Asymmetric Chemoenzymatic One-Pot Synthesis of α-Hydroxy Half-Esters

Abstract

A new chemoenzymatic one-pot strategy has been developed for the synthesis of α-hydroxy half-esters containing consecutive quaternary and tertiary stereocenters using asymmetric cascade catalysis. In this study, an asymmetric Ca²⁺-catalyzed [2,3]-Wittig rearrangement reaction was proven to be suitable for a combination with porcine liver esterase-mediated hydrolysis resulting in the enhanced enantiomeric purity of the obtained products in a one-pot synthesis compared to the stepwise method.

Introduction

An α-hydroxy-β-dicarbonyl moiety is a ubiquitous fragment in naturally occurring biomolecules and their precursors.¹ These densely functionalized motifs, including α-hydroxy half-esters are synthetically pliable building blocks and, therefore, are widely used as intermediates in numerous synthetic routes for the preparation of different pharmaceuticals.²

Despite the fact that stereoselective chemical transformations of α-hydroxy-β-dicarbonyl compounds have been intensively studied, most of the methods have been developed for preparation of α-hydroxy-β-ketoesters.³ There have been only limited studies on the asymmetric synthesis of α-hydroxy half-esters, especially of α-hydroxy malonate derivatives. Both metal-catalyzed^4,5 and organocatalytic⁶⁻⁸ methods have been exploited. The described methods show a synthetic path to obtain α-hydroxy diesters, which can be readily transformed to α-hydroxy half-esters by basic hydrolysis. On the other hand, biotransformations provide a wide range of opportunities to catalyze the desymmetrization of prochiral mono- and disubstituted diesters utilizing both transesterification reactions and hydrolysis. The most suitable and commonly used class of enzymes for these transformations are hydrolases. Broad substrate specificity and high stereoselectivity, in addition to the fact that hydrolases are mostly commercially available and do not require additional cofactors, have made hydrolases widely used catalysts in asymmetric synthesis.⁹ Despite all of these tremendous advantages of hydrolases, the literature currently lacks proper coverage of synthetic pathways for obtaining α-hydroxy half-esters enantioselectively utilizing enzymes. Among the few reported methods, α-hydroxy half-esters were prepared enantioselectively with methodologies proposed by the Tamm¹⁰ and Kikelj¹¹ groups. All of the described examples provide attractive methods to synthesize α-hydroxy half-esters, although containing only one stereogenic center.

One-pot reactions have been demonstrated to be environmentally sustainable and economically viable techniques as no isolation of the intermediate is required, which leads to increased efficiency due to the reduction of energy and time consumption and the production of waste.¹² Combinations of asymmetric organocatalytic or metal-catalytic reactions with biotransformation in a chemoenzymatic one-pot process offer a great potential to achieve this goal. However, the possible incompatibility of the different reagents or solvents used to catalyze one of the steps and the corresponding reaction conditions with an enzyme make chemoenzymatic one-pot reactions challenging.^13,14

To the best of our knowledge, a systematic study of the creation of multiple stereogenic centers containing α-hydroxy half-esters has not been reported in the literature. In our ongoing endeavor to achieve this goal, herein, we report the asymmetric chemoenzymatic one-pot strategy for the formation of α-hydroxy half-esters bearing vicinal quaternary and tertiary stereocenters. We combine our previous study of the metal-catalyzed asymmetric [2,3]-Wittig rearrangement of allyloxy malonates 1(15) with the following enzymatic desymmetrization of sterically demanding α-substituted diesters 2 (Scheme 1). The Wittig rearrangement affords α-hydroxy malonic derivative 2 with a single tertiary stereogenic center. In the second step of the sequence, a quaternary stereogenic center is formed affording product 3 with two contiguous highly crowded stereocenters.

Scheme 1. Chemoenzymatic Approach to Half-Esters 3.

Results and Discussion

In our initial study, we set the benchmarks for the asymmetric [2,3]-Wittig rearrangement reaction and enzymatic hydrolysis separately. As the asymmetric [2,3]-Wittig rearrangement reaction was based on our previous experience, we first investigated the enzymatic hydrolysis of the racemic [2,3]-Wittig rearrangement reaction product rac-2a (Figure 1). Of a number of widely used hydrolase family enzymes,¹⁶ we tested immobilized lipase B from Candida antarctica (CALB), esterase from porcine liver (PLE), lipase from porcine liver (PLL), lipase from Candida rugosa (CRL), and lipase from Rhizomucor miehei (RML). For the enantioselective desymmetrization of the racemic rearrangement product 2a, only PLE showed catalytic activity (the conversion of the starting material under unoptimized conditions in phosphate buffer was 34%) and it was used in all further experiments (see Table S1, Supporting Information (SI)). From the screening of the most commonly used buffers at different pH values, phosphate buffer (pH 8.0, 0.2 M), Tris buffer (pH 8.5, 0.2 M), and carbonate buffer (pH 8.5, 0.2 M) were selected as starting points for the optimization of the reaction conditions. Of them, Tris buffer showed a greater diastereoisomeric ratio (dr, 1:8) with comparably high conversion (91%), and therefore it was chosen as the primary buffer (see Table S2, SI). Additionally, the influence of organic co-solvents on the rate and the selectivity of the hydrolysis step was evaluated (Figure 1). In PLE-catalyzed hydrolysis, in general, the presence of the co-solvent inhibited the enzyme activity but the stereoselectivity was very dependent on the co-solvent used. The enantioselectivity of the minor diastereoisomer was high (>90%) in almost all cases, but the enantiomeric excess (ee) of the major diastereoisomer was influenced more. The addition of 20% of dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) gave a similar conversion, but the latter showed slightly higher enantioselectivity for the major diastereoisomer (ee 31%) (Figure 1A). The enantiomeric purity of the major diastereoisomer was increased using 40% of DMSO (ee 41%), although it decreased the reaction rate significantly. Upon further increasing the DMSO percentage to 60%, no reaction was observed. When alcohols were employed, there was a strong correlation between the reaction rate, selectivity, and the bulkiness of the alcohol used (Figure 1B). The reaction in MeOH proceeded with higher conversion, diastereoselectivity, and enantioselectivity of the minor diastereoisomer than the reaction in t-BuOH, although the enantioselectivity of the major diastereoisomer decreased significantly (ee 5%). Surprisingly, chlorinated solvents were found to be compatible with the biotransformation, although they led to longer reaction times (Figure 1C). Also, as in the case of DMSO, a larger amount of CHCl₃ caused a decrease in the rate of reaction, while it increased the ee of the major diastereoisomer (ee 55%). In combination with Tris buffer at pH 8.4, 20% of heptane was able to enhance the enzyme activity resulting in 78% of conversion in 2 h, but, unfortunately, the ee of the major diastereoisomer was only 17%. Other co-solvents or combinations of them (shown in Figure 1D) either showed a detrimental effect on the enantioselectivity of the hydrolysis or did not have a reaction at all.

Figure 1.

(A–F) Effect of co-solvents on the PLE-mediated hydrolysis of rac-2a.^a (^aReaction conditions: 0.1 mmol scale, 85 EU of crude PLE, solvent system (0.2 M). ^bConversion was determined by ¹H NMR analysis of the crude mixture and referred to the ratio of starting material and product. ^cDiastereoisomeric ratio was determined by ¹H NMR analysis of the crude mixture. ^dEnantiomeric excess was determined by chiral high-performance liquid chromatography (HPLC) analysis of the sample obtained by preparative thin-layer chromatography (TLC). ^eReaction was completed after 72 h. ^fHFIP, 1,1,1,3,3,3-hexafluoro-2-propanol).

Our preliminary results demonstrated that enzymatic reactions using an 8:2 ratio of Tris buffer/co-solvent (DMSO or CHCl₃) showed an optimal conversion-to-ee ratio. Thus, similar conditions were applied to phosphate (pH 8.0) and carbonate (pH 8.5) buffers (Figure 1E,F, respectively). The reaction in phosphate buffer/DMSO reached full conversion within 24 h without a decrease in the enantioselectivity of the major diastereoisomer compared to Tris buffer.

Since the desymmetrization of the racemic Wittig product rac-2a via enzymatic hydrolysis gave poor results, we studied a one-pot process where the first rearrangement reaction afforded an enantiomerically enriched rearranged product 2. This was achievable by exploiting our earlier study where we used either an organocatalytic approach or a Ca²⁺-catalyzed system in the [2,3]-Wittig rearrangement reaction (Scheme 2).¹⁵ Having observed the inhibition of enzyme with unacceptably low enantioselectivity (see Tables S3 and S4, SI) in a one-pot synthesis conducted using cyclopropenimine catalyst I in the first step, we moved on to the combination of the Ca²⁺-catalyzed [2,3]-Wittig rearrangement reaction with hydrolysis.

