displayed by TA_PDA_GDE_PEI can be tentatively attributed to a more favorable (hydrated) chemical microenvironment conferred by the PEI layer to the immobilized TAs . |
6661b56b91aefa6ce1eaad8b | 35 | systematically and optimized (see ESI, Figure -18) to lead to the results reported in Table . Overall, the catalytic performance of these membrane-immobilized TAs compares well with other immobilized TAs described in literature. Indeed, typical transaminase immobilization via covalent grafting on metal-derivatized epoxy resins yields only 30-50 % recovered specific activity . |
6661b56b91aefa6ce1eaad8b | 36 | In order to assess if the activity displayed by the developed immobilized TAs can only be attributed to heterogeneous catalysis, hot-filtration-tests were performed on both optimized TsRTA_HPAN3b and TsRTA_PP3 biocatalysts (Figure ). The TA-loaded membrane was removed from the reaction media after a short reaction time (15 minutes), and the activity was monitored and compared to a classical catalytic test (i.e. in which the membrane was not removed). Some residual activity could be detected after the removal of TsRTA_HPAN3b (Figure ), which highlights the fact that a small fraction of leached TsRTA contributed to the observed activity. Such leaching fraction was either estimated to 5.1 % (in the form of immobilized TsRTA) or 1.8 % (in the form of soluble TsRTA), based on the slope between 15and 60-minutes activity points. This can be explained by a slow hydrolysis of the imine bonds between GA and TsRTA or PEI. On the other hand, the hot-filtration test exhibited a completely flat profile after the removal of TsRTA_PP3 biocatalyst (Figure ), demonstrating that, in this case, only heterogeneous catalysis is involved in the observed activity. The two selected catalysts were also tested in 4 successive catalytic cycles to assess their recyclability. At the end of each cycle, the membrane-immobilized TAs were washed twice with 5 mL of buffer solution (i.e. HEPES 0.1 M pH 8 containing PLP 1 mM, pyruvate 10 mM), and then immersed into a fresh reaction medium. The obtained reaction profiles (Figure ) |
6661b56b91aefa6ce1eaad8b | 37 | unambiguously show that the membrane discs were recyclable. In all cases, the same final conversion (close to thermodynamic equilibrium) could be reached. More importantly, specific activity (approached by initial activity) was not affected throughout the successive catalytic cycles. This result paves the way toward a possible use of membrane-immobilized enzymes in continuous flow processes. Such robustness and recyclability was also confirmed with the Sselective HeWT enzyme (Figure ), as no significant decrease of specific activity could be observed throughout the cycles. Another interesting aspect to investigate was the ability of the biocatalytic membrane to work in the absence of externally added co-factor (PLP). Such ability has already been reported on PEI-coated supports, onto which both PLP and TA could be co-immobilized . This aspect would be particularly important in the perspective of a continuous flow membrane reactor, since it would allow to get rid of the costly PLP feed during the operation. Hence, TsRTA_PP3 (for which only the TA immobilization step is done in the presence of PLP) was tested in successive catalytic cycles without adding PLP to the reaction media (Figure ). In such case, the residual activity dropped after each reaction cycle (i.e. down from 78 % to 23 % after five cycles). This suggests a significant PLP leaching leading to immobilized TA deactivation. In order to overcome this problem, we slightly adapted the immobilization process. Inspired from López-Gallego et al. we implemented a two-step immobilization. |
6661b56b91aefa6ce1eaad8b | 38 | Briefly, after performing classical enzyme immobilization (as always, in presence of PLP, i.e. 0.1 mM or 1 mM), a subsequent step of PLP immobilization was performed at lower ionic force in order to favor the co-factor grafting at the PEI-coated membrane surface. The resulting membrane was tested in 8 successive catalytic cycles without PLP addition, and exhibited much higher stability as compared to TA_PP3. In particular, TA_PP3_SS0.1 did not show any activity drop. That remarkable stability displayed by TA_PP3_ SS0.1 might be explained by the higher PLP loading achieved for this catalyst (0.97 µmol as compared to TA_PP3_ SS1 (0.85 µmol) (Table ). These results suggest that upon this two-step immobilization strategy (and employing 0.1 mM PLP for TA immobilization step), PLP is suitably provided to the enzyme (i.e. available for the transamination catalytic act) in satisfying amounts. It is noteworthy that performing TA immobilization with 0.1 mM (instead of 1 mM) of PLP also enabled to boost the TA loading and increase the overall activity, but it lowered the specific activity of the catalyst (Table , line 3). Finally, chiral HPLC analyses allowed us to confirm that the immobilization process did not affect the biocatalysts enantioselectivity (Table ). To this aim, the produced BMBA enantiomers obtained when employing the two best-performing immobilization strategies (namely TA_HPAN3b and TA_PP3) were analyzed and quantified. For all four membraneimmobilized TAs studied, only one BMBA enantiomer was detected, suggesting that the obtained biocatalytic membranes are enantioselective. Since the investigated reaction is a kinetic resolution (i.e. starting from a racemic mixture), the unconverted BMBA enantiomer (e.g. R-BMBA for HeWT, S-BMBA for TsRTA) was always detected by chiral HPLC (Figure -22). |
6661b56b91aefa6ce1eaad8b | 39 | As important enzyme leaching was observed on the electrostatically immobilized TAs, post-treatment strategies of the electrostatically immobilized TAs were applied to improve TA anchoring at the functionalized PAN membrane surface. Among the developed strategies, the covalent binding of TAs and of the PEI layer using glutaraldehyde (GA) gave the most satisfying results (high specific activity, minor leaching). On the other hand, the TA covalent grafting on functionalized PP membranes yielded even more efficient biocatalysts (higher specific activity) displaying enhanced robustness (no leaching) and full recyclability. |
6661b56b91aefa6ce1eaad8b | 40 | Importantly, these two strategies allowed to efficiently immobilize two different TAs (the Sselective HeWT, and the R-selective TsRTA), resulting in stereo-divergent biocatalytic membranes. Additionally, co-immobilization of TA and PLP was also achieved on functionalized PP membranes by adapting the immobilization protocol, which resulted in highly reusable membrane-immobilized biocatalysts capable of catalyzing transaminations in absence of externally added PLP. Such self-sufficient ability should be attractive from an industrial point of view, since it should help increasing the cost-efficiency and reducing the Efactor of the transamination process. Thus, all in all, both studied routes to immobilize TAs on membranes led to functional biocatalytic materials exhibiting perfect enantioselectivity, high catalytic performance, minor leaching and excellent reusability. This paves the way to a future use in flow mode hybrid processes. |
6661b56b91aefa6ce1eaad8b | 41 | The transfer and implementation of such biocatalytic membranes in continuous flow (as a flat-sheet membrane reactor) has now to be carried out. Ultimately, more challenging transamination reactions (i.e. asymmetric synthesis) should be tackled with this immobilized TA, by taking benefit of the ability of the membrane carrier to act as a separation unit for (co)product removal. |