Scheme 2. Cyclopropenimine and Ca²⁺-Catalyzed [2,3]-Wittig Rearrangement Reaction.

Implementation of Ca²⁺-Catalyzed [2,3]-Wittig Rearrangement/PLE-Mediated Desymmetrization in a One-Pot Reaction

First, an investigation of the Ca²⁺-catalytic system compatibility with PLE-mediated hydrolysis was performed. In our earlier study,¹⁵ Ca²⁺-catalyzed [2,3]-Wittig rearrangement reactions were conducted in iPrOH. However, iPrOH caused transesterification of the starting material 1 and the intermediate 2, which also participated in the hydrolysis step, thereby complicating the purification process of product 3. To exclude the formation of these side products, we rescreened the solvents in the [2,3]-Wittig rearrangement reaction of 1a to 2a. As a result, MeOH showed equal activity to iPrOH, without the formation of any side products, although low enantioselectivity was observed (iPrOH showed 75% ee, and MeOH showed 24% ee; see Table S5, SI). Thus, to reach quantitative conversion and to avoid the formation of the transesterification products Ca(NTf₂)₂, (R,S)-inda-PyBox, and imidazole in MeOH were further used in the first step.

To this end, we carried out experiments to verify whether the Ca-inda-PyBox catalytic system used to perform the [2,3]-Wittig rearrangement reaction was compatible with PLE. For this, PLE-mediated hydrolysis was conducted in the selected solvent systems using the isolated racemic [2,3]-Wittig rearrangement reaction product rac-2a as a starting material in the presence of a catalytic amount of Ca(NTf₂)₂, (R,S)-inda-PyBox, and imidazole. The Tris buffer (pH 8.5)/CHCl₃ biphasic system was found to be ineffective by showing total inhibition of the enzyme activity (Table 1, entry 1; compare with Figure 1). We were delighted to find that PLE is sufficiently active in the phosphate buffer (Table 1, entries 2 and 3).

Table 1. Ca²⁺-Catalytic System Compatibility with PLE^a.

					ee (%)d
entry	solvent system	time (h)	conversion (%)b	drc	maj/min
1	Tris buffer (pH 8.5)/20% CHCl3	72	7	n.d.e	n.d.e
2	phosphate buffer (pH 8.0)	24	75	7:1	13/99
3	phosphate buffer (pH 8.0)/20% DMSO	24	100	4:1	30/99

^aReaction conditions: 0.1 mmol scale, 5 mol % Ca(NTf₂)₂, 5 mol % (R,S)-inda-PyBox, 5 mol % imidazole, 85 EU of crude PLE, and the solvent system (0.2 M).

^bConversion was determined by ¹H NMR analysis of the crude mixture and referred to the ratio of the starting material and the product.

^cDiastereoisomeric ratio was determined by ¹H NMR analysis of the crude mixture.

^dEnantiomeric excess was determined by the chiral HPLC analysis of the sample obtained by preparative TLC; maj, major diastereoisomer and min, minor diastereoisomer.

^en.d., not determined.

We then combined a Ca²⁺-catalyzed [2,3]-Wittig rearrangement reaction and hydrolysis in a consecutive one-pot process. The results presented in Table 2 show the influence of the solvent system used in the second step on the rate and the selectivity of the one-pot reaction. During the one-pot synthesis, intermediate 2a was not isolated, although solvent exchange was performed after the first step was complete (such a multistep process is still considered a one-pot reaction).^12,17 Hence, relying on our preliminary screening of buffers and co-solvents (see Table S2, SI; and Figure 1), we conducted the reactions in Tris (pH 8.5), carbonate (pH 8.5), and phosphate (pH 8.0) buffers. It was confirmed that the hydrolysis step could not be carried out in the biphasic system using a carbonate buffer (pH 8.5)/CHCl₃ solvent system (Table 2, entry 1). High conversion was achieved using Tris buffer (pH 8.5) without co-solvent as the reaction medium, as well as with DMSO, but in both cases product 3a was formed in moderate enantioselectivity of the major diastereoisomer (ee 51 and 64%, respectively) (Table 2, entries 2 and 3). The reaction carried out in a carbonate buffer (pH 8.5) showed a moderate reactivity reaching 86% conversion in 72 h with the enantioselectivity of the major diastereoisomer of 64% (Table 2, entry 4). In addition to the enhancement of enzyme activity, higher diastereoselectivity was observed in the reaction utilizing DMSO as a co-solvent. However, it led to a lower ee of the major diastereoisomer (Table 2, entry 5). Surprisingly, a decrease in temperature did not influence the results obtained (Table 2, entry 6). The use of a phosphate buffer (pH 8.0) and a phosphate buffer (pH 8.0)/DMSO system decreased the rate of the reaction. However, promising enantioselectivity of the major diastereoisomer (85%) was detected (Table 2, entries 7 and 8). Therefore, by increasing the temperature to 35 °C, we were now able to run the one-pot reaction with full conversion (Table 2, entry 9).

Table 2. Combination of a Ca²⁺-Catalyzed [2,3]-Wittig Rearrangement Reaction with PLE-Mediated Hydrolysis in a One-Pot Reaction^a.

		conversion (%)b			ee (%)d
entry	solvent system	24 h	72 h	drc	maj/min
1	carbonate buffer (pH 8.5)/20% CHCl3	7	8	4.5:1	n.d.e
2	Tris buffer (pH 8.5)	69	81	6.3:1	51/95
3	Tris buffer (pH 8.5)/20% DMSO	78	83	4:1	64/96
4	carbonate buffer (pH 8.5)	47	86	3:1	64/99
5	carbonate buffer (pH 8.5)/20% DMSO	79f		4.3:1	59/99
6	carbonate buffer (pH 8.5)/20% DMSO (5 °C)	72f		4.5:1	57/99
7	phosphate buffer (pH 8.0)	36	46	7.6:1	63/99
8	phosphate buffer (pH 8.0)/20% DMSO	9	43	3.6:1	85/99
9	phosphate buffer (pH 8.0)/20% DMSO (35 °C)	100		2.9:1	73/99

^aReaction conditions: 0.1 mmol scale, 5 mol % Ca(NTf₂)₂, 5 mol % (R,S)-inda-PyBox, 5 mol % imidazole, and MeOH (0.1 M) were stirred at 60 °C for 24 h, followed by solvent evaporation and addition of 85 EU of crude PLE; solvent system (0.2 M).

^bConversion was determined by ¹H NMR analysis of the crude mixture and referred to the ratio of starting material and the product.

^cDiastereoisomeric ratio was determined by ¹H NMR analysis of the crude mixture.

^dEnantiomeric excess was determined by chiral HPLC analysis of the sample obtained by preparative TLC; maj, major diastereoisomer and min, minor diastereoisomer.

^en.d., not determined.

^fReaction conversion did not change after 2 h.

To obtain a better understanding of the reaction, we decided to run the one-pot synthesis in optimal conditions and to follow the hydrolysis step using ¹H NMR. By monitoring the progress of the hydrolysis step (after the completion of the first step), we found that the reaction exhibited a sigmoidal curve leading to full conversion in 24 h (Figure 2a). For comparison, the kinetic study of the enzymatic reaction of isolated [2,3]-Wittig rearrangement product 2a (ee 25%) was carried out under the same reaction conditions (Figure 2b). In this case, the reaction reached full conversion in 3 h. The presence of a lag phase and extended reaction time (Figure 2a) suggests that the inhibition of the enzyme occurred during the hydrolysis step in the one-pot reaction. However, it promotes the stereoselectivity of the reaction affording the major diastereoisomer in noticeably higher enantiomeric purity than in the case of the hydrolysis of the isolated [2,3]-Wittig rearrangement product 2a (ee 73 and 52%, respectively).

Figure 2.

(a) Reaction profile for the hydrolysis step of the one-pot reaction without isolation of the [2,3]-Wittig rearrangement product 2a. Enantiomeric excess of the minor diastereoisomer remained 99% during the reaction. (b) Reaction profile for the hydrolysis reaction using isolated [2,3]-Wittig rearrangement product 2a as the starting material. Enantiomeric excess of the minor diastereoisomer remained 99% during the reaction.

Having successfully developed a one-pot procedure, the substrate scope was evaluated using Ca(NTf₂)₂, (R,S)-inda-PyBox, and imidazole in MeOH at 60 °C for the first step and PLE in a phosphate buffer (pH 8.0) with 20% of DMSO at 35 °C for the second step (Scheme 3). First, we used (R,S)- and (S,R)-inda-PyBox ligands for the consecutive one-pot reaction to gain access to both enantiomers of the rearrangement intermediate 2a. In the case of intermediate (S)-2a, the hydrolysis step was slightly slower than expected compared to the hydrolysis with its enantiomer (R)-2a. Additionally, the enantiomeric excess of the major diastereoisomer of product 3a from the reaction with intermediate (S)-2a dropped to 27% compared to 73% of R-enantiomer. From the obtained results it was clear that the PLE preferred R-enantiomer of compound 2a providing notably higher ee of the major diastereoisomer in a shorter time; thus, the following one-pot reactions were carried out using (R,S)-inda-PyBox in the first step.

Scheme 3. Scope of the Reaction^–

Reaction conditions: 0.3 mmol scale, 5 mol % Ca(NTf₂)₂, 5 mol % (R,S)-inda-PyBox, 5 mol % imidazole, and MeOH (0.1 M) were stirred at 60 °C for 20 h (if not stated otherwise), followed by solvent evaporation and addition of 255 EU of crude PLE, phosphate buffer (pH 8.0)/DMSO (8:2, 0.2 M). Diastereoisomeric ratio was determined by ¹H NMR analysis of the crude mixture. Enantiomeric excess was determined by chiral HPLC of the isolated product. Major diastereoisomers are depicted in the scheme.

Hydrolysis was completed after 24 h when the plateau phase was reached.

(S,R)-Inda-PyBox was used.

Hydrolysis was completed after 48 h when the plateau phase was reached.

Diastereoisomeric ratio of the isolated product is presented.

Reaction was conducted in 0.1 mmol scale.

The [2,3]-Wittig rearrangement reaction was completed after 7 days.

The [2,3]-Wittig rearrangement reaction was conducted in absolute EtOH.

The [2,3]-Wittig rearrangement reaction was conducted in iPrOH.

Conversion of the hydrolysis was determined after 72 h.

The [2,3]-Wittig rearrangement reaction was stopped after 5 days.

Conversion of the [2,3]-Wittig rearrangement reaction was determined after 48 h.

The aromatic substitution pattern in substrates 1a–i did not affect the results of the [2,3]-Wittig rearrangement step substantially (full conversion was achieved in 24 h, and the ee of the intermediates 2a–i were in the range 14–24%; detailed information in Table S7, SI), although the results of the hydrolysis step were dependent on it.

The hydrolysis step of o-chlorophenyl derivative 2b was less efficient compared to the reaction with unsubstituted 2a and reached only 56% of conversion in 48 h. Surprisingly, product 3b was isolated with excellent diastereoselectivity but with low enantiomeric excesses of both diastereoisomers. Reactions with m- and p-chlorophenyl substituted intermediates 2c and 2d stopped before reaching full conversion, affording products 3c and 3d with 68 and 64% yields, respectively. However, similar diastereo- and enantioselectivities were detected. Compounds 1e and 1f with electron-donating and electron-withdrawing substituents were tolerated under the reaction conditions, but in both cases, the hydrolysis step was slower. While the electron-donating substituent had no notable influence on the selectivities of the obtained product 3e, the electron-withdrawing nitro group containing intermediate 2f provided product 3f with a diminished enantiomeric purity of both diastereoisomers. A significant decrease in the rate of the hydrolysis step was detected using bulkier naphthyl- and indole-substituted intermediates 2g and 2h instead of the phenyl substituent. Surprisingly, the naphthyl-derived product 3g was obtained with even better selectivity, although in the case of the indole-derived product 3h, the enantioselectivities of both diastereoisomers dropped drastically. It was assumed that due to variation in the spatial arrangement of the substituents (2-naphtyl and 1-indolyl), substrates fit the active site of the enzyme differently, which led to differences in enantioselectivity. When a heteroaromatic derivative 1i was used as a starting material, a full conversion in the hydrolysis step was achieved within 24 h, resulting in product 3i with higher diastereoselectivity (6.7:1), although, unfortunately, the enantiomeric purity of the major diastereoisomer decreased. In the case of crotyl-substituted substrate 1j, an unreasonably long reaction time (7 days) was required for the [2,3]-Wittig rearrangement reaction. The conversion of the hydrolysis step reached a plateau at 93% in 24 h, but the one-pot product 3j was isolated with a moderate 62% yield due to its instability during workup. The absence of the aromatic ring strongly affected the selectivities of the obtained product, leading to diminished diastereo- and enantioselectivities.

The rearrangement-hydrolysis protocol of other malonic esters was studied briefly. Due to the formation of transesterification products in the [2,3]-Wittig rearrangement step, the solvents were rescreened with substrates 1k and 1l (see Table S6, SI), indicating EtOH and iPrOH as the optimal solvents, respectively. The reaction with diethyl malonic derivative 1k proceeded smoothly, providing half-ester 3k with high diastereo- and enantioselectivities. In this case, higher enantioselectivity (ee 55%) of the intermediate 2k was observed in the first step, which influenced the selectivities of the obtained one-pot product 3k. Bulky dibenzyl derivative 1l showed the highest enantioselectivity in the [2,3]-Wittig rearrangement, but unfortunately, only 17% of conversion was observed in the hydrolysis step within 72 h. The mixed ester 1m did not reach full conversion in the first step even in 5 days; therefore, a mixture of the starting material 1m and intermediate 2m was used in the hydrolysis step leading to no reaction. The additional substituent in the double bond suppressed the reactivity of the starting material 1n; thus, only 24% of conversion in the [2,3]-Wittig rearrangement reaction was detected.

Unfortunately, we were not able to determine the absolute configuration of product 3. All our attempts to crystallize product 3 or amides derived from them using various solvent systems at different temperatures, slow evaporation technique, vapor diffusion method, seeding procedure, as well as co-crystallization and the crystalline sponge method with metal–organic frameworks¹⁸ were unsuccessful. Also, we performed a conformational analysis and found that differences in energies of enantiomers were too small to determine the preferred configuration.

To show the synthetic utility of the proposed method, compound 3a was converted into amide with benzylamine in the presence of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) in nearly quantitative yield (Scheme 4). The diastereoisomers of the obtained amide were chromatographically separable affording single enantiomers in high-to-very-high enantiomeric purities.

Scheme 4. Amidation of Compound 3a.

Reaction conditions: 0.2 mmol scale, 1.1 equiv of BnNH₂, 1.1 equiv of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), and 1.1 equiv of N,N-diisopropylethylamine (DIPEA) in DMF (0.1 M) were stirred at room temperature (rt) for 20 min. Diastereoisomeric ratio was determined by ¹H NMR analysis of the crude mixture. Enantiomeric excess was determined by chiral HPLC of the isolated diastereoisomers of product 4a.

Conclusions

We developed an effective one-pot method by combining an asymmetric Ca²⁺-catalyzed [2,3]-Wittig rearrangement reaction with an enzymatic desymmetrization of malonic esters. Such an implementation of the cascade catalysis in a one-pot manner provided access for the synthesis of vicinal quaternary and tertiary stereogenic centers containing α-hydroxy half-esters in high yields and good enantioselectivities. Different α-branched sterically demanding esters could be easily desymmetrized to the corresponding half-esters using the same one-pot procedure. Due to the compatibility of the Ca²⁺-catalytic system with PLE, no isolation of the intermediate was required. In addition, the hydrolysis step is efficiently catalyzed by commercially available, crude PLE; thus, no expensive purified isoenzyme forms nor modification of the enzyme were needed.