670c327d12ff75c3a151ebfd | 0 | As we strive towards a sustainable energy economy, it becomes increasingly urgent to optimize electrochemical reaction rates at the practical device level. In heteroge-neous electrocatalysis, the reaction rate is a direct measure of the overall current which inexorably intertwines the kinetics of the catalytic surface reaction with mass transport. This crucial interplay manifests in a number of different phenomena including the diffusion-limited transfer of reactants to the active surface , ion crowding or so-called "local" pH effects , as well as the diffusion-controlled desorption/readsorption of surface-bound reaction intermediates . While surface kinetics are determined by atomistic processes over nanoscopic active sites, however, transport effects typically emerge over micrometer length scales. Bridging these two disparate scales into a (single) comprehensive reaction model is an ongoing, yet key, challenge both from a conceptual as well as a methodological point of view. |
670c327d12ff75c3a151ebfd | 1 | The actual coupling is then achieved through a mass flux boundary condition at the electrochemical interface which establishes an interdependence between the two processes. Solving the resulting multi-scale problem thus always requires that near-surface fluxes and concentrations are aligned between the kinetic and transport models. Depending upon how these two models are set up and combined in practice, however, we distinguish two basic modeling approaches in the current literature. The approaches generally differ in their primary focus and, hence, the level of theory used for the individual model components, as illustrated in Figure . The first approach uses phenomenological kinetics to approximate the partial current density J surf that is generated through the surface reaction and fed as input into a (usually) rather elaborate transport model. As a result, this approach often addresses more complex transport phenomena and mechanisms (diffusion, migration, convection) or design strategies at the device level through, e.g., simulations of realistic porous electrode morphologies, gas diffusion electrodes (GDEs), and even full electrolyzers (including cathode/anode and membrane) . Alternatively, the second approach is to put the stronger focus on the surface kinetics and couple to transport through a predictive-quality J surf that is computed from a first-principles based microkinetic model (1p-mkm). This approach will thus directly include the free energy profile of elementary surface reaction steps while integrating microscopic details of an assumed catalytic mechanism. At this point, it should be em-phasized that the above classification simply reflects our view of the prevailing status quo and that there is, in principle, no conceptual limitation to, e.g., coupling a 1p-mkm to a most elaborate mass transport model. This situation is analogous to that observed in the field of thermal heterogeneous catalysis. This short review aims to give a current overview over corresponding transportcoupled kinetic models for heterogeneous electrocatalysis. We first separately discuss the two aforementioned modeling approaches, distinguished by whether the surface reaction is included via a phenomenological or first-principles based kinetic model. |
670c327d12ff75c3a151ebfd | 2 | Focus is specifically put on some of the main conceptual and methodological differences between these two approaches, while demonstrating the advantages and value of each through notable examples in the recent literature. Finally, we comment upon insights realized, remaining challenges, as well as the prospects of combining the two "schools of thought" in future research. A phenomenological treatment of the surface kinetics, e.g. via a Butler-Volmer expression, generates an empirical J surf that is (usually) combined with modeling more complex transport phenomena and design strategies at the device level. On the other hand, a first-principles based microkinetic model (1p-mkm) integrates into J surf a predictive-quality description of the free energy reaction path with specific focus on elucidating the underlying microscopic catalytic mechanism. |
670c327d12ff75c3a151ebfd | 3 | A phenomenological treatment of surface kinetics is meant to provide a simple, yet effective, ansatz for the resulting current density J surf at the surface. Common formulations here include a potential-dependent Butler-Volmer (BV) expression, or just the simple assumption that this J surf is a constant. In the latter case, J surf is simply approximated based on a specific faradaic efficiency and directly fed as input into the transport model with no effect from the applied potential U or interfacial species' concentration C. On the other hand, the BV formalism (which simplifies into a Tafel expression at large overpotentials) represents the more sophisticated ansatz, but comes at the price of relying upon a set of empirical parameters that must be a priori defined. These parameters primarily include the exchange current density J 0 and cathodic/anodic transfer (or symmetry) coefficient α within the standard BV formalism. Most often the basic BV formulation is extended by an additional concentration-dependent term that allows for a more advanced, transport-aware model : |
670c327d12ff75c3a151ebfd | 4 | where U eq. is the equilibrium reaction potential, C ref is a reference concentration (usually set to 1 M), and n represents a specific reaction order which is either taken from the literature or treated as an additional fitted parameter. The typical fitting procedure of above BV parameters generally involves the following steps : a 1D transportcoupled kinetic model is solved first , while essentially assuming a planar electrode with J surf set equal to the experimental product J measured in the bulk electrolyte at a given U. This solution gives an initial potential-dependent C profile which is then used to fit the included model parameters. The resulting BV expression is finally solved iteratively within a 2D or 3D set-up (according to the surface geometry of interest), yielding a self-consistent solution for J surf and C as a function of U. Note that, while practical, this approach suffers from its inherent dependence upon the above fitted parameters which have been shown to sensitively change the model's results . |
670c327d12ff75c3a151ebfd | 5 | The efficiency of phenomenological surface kinetics is most usually paired with a more elaborate transport model, either in the underlying transport equations themselves, considered electrode geometry, or both. While early such models focused mainly on planar electrodes , the approach has since been extended to include also, e.g., macroscale so-called "volume averaged" simulations as well as the simulations of pores . We specifically highlight in Figure two recent studies on GDEs . These examples nicely demonstrate how elaborate transport models, coupled to BV surface kinetics, can predict complex concentration profiles in the diffusion layer; both as a function of electrolyte composition and electrode geometry. Such fundamental insight into transport processes within a GDE is key to its future design and optimization . Upon developing an elaborate 2D model for ion migration, Butt et al. investigated the influence of electrolyte composition on the local reaction environment within a GDE catalyst nanopore. Figure ) shows simulated concentration profiles for the reactant CO 2 as a function of its distance from the catalyst surface at different KHCO 3 buffer concentrations. Increasing K + concentration predicts lower CO 2 solubility (modeled here through a 'Sechenov-corrected' Henry's constant ), but also induces steric hindrance at the electric double layer due to ion crowding. Together these two effects limit the CO 2 that can reach the surface and react, thus affecting catalytic performance. |