Experimental Section

Full assignment of ¹H and ¹³C chemical shifts is based on the one-dimensional (1D) and two-dimensional (2D) Fourier transform (FT) NMR spectra measured on a Bruker Avance III 400 MHz instrument. Residual solvent signals were used [CDCl₃ δ = 7.26 (¹H NMR), 77.16 (¹³C NMR), and DMSO-d₆ δ = 2.50 (¹H NMR), 39.52 (¹³C NMR)] as internal standards. All peak assignments are confirmed by 2D experiments (¹H–¹H correlated spectroscopy (COSY), ¹H–¹³C heteronuclear single quantum coherence (HSQC), ¹H–¹³C heteronuclear multiple bond correlation (HMBC)). In ¹³C NMR, 2C in brackets refers to either two chemically equivalent or two overlapping unique carbon signals. The determination of the diastereomeric ratio in the reaction mixture was based on CH₂ integrals of the double bonds (¹H NMR in DMSO-d₆). If possible, the obtained ratio of diastereomers was also determined using the CH integrals of the double bonds or CHAr integrals. The determination of the diastereomeric ratio of the isolated products was based on COOCH₃ integrals (¹H NMR in CDCl₃). High-resolution mass spectra (HRMS) were recorded using an Agilent Technologies 6540 UHD Accurate-Mass Q-TOF LC/MS spectrometer and electrospray ionization (ESI). Chiral HPLC was performed using Chiralpak AD-H (250 × 4.6 mm) and Chiralcel OJ-H (250 × 4.6 mm) columns. In the case of carboxylic acids 3, trifluoroacetic acid (TFA) was used as a mobile phase additive in chiral HPLC. Absolute ethanol (ABSE) was used as received. Precoated silica gel 60 F254 plates were used for TLC. Column chromatography was performed on a Biotage Isolera Prime preparative purification system with silica gel Kieselgel 40–63 μm. Purchased chemicals and solvents were used as received. Petroleum ether (PE) had a boiling point of 40–60 °C. Esterase from porcine liver (PLE, lyophilized powder; 18 units/mg, 20 units/mg, 24 units/mg) was purchased from Sigma-Aldrich. The reactions were performed under an air atmosphere without additional moisture elimination unless stated otherwise.

Ligands (S,R)- and (R,S)-inda-PyBox were prepared according to the literature procedures.¹⁹

Synthesis of Starting Materials 1a–n

Compounds 1a–g, 1i, and 1l were synthesized according to the procedure previously described in the literature. The analytical data of compounds 1a–g, 1i, and 1l are in agreement with the literature data.¹⁵

The same general procedure was used for the synthesis of compounds 1h, 1j, 1k, 1m, and 1n, which is described below.

General Procedure

Rhodium(II) acetate dimer (0.005 equiv) and dichloromethane (DCM) (0.4 M) were added to an alcohol (1.2 equiv) under an argon atmosphere. The corresponding 2-diazomalonate (1 equiv) solution in DCM (0.4 M) was added for over 5 min at 0 °C. The reaction was stirred overnight at rt. The solvent was removed under reduced pressure, and the crude mixture was purified by column chromatography on silica gel.

Dimethyl (E)-2-((3-(1H-Indol-1-yl)allyl)oxy)malonate 1h

Indole (1171 mg, 10 mmol) and sodium tert-butoxide (481 mg, 5 mmol) were added to DMF (63 mL, 0.16 M) under an argon atmosphere for 30 min. Then, ethyl propionate (1.21 mL, 12 mmol) was added for over 2 min, and the reaction was stirred for 4 h at rt. The solvent was removed under reduced pressure and the crude mixture was purified twice by column chromatography on silica gel (3–5% EtOAc in DCM and 30–65% DCM in PE), providing ethyl (E)-3-(1H-indol-1-yl)acrylate (570 mg, 26%) as a dark yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.29 (d, J = 14.0 Hz, 1H), 7.64–7.56 (m, 2H), 7.38 (d, J = 3.5 Hz, 1H), 7.33 (ddd, J = 8.4, 7.2, 1.2 Hz, 1H), 7.23 (ddd, J = 7.9, 7.2, 0.9 Hz, 1H), 6.73 (dd, J = 3.7, 0.8 Hz, 1H), 5.96 (d, J = 14.0 Hz, 1H), 4.28 (q, J = 7.1 Hz, 2H), 1.35 (t, J = 7.1 Hz, 3H). Analytical data are in agreement with the literature data.²⁰

Diisobutylaluminum hydride (DIBAL-H) (1 M solution in toluene, 5.7 mL, 5.73 mmol) was added to an ethyl (E)-3-(1H-indol-1-yl)acrylate (561 mg, 2.61 mmol) solution in DCM (13 mL, 0.2 M) under an argon atmosphere at −78 °C. The reaction was stirred for 2 h at −78 °C. Then, the reaction mixture was quenched by 1 M aq HCl solution and extracted with DCM (6 × 15 mL). The combined organic phase was dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The crude mixture was purified by column chromatography on silica gel (3–8% EtOAc in DCM), providing (E)-3-(1H-indol-1-yl)prop-2-en-1-ol (247 mg, 55%) as a dark orange oil. ¹H NMR (400 MHz, CDCl₃) δ 7.64–7.60 (m, 1H, Ar–C4), 7.50–7.45 (m, 1H, Ar–C7), 7.39 (d, J = 3.4 Hz, 1H, Ar–C2), 7.30–7.22 (m, 2H, CHAr and Ar–C6), 7.17 (ddd, J = 8.0, 7.1, 1.0 Hz, 1H, Ar–C5), 6.63 (d, J = 3.4 Hz, 1H, Ar–C3), 5.92 (dt, J = 14.1, 6.6 Hz, 1H, CHCH₂), 4.36 (ddd, J = 6.7, 5.6, 1.3 Hz, 2H, CH₂OH), 1.47 (t, J = 5.7 Hz, 1H, OH). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 135.7, 129.2, 126.6, 124.0, 122.9, 121.4, 121.0, 112.1, 109.6, 105.3, 62.1. HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₁H₁₂NO 174.0913; found 174.0905.

Following the general procedure, starting from (E)-3-(1H-indol-1-yl)prop-2-en-1-ol (289 mg, 1.67 mmol) and dimethyl 2-diazomalonate (220 mg, 1.39 mmol), the crude product was purified by column chromatography (2–8% EtOAc in PE/DCM 3/1 mixture), providing dimethyl (E)-2-((3-(1H-indol-1-yl)allyl)oxy)malonate 1h (60 mg, 14%) as an orange oil. ¹H NMR (400 MHz, CDCl₃) δ 7.61 (dt, J = 7.9, 1.1 Hz, 1H, Ar–C4), 7.49–7.44 (m, 1H, Ar–C7), 7.38 (d, J = 3.5 Hz, 1H, Ar–C2), 7.31–7.23 (m, 2H, CHAr and Ar–C6), 7.17 (ddd, J = 8.0, 7.1, 1.0 Hz, 1H, Ar–C5), 6.64 (d, J = 3.4 Hz, 1H, Ar–C3), 5.82 (dt, J = 14.2, 7.2 Hz, 1H, CH₂CH), 4.68 (s, 1H, OCH), 4.37 (dd, J = 7.1, 1.1 Hz, 2H, CH₂CH), 3.82 (s, 6H, COOCH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 167.1 (2C), 135.6, 129.3, 129.1, 123.8, 123.1, 121.4, 121.2, 109.6, 106.8, 105.9, 77.4, 70.2, 53.1 (2C). HRMS (ESI) m/z: [M + Na]⁺ calcd for C₁₆H₁₇NaNO₅ 326.0999; found 326.0987.

Dimethyl (E)-2-(But-2-en-1-yloxy)malonate 1j

Following the general procedure, starting from (E)-but-2-en-1-ol (185 mg, 2.57 mmol) and dimethyl 2-diazomalonate (338 mg, 2.14 mmol), the crude product was purified by column chromatography (2–8% EtOAc in PE/DCM 3/1 mixture), providing dimethyl (E)-2-(but-2-en-1-yloxy)malonate 1j (176 mg, 41%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 5.82–5.72 (m, 1H, CHCH₃), 5.62–5.53 (m, 1H, CHCH₂), 4.57 (s, 1H, OCH), 4.09 (dt, J = 6.6, 1.0 Hz, 2H, CHCH₂), 3.81 (s, 6H, COOCH₃), 1.75–1.69 (m, 3H, CHCH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 167.3 (2C), 132.7, 125.8, 77.3, 71.9, 53.1 (2C), 17.9. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₉H₁₄NaO₅ 225.0733; found 225.0735.