670c327d12ff75c3a151ebfd | 6 | where the catalyst is modeled as a sinusoidal layer of varying thickness (l) and wave periodicity (P). The results show that CO production has a much more complex dependence on catalyst shape than what might be simply expected from estimates of the corresponding surface roughness (ρ), leading to e.g. a peculiar activity maximum at intermediate ρ values. |
670c327d12ff75c3a151ebfd | 7 | A mean-field microkinetic model of the catalytic surface reaction predicts J surf by breaking the process down to a series of consecutive elementary reaction steps, deriving a rate expression for each, and solving the resulting set of ordinary differential equations. Near-surface concentrations and turnover frequencies predicted in this way subsequently serve as boundary conditions when coupling to mass transport. While there are many excellent reviews on recent mkm developments , it is clear that the overall approach hinges upon the reliability of the assumed reaction mechanism and input kinetic parameters. The huge benefit, but also cost, of a 1p-mkm is its predictive quality through explicitly calculated reaction energies and barriers at an ab initio level. Such calculations are nowadays becoming increasingly tractable within density functional theory (DFT), at least within small simulation cells and simple solvation models. Including a more advanced description of the electrolyte as well as the effect of applied potential at the solid/liquid interface, however, represent the forefront in the field. The vast majority of studies currently still include U only at the level of thermodynamic reservoirs via the so-called computational hydrogen electrode while relying on BEP scaling relations, vanishingly small or fitted barriers for the reaction kinetics . Possibly more critical, however, is the almost standard approximation of a 1D transport-coupled model that assumes uniform concentration profiles regardless of the catalyst's 2D or 3D morphology. The selectivity is measured against further reduced carbon-coupled products and plotted as a function of applied potential U. Key here is the kinetic competition between the surface-and solution-reaction of a ketene (Kt) intermediate to form acetate (Ac -), as illustrated in the figure's inset. This competition is resolved via a diffusion-coupled 1p-mkm, predicting a complex "U-shaped" selectivity profile with U that shifts with pH and catalyst roughness (ρ). Adapted from the study by Heenen et al. . |
670c327d12ff75c3a151ebfd | 8 | Because of their predictive-quality description of the surface kinetics, transportcoupled 1p-mkm models can provide us with valuable (atomically-resolved) mechanistic insights. Early such coupled models, for example, already found some success in elucidating catalytic mechanisms by comparing simulated polarization curves or reaction orders against experiments . More recent models have now advanced to further include a more realistic description of the electrical double layer while explicitly including field-dependent DFT kinetics. Ringe et al., for example, simulated CO 2 electro-reduction over Au by (self-consistently) integrating such a 1pmkm with a 1D transport model that could account for diffusion, migration, and buffer reactions . Figure ) reproduces the resulting polarization curve which shows potential-dependent transitions between three distinct kinetic regimes: from a ratelimiting *COOH reduction step at low overpotentials, to the field-driven adsorption of CO 2 at intermediate ones, and finally CO 2 mass transport at high overpotentials. |
670c327d12ff75c3a151ebfd | 9 | Importantly, this work shows that the experimental Tafel slope is only captured here by explicitly including CO 2 dipole-field interactions when modeling the electrochemical kinetics. Another situation which requires the detailed accuracy of a 1p-mkm is the senstitive kinetic competition that often arises in questions of electrocatalytic selectivity. For example, one of us recently discussed such a competition within a "desorption-re-adsorption-reaction" mechanism to explain the selectivity towards acetate during CO electro-reduction over Cu . The mechanism focuses on a specific surface-bound reaction intermediate and competing routes forward: continued conversion at the surface vs desorption and a subsequent solution reaction to form an early partially-converted product. This competition can only be resolved via a diffusioncoupled 1p-mkm which, in the case of acetate, yielded an intricate dependence upon potential, pH, and catalyst roughness ρ. The simulated selectivity is plotted in Fig- |
670c327d12ff75c3a151ebfd | 10 | In summary, we discuss two prevailing trends for modeling transport-coupled kinetics in heterogeneous electrocatalysis. The two approaches generally differ in the target scientific questions that they aim to answer and, hence, in whether the larger focus is put on the description of the catalytic surface kinetics or mass transport. While the value of each of these models is easily demonstrated through many notable studies in the existing literature, remaining practical and methodological challenges are cur-rently the object of ongoing research. Such efforts include, for example, developments towards a more systematic fitting of BV parameters as well as the exploration of improved phenomenological kinetic models such as, e.g., the Marcus-Hush-Chidsey model . Simultaneously, in the realm of 1p-mkm, extensive methodological work is presently being devoted to advancing simulations of the working electrochemical interface while providing constant-potential energies and barriers . Most noteworthy are also efforts toward an automatic exploration of reaction networks using machine learning . Promising research along this direction aims to lift the bias in assuming a catalytic mechanism which is usually based on chemical intuition. |
670c327d12ff75c3a151ebfd | 11 | One of the more natural directions forward of course involves the coupling of 1pmkm with elaborate transport models, in an obvious attempt to exploit the "best of both worlds". Although there is no real practical bottleneck to conducting such simulations, it represents an interdisciplinary challenge, closely intertwining the fields of theoretical ) with a 2D flow cell geometry that treats the catalyst as an active strip . The resulting mechanistic insight was found similar to that obtained in the original study by Ringe , thus highlighting the potential to further explore the effect of different geometries and flow conditions. Also within our group, efforts are already underway to expand the 1D concept of surface roughness to an explicit 2D or 3D geometry within the "desorption-re-adsorption-reaction" mechanism. Similarly, the concepts to couple spatially resolved kinetic Monte Carlo mkms to transport have also been established years ago for thermal catalysis and only wait to be employed within the context of heterogeneous electrocatalysis. These and similar advances will undoubtedly strengthen our understanding of detailed reaction mechanisms under (practical) operating conditions, while simultaneously allowing to gauge the level of detail that is required to be accounted for. The continued development of improved transport and kinetic models, along with accelerated ways to couple the two, thus offers a lot of promise in bridging the gap between atomistic processes at the electrified interface and mesoscale transport processes. |