Diethyl 2-(Cinnamyloxy)malonate 1k

Following the general procedure, starting from (E)-3-phenylprop-2-en-1-ol (259 mg, 1.93 mmol) and diethyl 2-diazomalonate (300 mg, 1.61 mmol), the crude product was purified by column chromatography (0–2% diethyl ether in toluene), providing diethyl 2-(cinnamyloxy)malonate 1k (136 mg, 29%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.41–7.36 (m, 2H), 7.35–7.29 (m, 2H), 7.28–7.23 (m, 1H), 6.63 (d, J = 16.1 Hz, 1H), 6.29 (dt, J = 15.8, 6.5 Hz, 1H), 4.59 (s, 1H), 4.34 (dd, J = 6.5, 1.2 Hz, 2H), 4.32–4.22 (m, 4H), 1.29 (t, J = 7.2 Hz, 6H). Analytical data are in agreement with the literature data.²¹

1-(tert-Butyl) 3-Methyl 2-(cinnamyloxy)malonate 1m

To a solution of tosyl azide (374 mg, 1.89 mmol) and triethylamine (0.3 mL, 2.15 mmol) in acetonitrile (ACN) (1.72 mL, 1 M) was added tert-butyl methyl malonate (300 mg, 1.72 mmol) in ACN (1.72 mL, 1 M) at 0 °C. The reaction mixture was warmed to rt and stirred for 24 h. After evaporating the solvent, the crude mixture was purified by column chromatography on silica gel (3–7% EtOAc in PE) providing 1-(tert-butyl) 3-methyl 2-diazomalonate (279 mg, 81%) as a light yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 3.83 (s, 3H), 1.51 (s, 9H). Analytical data are in agreement with the literature data.²²

Following the general procedure, starting from (E)-3-phenylprop-2-en-1-ol (224 mg, 1.67 mmol) and 1-(tert-butyl) 3-methyl 2-diazomalonate (279 mg, 1.39 mmol), after evaporating the solvent, the crude mixture was purified by column chromatography on silica gel (3% EtOAc in PE/DCM 3/1 mixture). The impure fractions were repurified by column chromatography on silica gel (2–4% EtOAc in PE/DCM 3/1 mixture), providing 1-(tert-butyl) 3-methyl 2-(cinnamyloxy)malonate 1m (total yield: 203 mg, 48%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.41–7.36 (m, 2H, ArH), 7.35–7.29 (m, 2H, ArH), 7.28–7.22 (m, 1H, ArH), 6.63 (d, J = 15.9 Hz, 1H, CHAr), 6.29 (dt, J = 15.9, 6.4 Hz, 1H, CH₂CH), 4.50 (s, 1H, OCH), 4.37–4.28 (m, 2H, CH₂CH), 3.79 (s, 3H, COOCH₃), 1.48 (s, 9H, tBu). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 167.6, 165.7, 136.4, 134.7, 128.7 (2C), 128.2, 126.8 (2C), 124.3, 83.4, 78.3, 71.7, 52.8, 28.0 (3C). HRMS (ESI) m/z: [M + Na]⁺ calcd for C₁₇H₂₂NaO₅ 329.1359; found 329.1350.

Dimethyl (E)-2-((2-Methyl-3-phenylallyl)oxy)malonate 1n

Following the general procedure, starting from (E)-2-methyl-3-phenylprop-2-en-1-ol (274 mg, 1.85 mmol) and dimethyl 2-diazomalonate (244 mg, 1.54 mmol), the crude mixture was purified by column chromatography (3–7% EtOAc in PE/DCM 3/1 mixture), providing dimethyl (E)-2-((2-methyl-3-phenylallyl)oxy)malonate 1n (200 mg, 47%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.37–7.31 (m, 2H), 7.30–7.21 (m, 3H), 6.52 (s, 1H), 4.61 (s, 1H), 4.22 (d, J = 1.1 Hz, 2H), 3.82 (s, 6H), 1.93 (d, J = 1.4 Hz, 3H). Analytical data are in agreement with the literature data.²¹

General Procedure for the Chemoenzymatic One-Pot Synthesis of α-Hydroxy Half-Esters 3

To a solution of allyloxy 1,3-dicarbonyl compound 1 (0.3 mmol) in methanol (unless stated otherwise, 3 mL), Ca(NTf₂)₂ (0.015 mmol, 9 mg), (R,S)-inda-PyBox (0.015 mmol, 5.9 mg), and imidazole (0.015 mmol, 1 mg) were added. The reaction mixture was stirred at 60 °C until full conversion to the [2,3]-Wittig rearrangement product 2 was observed (¹H NMR). After evaporation of the solvent, the crude intermediate was suspended in DMSO (0.3 mL) and sodium phosphate buffer (pH 8.0, 1.2 mL). PLE (255 EU) was added, and the mixture was stirred at 35 °C until the plateau phase was reached. It was then acidified to pH 2 with 1 M aq HCl solution and extracted with diethyl ether (a drop of ethanol can be used to prevent emulsion formation). The combined organic layers were dried over MgSO₄. After filtration, the solvent was removed in vacuo, and the crude carboxylic acid was purified by column chromatography on silica gel (2% EtOAc in DCM (to elute the possible residual starting material), followed by 2% EtOAc in the DCM/formic acid 99/1 mixture), providing the desired product 3. Since the diastereoisomers were chromatographically inseparable, a diastereomeric ratio after purification is shown. The enantioselectivities of intermediates 2 (see Table S7, SI) were determined by HPLC analysis according to the literature procedure¹⁵ unless stated otherwise. The enantioselectivities were determined by HPLC analysis of the purified products 3.

2-Hydroxy-2-(methoxycarbonyl)-3-phenylpent-4-enoic Acid 3a

The title compound was synthesized following the general procedure from dimethyl 2-(cinnamyloxy)malonate 1a (79 mg). After full conversion was achieved in the [2,3]-Wittig rearrangement reaction within 20 h, the enzymatic hydrolysis reaction mixture was stirred for an additional 24 h. Compound 3a was obtained as an off-white solid in 85% yield (64 mg); dr 2.9:1 (¹H NMR).

(3R)-Major Diastereoisomer

ee 73% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 95:5, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 39.0 min and t_R (minor) = 34.9 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.35–7.21 (m, 5H, ArH), 6.15 (ddd, J = 17.1, 10.2, 9.0 Hz, 1H, CHCH₂), 5.30–5.19 (m, 2H, CH₂), 4.32 (d, J = 9.0 Hz, 1H, CHAr), 3.69 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 169.8, 169.5, 137.3, 134.0, 129.0 (2C), 128.7 (2C), 128.0, 119.8, 83.1, 55.3, 54.3.

(3S)-Minor Diastereoisomer

ee 99% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 95:5, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 68.7 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.41–7.20 (m, 5H, ArH), 6.21–6.07 (m, 1H, CHCH₂), 5.22–5.17 (m, 2H, CH₂), 4.31 (d, J = 9.3 Hz, 1H, CHAr), 3.92 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 170.4, 169.3, 137.0, 134.7, 129.4 (2C), 128.6 (2C), 127.9, 119.1, 82.9, 55.2, 54.7.

HRMS (ESI) m/z: [M – H]⁻ calcd for C₁₃H₁₃O₅ 249.0768; found 249.0772.

3-(2-Chlorophenyl)-2-hydroxy-2-(methoxycarbonyl)pent-4-enoic Acid 3b

The title compound was synthesized following the general procedure from dimethyl (E)-2-((3-(2-chlorophenyl)allyl)oxy)malonate 1b (90 mg). After full conversion was achieved in the [2,3]-Wittig rearrangement reaction within 20 h, the enzymatic hydrolysis reaction mixture was stirred for an additional 48 h. Compound 3b was obtained as an off-white solid in 46% yield (39 mg); dr 20:1 (¹H NMR).

(3R)-Major Diastereoisomer

ee 31% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 9:1, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 16.3 min and t_R (minor) = 20.7 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.69 (dd, J = 7.7, 1.9 Hz, 1H, ArH), 7.37 (dd, J = 7.7, 1.6 Hz, 1H, ArH), 7.26–7.15 (m, 2H, ArH), 6.01 (ddd, J = 17.1, 10.1, 8.5 Hz, 1H, CHCH₂), 5.31–5.20 (m, 2H, CH₂), 5.09 (d, J = 8.5 Hz, 1H, CHAr), 3.66 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 170.2, 169.8, 135.3, 134.1, 133.7, 130.1, 129.9, 128.9, 127.2, 120.1, 82.5, 54.3, 49.5.

(3S)-Minor Diastereoisomer

ee 46% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 9:1, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 26.0 min and t_R (minor) = 24.1 min].

HRMS (ESI) m/z: [M – H]⁻ calcd for C₁₃H₁₂ClO₅ 283.0379; found 283.0384.

3-(3-Chlorophenyl)-2-hydroxy-2-(methoxycarbonyl)pent-4-enoic Acid 3c

The title compound was synthesized following the general procedure from dimethyl (E)-2-((3-(3-chlorophenyl)allyl)oxy)malonate 1c (90 mg). After full conversion was achieved in the [2,3]-Wittig rearrangement reaction within 20 h, the enzymatic hydrolysis reaction mixture was stirred for an additional 24 h. Compound 3c was obtained as a yellow solid in 68% yield (58 mg); dr 2.5:1 (¹H NMR).