62ea4f5f04c85faeb582173b | 0 | In the last decades, the alkynylation of nitrogen-based nucleophiles has been the focus of intensive research due to the synthetic versatility of the formed ynamines/ynamides products. Most methods relied on copper-catalysis with free or pre-activated alkynes (including halides, carboxylic acid, organometallic reagents and hypervalent iodine reagents (HIR), Scheme 1A). 1 Among the potential coupling partners capable of transferring alkynes, HIR particularly attracted attention as they allowed the use of milder reaction conditions. While alkynylation using mononitrogen-based nucleophiles is well established, only few examples of the synthesis of ynehydrazides are reported. Initial attempts to alkynylate hydrazides with alkynyl HIR or bromalkynes required harsh conditions and/or afforded the products in poor yields. To circumvent this issue, Batey and Beveridge followed an Umpolung strategy using azodicarboxylates as hydrazide precursors and acetylides nucleophiles (Scheme 1B). The obtained products could then be used in diverse applications. 3e-h However, this transformation was limited to symmetrical azodicarboxylates and required a strong base. Therefore, the development of a milder protocol suitable for non-symmetrical hydrazides would be beneficial. |
62ea4f5f04c85faeb582173b | 1 | Among hydrazide nucleophiles, azaglycine derivatives constitute a unique class (Scheme 1C). These amino acid analogs, in which the α-carbon is replaced by a nitrogen atom, enable the fine-tuning of the structural and conformational features of bioactive peptides. Notably, azapeptides exhibit enhanced protease stability, 5 greater H-bonding capability, and usually favor β-turn. While N-alkylation and N-arylation of azaglycine have been reported, to the best of our knowledge, N-alkynylation remains unexplored. Moreover, α-alkynyl amino acids have been shown to be irreversible enzyme inhibitors and versatile building blocks in the synthesis of bioactive compounds. An easy access to their α-alkynyl azaglycine analogues would therefore be valuable. Scheme 1. (A) Alkynylation of nitrogen-based nucleophiles, (B) Current method to access ynehydrazides, (C) Towards -alkynyl azaglycine derivatives, (D) Our work. |
62ea4f5f04c85faeb582173b | 2 | Our group and others have shown that HIR were ideal reagents for the selective functionalization of peptides. Herein, we report the successful copper-catalyzed alkynylation of azadipeptides (Scheme 1D). The mild reaction conditions allowed a broad functional group tolerance on the side chain of the peptides. Various types of alkynes could be transferred and undergo further transformations, enabling an easy access to functionalized azadipeptides. |
62ea4f5f04c85faeb582173b | 3 | We started our investigation using azadipeptide 2a derived from proline with the C-terminal protected with a tert-butyl group as a model subtrate. Reaction of the later with TIPS-EBX (3a) for 1 hour in the presence of Cu(CH3CN)4BF4 and potassium tert-butoxide led to the formation of 4a in an encouraging 20% yield (Table , entry 1). A similar result could be obtained using Cs2CO3 as a milder base (entry 2). Increased formation of the desired product was observed when the reaction was heated to 40 °C (entry 3). Replacing acetonitrile by i-PrOH or DCE increased the yield to 48% and 60% respectively (entries 4 and 5). Several copper catalysts can promote the reaction, with the best result obtained using CuI, affording 76% of 4a (entries 6-8). No difference could be observed when the transformation was carried out with an excess of TIPS-EBX (3a) (entry 9). Control reactions without base or copper catalyst resulted in, respectively, no reaction or degradation of the azapeptide (entries 10 and 11). Finally, DCM could be used as an alternative to DCE without impacting the reaction outcome (entry 12). a Reaction conditions: azapeptide 2a (1.0 equiv.), TIPS-EBX (3a) (1.0 equiv.), catalyst (5 mol%), base (1.5 equiv.), solvent (0.1 M), reactions were carried out under air on a 0.05 mmol scale. Isolated yields are reported. b TIPS-EBX (3a) (1.5 equiv.) |
62ea4f5f04c85faeb582173b | 4 | With the optimized conditions in hand we started to explore the scope of amino acids present on the urea. Carrying out the reaction on scope scale (0.3 mmol) using the model substrate afforded 4a in 76% yield. Simple glycine or alanine gave the corresponding alkynylated azapeptides 4b and 4c in good yields. Product 4b could be obtained in 97% yield on a 1 mmol scale. More sterically demanding valine only afforded 38% of 4d. Aromatic residues are tolerated in the reaction and good yields can be obtained for phenylalanine (2e) and tryptophan (2f). However, lower efficiency was observed with tyrosine (2g), probably due to the presence of an unprotected phenol. Methionine derived azapeptide (2h) could be alkynylated in 58% yield with no side reactivity of the sulfur atom with the hypervalent iodine reagent. 11 With serine, product 4i could be obtained in a moderate 29% yield. Amino acids bearing additional nitrogen atoms, such as protected lysine (2j) or asparagine (2k) were alkynylated selectively on the azaglycine affording 4j and 4k in 76% and 56% yields, respectively. Protected glutamic acid (2l) was well tolerated in the reaction. Finally, using a methyl carbamate protected azaglycine, we could access 4m in 60% yield. Replacing the protecting group with a bulkier tert-butyl carbamate (2n) led to a drop in yield. Scheme 2. Scope of amino acids. a a conditions: azapeptide 2a-n (1.0 equiv.), TIPS-EBX (3a) (1.0 equiv.), CuI (5 mol%), Cs2CO3 (1.5 equiv.), DCM (0.1 M), 40 °C, 1 h, reactions were carried out under air on a 0.3 mmol scale. Reaction was performed on 1 mmol scale. |
62ea4f5f04c85faeb582173b | 5 | Having established the compatibility of the reaction with different amino acids, we next explored the variety of alkynes that could be transferred with azapeptide 2b as partner. A variety of EBXs could be easily prepared by using established procedures or the most recent protocol developed by our group (1 h reaction time, no additives, no purification). High yields of alkynylated azapeptides could be maintained when replacing the TIPS group by a simple phenyl (3b) or mesityl (3c) substituent. The structure of 5b was determined by X-ray diffraction and displayed a trans-amide geometry in the solid state. Electron-withdrawing substituent on the aryl ring such as fluoride or bromide were well tolerated affording 5c and 5d with only a slight decrease in yield. Alkyl substituted alkynes could also be transferred. Alkynylated azapeptide 5e bearing a methyl group was obtained in 40% yield, and higher yield could be obtained with a cyclopropyl group (5f). Having a chloride on the alkyl chain was tolerated and 5g could be obtained in 53% yield. Scheme 3. Scope of EBX reagents. |
62ea4f5f04c85faeb582173b | 6 | Finally, we explored transformations of the alkynylated azapeptides. Hydration of the alkyne using PTSA afforded the corresponding amide moiety 6 bearing an α-silyl group. To the best of our knowledge, acylation of azaglycine has not been reported so far. For instance, chloroacetyl chloride has been shown to react with the hydrazone C=N bond. 5-Cyclization of the second nitrogen of the urea onto the alkyne afforded the product 7 in 63%. Similar cyclic scaffolds have been shown to induce -turn conformations in peptides. This type of cyclized product could only be observed as traces (<5%) during the formation of alkynylated azapeptides, which proceeded at lower reaction temperature with a shorter reaction time. Attempts to deprotect the TIPS substituted product 4a showed that the resulting free alkyne was too unstable to be purified. To circumvent this issue, the unsubstituted alkyne was directly engaged in a copper catalyzed alkyne-azide cycloaddition affording triazole 8 in 76% yield over 2 steps. Furthermore, N-terminus deprotection using hydroxylamine afforded the free hydrazide 9 as the TFA salt in 32% isolated yield after purification by reverse-phase HPLC. In summary, we have developed conditions to perform the alkynylation of azadipeptides using EBX reagents and an inexpensive copper catalyst. The reaction is selective to the nitrogen of the azaglycine residue and tolerate a large variety of amino acids affording the desired alkynylated azadipeptides in moderate to good yields. The transformation is not limited to silyl protected alkynes and both aryl and alkyl acetylenes can be transferred. The products could be further functionalized using classical alkyne reactivity affording different azadipeptide derivatives. |
67da98eefa469535b9c8b1cd | 0 | ABSTRACT: Metathesis and reversible catalytic reactions are fundamentally intriguing and powerful tools in modern synthetic chemistry. While most reversible catalytic reactions are predicated on breaking and forming reactive functional groups, the ability to leverage the C-H bond as a functional group into metathesis reactions has proved to be exceptionally challenging. Here, we develop a C-H/C-X metathesis reaction through a radical swapping protocol where a hydrogen and halogen are traded between molecules via reversible hydrogen atom transfer (HAT) and halogen atom transfer (XAT) that allows for mild C-H halogenation. The reversibility of this process allows for selective dehalogenation of polyhalogenated products to form monohalogenated products. Leveraging the reversibility of this process, halogenated organic pollutants can also serve as a halogen source for C-H halogenation. In the broader context, this work establishes that incorporating reversible metathesis logic in C-H bond functionalization can provide complementary advantages in synthetic strategies. |
67da98eefa469535b9c8b1cd | 1 | Metathesis reactions represent a mild, and complementary synthetic approach towards constructing complex molecules and polymers. The ability to run the reaction in the forward or reverse direction to construct or deconstruct the bonds using the same catalytic system are attractive features which allows for reversible reorganization of molecular fragments that are not possible using conventional reactions. Olefin metathesis (Figure ) is one of the most prominent examples of reversible metathesis reactions, and has led to exciting applications such diverse fields as biomass valorization, commodity and fine chemical synthesis, waste upcycling, supramolecular assembly, and recyclable polymer synthesis. Using the metathesis strategy, our group and others have developed catalytic functional group metathesis reactions between two different reactive functional groups. However, the broader applicability of these new methods, as well as the established applications above, is clearly limited as two preinstalled reactive functional groups are required on both reaction partners. On the other hand, C-H functionalization makes use of ubiquitous C-H bonds to directly form elaborated products without the need for prefunctionalization. A reversible metathesis approach to C-H bond functionalization would thus merge two attractive concepts and provide exciting prospects for synthetic chemistry, however, this approach has remained considerably underexplored. Among the rare examples of C-H bonds being used in reversible metathesis reactions, the most notable are C(sp 2 )-H to C(sp 2 )borylation/iodination where self-correcting abilities have led to interesting selectivities. In addition to these methods, an important industrial application of C-H metathesis is the C-H/C-Me metathesis of toluene to form xylenes and benzene. Given these precedents, it would be particularly attractive to incorporate native C(sp 3 )-H bonds into metathesis strategies to expand the capabilities of C-H functionalization, and enable a new class of metathesis reactions (Figure ). Using the conceptual appeal of a C-H bond metathesis (Figure ,D), we believed a C(sp 3 )-H benzylic halogenation would be attractive and complementary to traditional methods. Modern reagents have been developed for unactivated C(sp 3 )-H halogenation, but benzylic C(sp 3 )-H positions still largely rely on classical reagents. These reagents (X2: X = Br or Cl, or AIBN and NBS/NCS) often lead to overhalogenation, and require tailored optimization for individual substrates. The reverse reaction, i.e. dehalogenation, cannot be achieved using these methods and requires different conditions which commonly employ toxic tin reagents, although modern methods offer some alternatives. "Overhalogenation" is another common problem associated with classical methods where a metathesis approach could offer the prospect to regenerate a desired monohalogenated product by reacting the overhalogenated material in reverse. First, we sought to identify an HAT catalyst and XAT catalyst which could reversibly cleave and form a C-H and C-X bond and proceed through a common intermediate, in this case a free radical. Here a diverse range of catalysts that have been utilized in deracemization, epimerization, functional group migration, and "radical sampling" 53 could be surveyed. Reversible halogen atom transfer (XAT), as employed in controllable radical polymerization like atom transfer radical polymerization (ATRP) serves as a platform for evaluating different XAT catalysts. Despite separate applications of reversible HAT and XAT, identifying two catalysts that can simultaneously, yet independently, activate a C-H bond (through HAT) and a C-X bond (through XAT) has remained challenging. This is likely due to the challenges of matching the kinetics of both cycles, and preventing the catalysts from interfering with each other. We began our investigation by surveying a panel of Culigand combinations commonly employed in ATRP in combination with HAT catalysts (Figure ). Despite reports of dual catalytic cycles with decatungstate (DT) and copper, only dehalogenation and radical dimerization were observed clearly demonstrating the challenges of matching activation kinetics and catalyst compatibility. We then shifted our focus to aryl ketone HAT catalysts. After evaluating several HAT catalysts (entries 2-3), 5,7,12,14pentacenetetrone (PT) proved uniquely effective for this transformation (entries 4-8). The preference of excited state PT to undergo HAT instead of electron transfer, or energy transfer pathways led the success of this catalyst and allowed us to overcome the above-mentioned challenges (vide supra). To test the efficacy of the system for selective halogen sorting, a mixture (~7:1) of monohalogenated 3a (0.50 mmol), dihalogenated 3a' (0.075 mmol), and non-halogenated 1a (0.15 mmol) were subjected to the optimized reaction conditions. After full conversion of 3a', 3a (0.57 mmol) was obtained