(3R)-Major Diastereoisomer

ee 73% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 97:3, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 89.6 min and t_R (minor) = 118.9 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.36–7.31 (m, 1H, CCHCCl), 7.29–7.19 (m, 3H, ArH), 6.10 (ddd, J = 16.9, 10.2, 9.0 Hz, 1H, CHCH₂), 5.31–5.20 (m, 2H, CH₂), 4.30 (d, J = 9.0 Hz, 1H, CHAr), 3.72 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 169.7, 169.6, 139.4, 134.4, 133.5, 129.9, 129.2, 128.1, 127.2, 120.3, 82.8, 54.7, 54.4.

(3S)-Minor Diastereoisomer

ee 99% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 97:3, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 77.4 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.38–7.41 (m, 1H, CCHCCl), 7.29–7.19 (m, 3H, ArH), 6.12–6.00 (m, 1H, CHCH₂), 5.26–5.15 (m, 2H, CH₂), 4.29 (d, J = 9.4 Hz, 1H, CHAr), 3.91 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 170.1, 169.4, 139.2, 134.3, 134.2, 129.85, 129.6, 128.0, 127.8, 119.6, 82.6, 54.8, 54.6.

HRMS (ESI) m/z: [M – H]⁻ calcd for C₁₃H₁₂ClO₅ 283.0379; found 283.0383.

3-(4-Chlorophenyl)-2-hydroxy-2-(methoxycarbonyl)pent-4-enoic Acid 3d

The title compound was synthesized following the general procedure from dimethyl (E)-2-((3-(4-chlorophenyl)allyl)oxy)malonate 1d (90 mg). After full conversion was achieved in the [2,3]-Wittig rearrangement reaction within 20 h, the enzymatic hydrolysis reaction mixture was stirred for an additional 24 h. Compound 3d was obtained as a white solid in 64% yield (55 mg); dr 2.4:1 (¹H NMR).

(3R)-Major Diastereoisomer

ee 75% [Chiralpak OJ-H, hexane (TFA 0.01%)/iPrOH = 97:3, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 35.1 min and t_R (minor) = 32.4 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.35–7.21 (m, 4H, ArH), 6.08 (ddd, J = 16.9, 10.2, 8.9 Hz, 1H, CHCH₂), 5.27–5.18 (m, 2H, CH₂), 4.29 (d, J = 8.9 Hz, 1H, CHAr), 3.68 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 170.2, 169.6, 135.9, 133.9, 133.82, 130.4 (2C), 128.9 (2C), 120.0, 82.8, 54.4 (2C).

(3S)-Minor Diastereoisomer

ee 96% [Chiralpak OJ-H, hexane (TFA 0.01%)/iPrOH = 97:3, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 30.0 min and t_R (minor) = 22.2 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.35–7.21 (m, 4H, ArH), 6.10–5.99 (m, 1H, CHCH₂), 5.21–5.14 (m, 2H, CH₂), 4.28 (d, J = 9.3 Hz, 1H, CHAr), 3.89 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 170.1, 169.9, 135.7, 134.4, 133.77, 130.9 (2C), 128.8 (2C), 119.4, 82.6, 54.7, 54.3.

HRMS (ESI) m/z: [M – H]⁻ calcd for C₁₃H₁₂ClO₅ 283.0379; found 283.0383.

2-Hydroxy-2-(methoxycarbonyl)-3-(4-methoxyphenyl)pent-4-enoic Acid 3e

The title compound was synthesized following the general procedure from dimethyl (E)-2-((3-(4-methoxyphenyl)allyl)oxy)malonate 1e (88 mg). After full conversion was achieved in the [2,3]-Wittig rearrangement reaction within 20 h, the enzymatic hydrolysis reaction mixture was stirred for an additional 48 h. Compound 3e was obtained as an off-white solid in 82% yield (69 mg); dr 2.5:1 (¹H NMR).

(3R)-Major Diastereoisomer

ee 72% [Chiralpak OJ-H, hexane (TFA 0.01%)/iPrOH = 95:5, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 50.2 min and t_R (minor) = 57.2 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.24 (d, J = 8.9 Hz, 2H, ArH), 6.83 (d, J = 8.9 Hz, 2H, ArH), 6.11 (ddd, J = 16.9, 10.2, 8.8 Hz, 1H, CHCH₂), 5.27–5.16 (m, 2H, CH₂), 4.28 (d, J = 8.8 Hz, 1H, CHAr), 3.78 (s, 3H, CH₃), 3.71 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 169.9, 169.0, 159.24, 134.2, 130.0 (2C), 129.2, 119.5, 114.1 (2C), 83.2, 55.3, 54.6, 54.4.

(3S)-Minor Diastereoisomer

ee 97% [Chiralpak OJ-H, hexane (TFA 0.01%)/iPrOH = 95:5, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 61.9 min and t_R (minor) = 36.6 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.29 (d, J = 8.9 Hz, 2H, ArH), 6.86–6.80 (m, 2H, ArH), 6.14–6.05 (m, 1H, CHCH₂), 5.20–5.15 (m, 2H, CH₂), 4.26 (d, J = 9.5 Hz, 1H, CHAr), 3.92 (s, 3H, CH₃), 3.77 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 170.5, 170.2, 159.22, 134.8, 130.5 (2C), 128.9, 118.8, 114.05 (2C), 83.0, 55.3, 54.7, 54.5.

HRMS (ESI) m/z: [M – H]⁻ calcd for C₁₄H₁₅O₆ 279.0874; found 279.0875.

2-Hydroxy-2-(methoxycarbonyl)-3-(4-nitrophenyl)pent-4-enoic Acid 3f

The title compound was synthesized following the general procedure from dimethyl (E)-2-((3-(4-nitrophenyl)allyl)oxy)malonate 1f (93 mg). After full conversion was achieved in the [2,3]-Wittig rearrangement reaction within 20 h, the enzymatic hydrolysis reaction mixture was stirred for an additional 48 h. Compound 3f was obtained as an off-white solid in 71% yield (63 mg); dr 2.7:1 (¹H NMR).

(3R)-Major Diastereoisomer

ee 65% [Chiralpak AD-H, hexane (TFA 0.01%)/ABSE/iPrOH = 95:4:1, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 84.7 min and t_R (minor) = 97.4 min]. ¹H NMR (400 MHz, CDCl₃) δ 8.17 (d, J = 8.8 Hz, 2H, ArH), 7.55 (d, J = 8.8 Hz, 2H, ArH), 6.17–6.02 (m, 1H, CHCH₂), 5.28–5.32 (m, 1H, CH₂), 5.28–5.25 (m, 1H, CH₂), 4.45 (d, J = 8.9 Hz, 1H, CHAr), 3.71 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 169.8, 169.3, 147.58, 145.0, 133.0, 130.1 (2C), 123.8 (2C), 120.9, 82.4, 54.5, 54.5.

(3S)-Minor Diastereoisomer

ee 91% [Chiralpak AD-H, hexane (TFA 0.01%)/ABSE/iPrOH = 95:4:1, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 76.4 min and t_R (minor) = 153.0 min]. ¹H NMR (400 MHz, CDCl₃) δ 8.21–8.11 (m, 2H, ArH), 7.58 (d, J = 8.8 Hz, 2H, ArH), 6.17–6.02 (m, 1H, CHCH₂), 5.28–5.25 (m, 1H, CH₂), 5.25–5.22 (m, 1H, CH₂), 4.45 (d, J = 9.2 Hz, 1H, CHAr), 3.94 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 169.7, 169.6, 147.55, 144.7, 133.5, 130.5 (2C), 123.7 (2C), 120.4, 82.3, 54.9, 54.4.

HRMS (ESI) m/z: [M – H]⁻ calcd for C₁₃H₁₂NO₇ 294.0619; found 294.0626.

2-Hydroxy-2-(methoxycarbonyl)-3-(naphthalen-2-yl)pent-4-enoic Acid 3g

The title compound was synthesized following the general procedure from dimethyl (E)-2-((3-(naphthalen-2-yl)allyl)oxy)malonate 1g (94 mg). After full conversion was achieved in the [2,3]-Wittig rearrangement reaction within 20 h, the enzymatic hydrolysis reaction mixture was stirred for an additional 48 h. Compound 3g was obtained as an off-white solid in 43% yield (39 mg); dr 2.3:1 (¹H NMR).