in 86% yield (yield based on mols of Cl). Besides its mechanistic value, this experiment also indicates the possibility to use halogen sorting to convert a mixture of polyhalogenated isomers to a single product. We propose that the HAT and XAT catalysts are working in concert to predictably and selectively control the metathesis products. The HAT catalyst operates under kinetic control (consistent with the observed primary KIE of 1.9, Fig. ) and cleaves the most electron-rich sterically accessible C-H bonds in the forward direction. In the reverse direction, it prefers to form electron-poor C-H bonds through proton and electron transfer to electronpoor radicals. The XAT catalyst can activate both electronpoor and electron-rich C-X bonds, although activation of electron-poor C-X bonds is faster. The electron-poor Cu-II intermediate then combines faster with the more electronrich radical over the electron-poor cyanomethyl radical. The activation of all C-X bonds is fully reversible, however due to other irreversible or slow steps in the HAT cycle, benzylic C-X/C-H bonds can only exchange very slowly (See supplementary information for benzyl C-X/C-H exchange control experiments). Running the reaction in the forward direction, simple benzyl and napthyl deratives (2ab; 3a-b)and 2c-e and 3c-e which contain typical crosscoupling handles could be halogenated in good yield. Secondary C-H bonds (1f-i) also worked in good yield for bromination and chlorination, although attempted bromination of cyclic systems led to desaturation forming styrenes (2f). The unprotected drug molecule Ibuprofen provided the desired product 2h and 3h with complete selectivity for the more electron rich benzylic C-H bond, and no competing decarboxylation. Protection of Ibuprofen as the methyl ester increased the yield significantly for bromination product 2i. An electron-donating group in the para position worked well for chlorination (3j), although the brominated product 2j was lower yielding and unstable towards isolation. Ortho substituents on 1k-l resulted in lower yields for both bromination and chlorination with significant amounts of unreacted started material. Strong electron withdrawing groups and electron-poor heterocycles (pyridines, or Ar-NO2/-CF3) in any position of the ring completely shut down the reaction suggesting the C-H bonds are too electron-poor to be activated. The use of the bulky PT catalyst allowed us to overcome classical selectivity of C-H halogenation and preferentially functionalize the sterically most accessible C-H bond over the weaker C-H bond. P-cymene 1m, gave complete selectivity for the methyl group, which contrasts with the selectivity of NBS or Cl2 . The methyl group could even be discriminated over a secondary benzylic position, giving a modest selectivity of 1.7:1 for 1n. This inverts selectivity trends observed with modern methods which give >20:1 selectivity for secondary bromination or 2.4:1 for secondary chlorination. When applying the conditions to unactivated aliphatic C-H bonds, lower yields were obtained when using one equivalent of C-H starting material. This is consistent with previous reports where PT is employed, and is consistent with Stern-Volmer quenching experiments showing less-efficient quenching of the excited photocatalyst by aliphatic substrates compared to benzylic substrates (See supplementary information). Using an excess of C-H partner, cyclooctane gave good yields to form 2r and 3r, and adamantane gave good yields with complete selectivity for 1-halo adamantanes 2s and 3s. Using the natural products (+)-sclareolide, and isosteviol methyl ester which are benchmarked substrates for selectivity in C-H HAT processes, we obtained 2t/3t and 2u/3u. Although 2t/3t are known in the literature, 2u/3u have not been reported, and the regioselectivity of the product 2u was unambiguously assigned by X-ray diffraction analysis. The use of the C-H metathesis reaction was then evaluated as a tool for transferring halogens from halogenated persistent organic pollutants in an upcycling reaction. With millions of tons of chlorinated POPs remaining stockpiled, current protocols for their disposal involve expensive transport to specialized incinerators, thereby making halogen upcycling an increasingly attractive approach. Using DDT or lindane as the limiting reagent, toluene (1a) could be chlorinated in high yield giving 3a, while aliphatic substrates such as adamantane (1s) provided moderate yields of 3s. Utilizing brominated flame retardant HBCD as a brominating source, cyclooctane provided moderate yields, however, the benzylic bromination performed significantly worse than the chlorinated POPs. In this case, we attribute these low yields to strong product inhibition of 2a which may react with the Cu-I orders of magnitude faster than HBCD thereby shutting down the reaction. Similar to other reports which report incomplete conversion of halogenated pollutants, this strategy may be use in synthetic chemistry, but challenging to employ as a large-scale remediation strategy. |
67da98eefa469535b9c8b1cd | 2 | Next, we sought to utilize the forward reaction and reversible halogenation for a halogen sorting reaction. In the original manufacturing process of Valsartan reported by Novartis, the C-H bromination of 1v was performed with NBS and AIBN in chlorobenzene at 100 °C. To prevent excessive product loss through overhalogenation, the reaction was carefully monitored and stopped at 70% conversion, the chlorobenzene evaporated, and the overhalogenated 2v' was removed by solvent washes, followed by addition of more NBS and resubjecting the remaining starting material. Using our method, we stopped the reaction at ~80% conversion, then resubjecting the mixture (0.42 mmol of 2v, 0.075 mmol of 2v', 0.125 mmol of 1v) to the catalytic Figure . Figure . A. Increasing the yield of the first step in synthesis of Valsartan by halogen sorting of the mixture of brominated products, and directly by catalytic C-H bromination. B. [2+2] cycloaddition of in situ generated dichloroketene with indene to afford 3ad followed by selective monodechlorination. chlorinating conditions. 2v' could be reconverted to the desired 2v, boosting the total amount of 2v to 0.48 mmol. As an alternative to the halogen sorting process, 1v can also directly be brominated to exclusively form 2v in 71% yield using our standard catalytic brominating conditions. In addition to halogen sorting from overhalogenation, we envisioned synthetic contexts where the overall reversibility of our process could be used for selective monodehalogenation. Using the product of a [2+2] dichloroketene-alkene cycloaddition 3w', we observed selective monodechlorination of 3w' to 3w as a single diastereomer in high yield. The high chemoselectivity of Cu-1 for the most electron poor halide, and the formation of benzyl chloride (3a) likely prevent complete dechlorination and allow controlled monodechlorination. Indeed, attempts to form the same versatile synthetic intermediate 3w using classical Zn/H+ conditions only provided mixtures of 3w, 3w', and 1w (See SI for full experimental details). |
67da98eefa469535b9c8b1cd | 3 | Leveraging the concept of metathesis, a new catalytic strategy for C-H functionalization was discovered. This C-H metathesis reaction takes ubiquitous C-H bonds and swaps single atoms (H and X) between two molecules to form versatile halogenated products. Lastly, the modular design of this reaction should allow for further applications and incorporation of other functional groups into the C-H metathesis paradigm. |