(3R)-Major Diastereoisomer

ee 81% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 95:5, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 66.9 min and t_R (minor) = 60.1 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.80–7.72 (m, 4H, ArH), 7.50–7.39 (m, 3H, ArH), 6.25 (ddd, J = 17.2, 10.1, 8.7 Hz, 1H, CHCH₂), 5.34–5.19 (m, 2H, CH₂), 4.51 (d, J = 8.8 Hz, 1H, CHAr), 3.64 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 169.9, 169.8, 134.9, 134.1, 133.5, 133.0, 128.3, 128.1, 128.0, 127.8, 126.9, 126.3, 126.23, 119.9, 83.2, 55.3, 54.3.

(3S)-Minor Diastereoisomer

ee 99% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 95:5, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 130.4 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.89–7.72 (m, 4H, ArH), 7.59–7.39 (m, 3H, ArH), 6.33–6.15 (m, 1H, CHCH₂), 5.34–5.19 (m, 2H, CH₂), 4.50 (d, J = 9.2 Hz, 1H, CHAr), 3.93 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 170.4, 169.6, 134.8, 134.6, 133.4, 132.9, 128.6, 128.2, 128.18, 127.7, 127.3, 126.16 (2C), 119.2, 83.0, 55.1, 54.7.

HRMS (ESI) m/z: [M – H]⁻ calcd for C₁₇H₁₅O₅ 299.0925; found 299.0928.

2-Hydroxy-3-(1H-indol-1-yl)-2-(methoxycarbonyl)pent-4-enoic Acid 3h

The title compound was synthesized following the general procedure in a 0.1 mmol scale from dimethyl (E)-2-((3-(1H-indol-1-yl)allyl)oxy)malonate 1h (35 mg). After full conversion was achieved in the [2,3]-Wittig rearrangement reaction within 20 h, the enzymatic hydrolysis reaction mixture was stirred for an additional 48 h. Compound 3h was obtained as an off-white solid in 45% yield (15 mg); dr 2.6:1 (¹H NMR).

(3S)-Major Diastereoisomer

ee 28% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 95:5, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 47.9 min and t_R (minor) = 40.1 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.60 (dt, J = 7.9, 1.0 Hz, 1H, Ar–C4), 7.47 (d, J = 8.4 Hz, 1H, Ar–C7), 7.38 (d, J = 3.4 Hz, 1H, Ar–C2), 7.25–7.19 (m, 1H, Ar–C6), 7.13–7.08 (m, 1H, Ar–C5), 6.54 (d, J = 3.3 Hz, 1H, Ar–C3), 6.16 (ddd, J = 17.2, 10.4, 7.0 Hz, CHCH₂), 5.95 (d, J = 6.7 Hz, 1H, CHAr), 5.38–5.26 (m, 2H, CH₂), 3.40 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 168.5, 168.4, 136.0, 130.4, 128.2, 126.1, 121.79, 121.2, 121.0, 120.0, 109.4, 103.4, 82.5, 61.1, 54.3.

(3R)-Minor Diastereoisomer

ee 39% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 95:5, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 61.1 min and t_R (minor) = 101.3 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.57 (d, J = 7.8 Hz, 1H, Ar–C4), 7.51 (d, J = 8.2 Hz, 1H, Ar–C7), 7.48 (d, J = 3.3 Hz, 1H, Ar–C2), 7.23–7.16 (m, 1H, Ar–C6), 7.12–7.06 (m, 1H, Ar–C5), 6.52 (d, J = 3.3 Hz, 1H, Ar–C3), 6.17–6.06 (m, 1H, CHCH₂), 5.94 (d, J = 6.1 Hz, 1H, CHAr), 5.34–5.28 (m, 1H, CH₂), 5.20 (dt, J = 16.9, 1.1 Hz, 1H, CH₂), 3.94 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 168.8, 168.1, 136.6, 131.2, 128.2, 126.9, 121.77, 120.8, 120.6, 119.9, 109.9, 103.3, 82.3, 60.9, 54.5.

HRMS (ESI) m/z: [M + Na]⁺ calcd for C₁₅H₁₅NaNO₅ 312.0842; found 312.0833.

2-Hydroxy-2-(methoxycarbonyl)-3-(thiophen-2-yl)pent-4-enoic Acid 3i

The title compound was synthesized following the general procedure from dimethyl (E)-2-((3-(thiophen-2-yl)allyl)oxy)malonate 1i (81 mg). After full conversion was achieved in the [2,3]-Wittig rearrangement reaction within 20 h, the enzymatic hydrolysis reaction mixture was stirred for an additional 48 h. Compound 3i was obtained as an off-white solid in 86% yield (66 mg); dr 6:1 (¹H NMR).

(3S)-Major Diastereoisomer

ee 40% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 93:7, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 33.4 min and t_R (minor) = 28.5 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.22 (dd, J = 4.9, 1.5 Hz, 1H, ArH), 6.99–6.91 (m, 2H, ArH), 6.06 (ddd, J = 17.0, 10.1, 8.9 Hz, 1H, CHCH₂), 5.33–5.25 (m, 1H, CH₂), 5.22 (dd, J = 10.1 Hz, 1H, CH₂), 4.69 (d, J = 8.9 Hz, 1H, CHAr), 3.77 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 169.9, 169.4, 138.8, 134.0, 126.8, 126.5, 125.5, 119.8, 82.7, 54.5, 50.85.

(3R)-Minor Diastereoisomer

ee 99% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 93:7, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 59.9 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.25–7.16 (m, 1H, ArH), 7.04–7.00 (m, 1H, ArH), 6.99–6.91 (m, 1H, ArH), 6.12–5.97 (m, 1H, CHCH₂), 5.33–5.16 (m, 2H, CH₂), 4.68 (d, J = 9.2 Hz, 1H, CHAr), 3.90 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 170.2, 169.7, 138.7, 134.7, 127.0, 126.7, 125.6, 119.2, 82.5, 54.6, 50.88.

HRMS (ESI) m/z: [M – H]⁻ calcd for C₁₁H₁₁O₅S 255.0333; found 255.0334.

2-Hydroxy-2-(methoxycarbonyl)-3-methylpent-4-enoic Acid 3j

The title compound was synthesized following the general procedure from dimethyl (E)-2-(but-2-en-1-yloxy)malonate 1j (61 mg). After full conversion was achieved in the [2,3]-Wittig rearrangement reaction within 7 days, the enzymatic hydrolysis reaction mixture was stirred for an additional 24 h. Compound 3j was obtained as a yellow oil in 62% yield (35 mg); dr 1.4:1 (¹H NMR).

(3S)-Major Diastereoisomer

ee 64% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 93:7, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 30.5 min and t_R (minor) = 18.9 min]. ¹H NMR (400 MHz, CDCl₃) δ 5.77–5.67 (m, 1H, CHCH₂), 5.24–5.11 (m, 2H, CH₂), 3.91 (s, 3H, COOCH₃), 3.25–3.12 (m, 1H, CHCH₃), 1.02 (d, J = 6.8 Hz, 3H, CHCH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 171.0, 170.6, 136.2, 118.4, 82.33, 54.4, 43.9, 14.37.

(3R)-Minor Diastereoisomer

ee 61% [Chiralpak AD-H, hexane (TFA 0.01%)/iPrOH = 93:7, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 25.0 min and t_R (minor) = 22.0 min]. ¹H NMR (400 MHz, CDCl₃) δ 5.67–5.59 (m, 1H, CHCH₂), 5.19–5.07 (m, 2H, CH₂), 3.86 (s, 3H, COOCH₃), 3.25–3.12 (m, 1H, CHCH₃), 1.09 (d, J = 6.8 Hz, 3H, CHCH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 170.7, 170.5, 136.6, 118.0, 82.26, 54.4, 44.3, 14.43.

HRMS (ESI) m/z: [M – H]⁻ calcd for C₈H₁₁O₅ 187.0612; found 187.0615.

2-(Ethoxycarbonyl)-2-hydroxy-3-phenylpent-4-enoic Acid 3k

HPLC analysis conditions for the [2,3]-Wittig rearrangement intermediate 2k: ee 55% [Chiralpak AD-H, hexane/ABSE/iPrOH = 95:3:2, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 12.0 min and t_R (minor) = 10.8 min].

The title compound was synthesized following the general procedure from diethyl 2-(cinnamyloxy)malonate 1k (88 mg). After 20 h, full conversion was achieved in the [2,3]-Wittig rearrangement reaction conducted in ethanol, then the enzymatic hydrolysis reaction mixture was stirred for 48 h. Compound 3k was obtained as an off-white solid in 85% yield (68 mg); dr 8.3:1 (¹H NMR).