662155c691aefa6ce1d61c7b | 0 | Acquiring mechanistic reasoning skills to explain how and why reactions occur is a central learning outcome in organic chemistry. The Morrison and Boyd 1 electron pushing formalism (EPF) representations have been widely adopted in undergraduate organic chemistry since their textbook was first published in 1959, and EPFs are perhaps the most important representation in organic chemistry. EPFs illustrate the movement of electrons to explain the lowest energy and most kinetically favored pathway. Despite the goal of EPFs to promote understanding and the development of mechanistic reasoning skills, students often memorize EPF representations and attempt to regurgitate these representations on formative and summative assessments. Students' reliance on memorization of EPFs is 50 further supported by findings reporting students are unsure of the physical meaning and interpretation of EPFs. Students' understanding of the physical meaning of EPFs is improved by incorporating kinetics and thermodynamics when discussing reaction mechanisms. Kinetic and thermodynamic arguments can be coupled with EPFs using Reaction Coordinate Diagrams (RCDs), which are two-dimensional graphical representations 55 of the energy changes as a function of structure. Coupling EPFs and RCDs provides a multidimensional representation depicting the sequence in which bonds are broken and formed and the relative energy changes of a structure over a reaction. This research study expands on existing research on RCDs in general chemistry and was specifically guided by Popova's research in organic chemistry. The general chemistry 60 studies provided insight into how students interpret and analyze RCDs. Parobek et al. analyzed students' conceptions of the x-axis on RCDs and associated challenges with making inferences about reaction rates. Parobek et al. emphasized the importance of using RCDs across the curriculum, such as while discussing organic mechanisms. Lamichhane et al. likewise identified students' challenges with interpreting the x-axis. Atkinson developed a 65 Reaction Coordinate Diagram (RCD) Inventory that analyzed students' understanding and confidence in interpreting RCDs. Atkinson reported an improvement in interpreting RCDs as students progress across the chemistry curriculum. Additionally, Atkinson reported a correlation between performance and student confidence. starting and ending points of RCDs, the chemical interpretation of peak height and peak width, and the meaning of peaks and valleys in RCDs. The study provided insight into common challenges that organic chemistry students face when analyzing RCDs. Popova 75 reported that students tend to focus predominately on the major product when analyzing RCDs and ignore nuanced details, which is a key finding because of the significance of the nuanced information in explaining kinetics and thermodynamics. This study extends on Popova's studies by considering how students rationalize the major and minor products formed in different reaction contexts -specifically when the product distribution must be 80 rationalized by a careful analysis of the kinetics and thermodynamics. |
662155c691aefa6ce1d61c7b | 1 | Research Question 1 (RQ1): What resources are activated and productively applied when constructing RCDs across organic reactions with differing thermodynamic and kinetic favorability? on chemical phenomena." To construct RCDs, students must demonstrate competence in understanding the information provided by RCDs and how this information is graphically 100 conveyed. For example, a concerted reaction has only one energy maxima versus a stepwise reaction with more than one energy maxima. More detailed information is provided that illustrates the relationship between RC and the resources framework in Figures and. |
662155c691aefa6ce1d61c7b | 2 | The research was further guided by the resources framework developed by Hammer 105 and Elby . Wittman, Parobek et al., and Rodriguez define a resource as "fine-grained knowledge" elements that are activated and contribute productively or unproductively in problem-solving. Modir et al. further extended the definition of resource to "a manifold of knowledge pieces." The manifold of knowledge pieces is context-dependent, with resources being activated to differing extents based on the prompt or other cues, and resources 110 activated will evolve with student experiences.The grain size of a resource varies widely based from fundamental phenomenological primitives (p-prims), which are accepted facts used in explanations to complex theories. Research question 1 compares the resources activated when constructing RCDs for reactions having differing mechanisms and kinetic and thermodynamic favorability. Research question 115 2 compares and contrasts the resources activated as students construct RCDs before and after a set of perceptual cues. These multidimensional research questions are key because researchers and practitioners can use the findings to develop scaffolds to better instruct students in constructing RCDs and rationalizing them to organic mechanisms, ultimately leading to a deeper understanding of the logic behind chemical reactions. The resources 120 framework aligns with scaffolding activities because the framework asserts that conceptual understanding is not stable but dynamic depending on the resources activated, which likewise depends on the context, such as the type of reaction. For organic chemistry, EPFs are used to explain the pathway reactions that follow, and RCDs are used to explain the energetics of these pathways. To successfully write EPFs 125 and construct RCDs, resources must be activated and productively applied to different representations. Specifically, the activation of the resource, "the reaction is stepwise" can only be applied productively if students have RC with RCDs. |
662155c691aefa6ce1d61c7b | 3 | The research was conducted at a private research-intensive R1 university in the Southern United States. The university uses a 1-2-1 model for chemistry in which students take General Chemistry I, then a year of organic chemistry, and complete General Chemistry II as the fourth course in the sequence. The institutional review board (IRB) approved the data collection using a Qualtrics multiple choice survey (IRB #2022-0203, IRB #2022-0270). |
662155c691aefa6ce1d61c7b | 4 | The survey was implemented in two Organic Chemistry II courses: (1) Spring 2022 (N=46, at the end of the course) and (2.) Summer 2022 (N = 46, at the beginning of the course). The participants enrolled in Organic Chemistry were predominantly sophomores interested in pre-health and medical school. The demographics of the class were representative of the tabulated student population. Data were discarded for participants less than 18 years of 140 age or who declined to have their data included in the study. |
662155c691aefa6ce1d61c7b | 5 | The interview protocol and prompts were approved by the IRB (IRB #2022-0366). Nine participants were recruited from Organic Chemistry I, a separate course from the ones surveyed, in the spring of 2022. There were two sections of Organic Chemistry I taught by different instructors, but the content and textbook 41 were shared across both classes. All 145 participants were first-year students, and as first years, they had not officially declared majors. Of the nine participants, seven identified as female, and two identified as male. The interviews were conducted in the 12 th and 13 th weeks of the semester. Addition, substitution, and elimination reactions were taught and assessed in both sections before the interviews. |
662155c691aefa6ce1d61c7b | 6 | The semi-structured , think-aloud interview format was tailored based on Popova's study. Two researchers conducted the interviews using a semi-structured interview guide, and two trial interviews with volunteers were conducted before starting the research interviews. Participants were instructed to analyze the reaction and construct RCDs that best 160 explain the product distribution. Students were provided an iPad and an Apple pencil and were asked to draw structures, transition states, EPF mechanisms, or other figures of importance while verbally outlining their thought processes. The iPad screen was recorded to capture drawings and audio. Zoom was used as a backup to capture the voice recording with the video capture turned off. |
662155c691aefa6ce1d61c7b | 7 | A multiple-choice Qualtrics assessment was developed using common reactions presented in Organic Chemistry I. The survey consisted of ten multiple-choice items -three of which will be discussed in this study. The three items are shown in Figures and. The same questions were used for the interview prompts, but participants were instructed to construct keep their original RCD. Again, they were instructed to provide an explanation to support their decision. |
662155c691aefa6ce1d61c7b | 8 | The three items were designed to illustrate reaction scenarios in which the major and minor products have different kinetic pathways but are similar in terms of thermodynamic favorability (Figure , Problem 1), in which the major product is both the kinetic and thermodynamic product (Figure , Problem 2), and in which the major product is the kinetic product but not the thermodynamic product (Figure , Problem 3). The resources framework and RC were used to guide the data collection. Figures and illustrates an expert resource map and RCD for the Markovnikov Addition and Zaitsev 235 Elimination in which the resources are denoted in circles. Using a methodology similar to Braun and Graulich , the specific information for constructing RCD, which could extracted from a given resource, was identified. The four pieces of information include (1.) structure, (2.) the type of reaction, (3.) kinetic, and (4.) thermodynamic. The extracted information provides key insight into the energetic information used to construct the RCD. The block 240 arrow denotes how students transitioned between a resource and the graphical RCD. This transition between the representational competence (RC) aspect is associated with the analyses. |
662155c691aefa6ce1d61c7b | 9 | The qualitative interview data were transcribed verbatim and analyzed individually by three research team members. Each researcher reviewed and coded the transcripts to 245 identify the specific resources activated as students constructed the RCDs de novo and the resources activated when the multiple-choice items were provided. Resources were coded based on explicit statements from students -if it was not explicitly stated, the resource was coded as not activated. The three researchers analyzed the constructed RCDs from the resources activated to evaluate whether the resources were productively applied into an RCD 250 feature. Four codes emerged from the discussions: (1.) activated and productively applied , (2.) activated and not productively applied, (3.) not activated, and (4.) activated once the multiple-choice options were provided. Activated and productively applied denotes scenarios where the resource was activated and represented accurately in the RCD. This is linked to the selection of the resources and RC frameworks. Activation of the resource represents part 255 of the challenge, with the second requiring accurate representation in an RCD. Activated and not productively applied denotes scenarios in which resources were activated but not productively applied in constructing the RCD. The researchers met and discussed each coding scheme until reaching an agreement on the final codes reported in the manuscript. |
662155c691aefa6ce1d61c7b | 10 | The survey was used to gain insight into student performance before the interviews. Three reactions were analyzed, which required differing use of resources. The z-scores for two population proportions were calculated to compare student success on each problem within the spring and summer cohorts. The proportion of correct responses for the Zaitsev elimination was statistically higher using a 95% level of confidence in both the spring and 290 summer cohorts. There was no statistical difference between the Markovnikov and Hofmann proportion correct for either cohort. The association between the extent of substitution and stability is a heuristic introduced for students to gauge the reactivity and stability of alkenes. |
662155c691aefa6ce1d61c7b | 11 | Unlike the Zaitsev elimination, in which analyses of alkene substitution can afford the correct conclusion, there is not a lone heuristic for the Markovnikov addition and Hofmann elimination. The Markovnikov reaction is kinetically favored, but the products have roughly the same thermodynamic favorability. With the Hofmann elimination, the less substituted 300 alkene, the less thermodynamically favored product, is formed as the major product. |
662155c691aefa6ce1d61c7b | 12 | The survey data provided insight into students' abilities to correctly identify RCDs, 305 with the data specifically providing insight into the proportion correct and the number of resources that must be activated, and greater RC is needed for the productive application of the resources. For example, for the Markovnikov addition, RC depicts stepwise reactions with intermediates, elementary steps having differing activation energies, and the relationship between the energies of the major and minor products. Likewise, for the Hofmann 310 elimination, the productive application required RC to depict kinetic versus thermodynamic favorability. The survey data is a precursor to the interview data, which explores more closely the specific resources activated for each problem and whether these resources are used productively. Although carbocation resources were activated in several interviews, from Figure , the resources comparing the carbocation stability were generally either not activated or activated and unproductively applied. For mechanistic reasoning, carbocation formation is important; However, comparing two pathways and accounting for the differences is the more important learning outcome that extends beyond rote memory. For assessments, EPFs focus 355 on explaining how a specific product is formed from a given reactant or identifying the major product formed given specific reaction conditions. Therefore, similar to Popova's finding, the focus on the minor product was a challenging task for students during the interview. The resources activated and productively applied focused on the analysis of the major product and not the comparison of the two products. |
662155c691aefa6ce1d61c7b | 13 | Additionally, the extent of alkyl substitution was used by P3. : P3: I'm thinking the most 370 substituted one would be lower energy [the tertiary product]. For alkenes, the extent of alkyl substitution is correlated with stability. That is a productive resource when considering alkene stability and reactivity. However, it is not productive in terms of comparing the stability of alkyl halides. |
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