(3R)-Major Diastereoisomer

ee 73% [Chiralpak OJ-H, hexane (TFA 0.01%)/iPrOH = 95:5, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 19.2 min and t_R (minor) = 23.9 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.41–7.33 (m, 2H, ArH), 7.32–7.22 (m, 3H, ArH), 6.15 (ddd, J = 17.1, 10.0, 9.0 Hz, 1H, CHCH₂), 5.29–5.23 (m, 1H, CHCH₂), 5.21 (dd, J = 10.1 Hz, 2.1 Hz, 1H, CHCH₂), 4.33 (d, J = 8.9 Hz, 1H, CHAr), 4.11 (q, J = 7.0 Hz, 2H, CH₂CH₃), 1.16 (t, J = 7.2 Hz, 3H, CH₂CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 170.1, 169.3, 137.4, 134.4, 129.2 (2C), 128.6 (2C), 127.9, 119.5, 82.8, 64.1, 55.1, 13.9.

(3S)-Minor Diastereoisomer

ee 99% [Chiralpak OJ-H, hexane (TFA 0.01%)/iPrOH = 95:5, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 29.7 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.41–7.33 (m, 2H, ArH), 7.32–7.22 (m, 3H, ArH), 6.18–6.08 (m, 1H, CHCH₂), 5.25–5.17 (m, 2H, CHCH₂), 4.37 (q, J = 7.2 Hz, 2H, CH₂CH₃), 4.32 (d, J = 9.4 Hz, 1H, CHAr), 1.38 (t, J = 7.2 Hz, 3H, CH₂CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 170.0, 169.9, 137.2, 134.6, 129.4 (2C), 128.6 (2C), 127.8, 119.1, 82.5, 64.4, 55.0, 14.2.

HRMS (ESI) m/z: [M – H]⁻ calcd for C₁₄H₁₅O₅ 263.0925; found 263.0926.

1 mmol Scale Chemoenzymatic One-Pot Synthesis of 2-Hydroxy-2-(methoxycarbonyl)-3-phenylpent-4-enoic Acid 3a

The experiment was conducted following the general procedure in a 1 mmol scale from dimethyl 2-(cinnamyloxy)malonate 1a (264 mg, 1 mmol). After full conversion was achieved in the [2,3]-Wittig rearrangement reaction within 20 h, the enzymatic hydrolysis reaction mixture was stirred for an additional 24 h. Compound 3a was obtained as an off-white solid in 83% yield (208 mg); dr 2.2:1 (¹H NMR), ee 76/99% (HPLC).

General Procedure for the Synthesis of Racemic α-Hydroxy Half-Esters 3a–k

To a solution of allyloxy 1,3-dicarbonyl compound 1 (1 equiv) in CHCl₃ (0.2 M), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (0.2 equiv) was added. After stirring at rt for 5 min, the solvent was evaporated. The crude mixture was purified by column chromatography on silica gel (4–10% EtOAc in PE/DCM 3/1) providing racemic homoallyl alcohol 2.

To a solution of homoallyl alcohol 2 (1 equiv) in MeOH (0.1 M), KOH (1 equiv) was added. The reaction mixture was stirred at rt until the completion of the reaction, monitored by TLC. Then, the pH of the mixture was adjusted to 9–10 using 1 M aq NaOH solution and extracted twice with diethyl ether. The water layer was then acidified to pH 2 with 1 M aq HCl solution and extracted with diethyl ether. The combined organic layers (extracted at pH 2) were dried over MgSO₄. Evaporation of the solvent provided the racemic product 3. If necessary, additional purification by column chromatography was performed using 2% EtOAc in DCM/formic acid 99/1 mixture.

Methyl 2-(Benzylcarbamoyl)-2-hydroxy-3-phenylpent-4-enoate 4a

To a solution of 2-hydroxy-2-(methoxycarbonyl)-3-phenylpent-4-enoic acid 3a (50 mg, 0.2 mmol, dr 2.9:1) and HATU (84 mg, 0.22 mmol) in DMF (2 mL, 0.1 M), DIPEA (38 μL, 0.22 mmol) and benzylamine (24 mg, 0.22 mmol) were added. The reaction was stirred for 20 min at rt. Then, the reaction mixture was extracted once with distilled water and EtOAc. The organic phase was collected, dried (MgSO₄), filtered, and concentrated under reduced pressure. The crude mixture was purified twice by column chromatography on silica gel (5–20% EtOAc in PE), providing the major diastereoisomer (46 mg, 68%) and the minor diastereoisomer (18 mg, 27%) of compound 4a as white solids.

(3R)-Major Diastereoisomer

ee 72% [Chiralpak AD-H, hexane/iPrOH = 9:1, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 19.1 min and t_R (minor) = 22.2 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.43 (t, J = 5.9 Hz, 1H, NH), 7.37–7.20 (m, 10H, Ar), 6.11 (ddd, J = 17.1, 10.2, 8.8 Hz, 1H, CHCH₂), 5.19 (dt, J = 17.1, 1.3 Hz, 1H, CHCH₂), 5.14 (dd, J = 10.3, 1.6 Hz, 1H, CHCH₂), 4.56 (dd, J = 14.8, 6.3 Hz, 1H, NHCH₂Ar), 4.37 (d, J = 8.8 Hz, 1H, CHAr), 4.36 (dd, J = 14.8, 5.5 Hz, 1H, NHCH₂Ar), 4.29 (s, 1H, OH), 3.64 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 171.9, 167.5, 138.3, 137.7, 134.8, 129.1 (2C), 128.8 (2C), 128.5 (2C), 128.0 (2C), 127.8, 127.6, 118.9, 82.9, 55.5, 54.0, 44.0.

(3S)-Minor Diastereoisomer

ee 99% [Chiralpak AD-H, hexane/iPrOH = 9:1, flow rate = 1.0 mL/min, 25 °C, λ = 210 nm; t_R (major) = 15.5 min]. ¹H NMR (400 MHz, CDCl₃) δ 7.41–7.36 (m, 2H, CH-m-Ar), 7.31–7.25 (m, 4H, CH-o-Ar, CH-p-Ar, NHCH₂-p-Ar), 7.21–7.14 (m, 2H, NHCH₂-o-Ar), 7.12–7.01 (m, 1H, NH), 6.76–6.64 (m, 2H, NHCH₂-m-Ar), 6.14 (dt, J = 17.1, 9.9 Hz, 1H, CHCH₂), 5.21 (ddd, J = 17.1, 1.6, 0.8 Hz, 1H, CHCH₂), 5.15 (dd, J = 10.2, 1.8 Hz, 1H, CHCH₂), 4.40 (dd, J = 15.0, 7.3 Hz, 1H, NHCH₂Ar), 4.38 (s, 1H, OH), 4.39–4.33 (m, 1H, CHAr), 4.00 (dd, J = 15.0, 4.6 Hz, 1H, NHCH₂Ar), 3.92 (s, 3H, CH₃). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 172.5, 167.1, 137.9, 137.4, 135.6, 129.7 (2C), 128.6 (2C), 128.4 (2C), 127.5 (2C), 127.4 (2C), 118.5, 83.0, 55.4, 54.5, 43.4.

HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₀H₂₁NO₄ 340.1543; found 340.1536.

General Procedure for the Kinetic Study

Reaction Profile for the Hydrolysis Step of One-Pot Reaction without Isolation of the [2,3]-Wittig Rearrangement Product 2a

The experiment was conducted following the general procedure for the chemoenzymatic one-pot synthesis of α-hydroxy half-esters 3, starting from dimethyl 2-(cinnamyloxy)malonate 1a (79 mg). After full conversion was achieved in the [2,3]-Wittig rearrangement reaction within 20 h, the enzymatic hydrolysis reaction mixture was stirred for an additional 24 h. During this period, samples were taken from the reaction mixture for ¹H NMR (20 μL in 600 μL DMSO-d₆) and HPLC (30 μL). HPLC samples were acidified to pH 2 with 1 M aq HCl solution and extracted with diethyl ether. Enantiomeric excess was determined by chiral HPLC analysis of the sample obtained by preparative TLC.

Reaction Profile for the Hydrolysis Reaction Using Isolated [2,3]-Wittig Rearrangement Product 2a as a Starting Material

To a solution of dimethyl 2-(cinnamyloxy)malonate 1a (53 mg, 0.2 mmol) in methanol (2 mL), Ca(NTf₂)₂ (0.01 mmol, 6 mg), (R,S)-inda-PyBox (0.01 mmol, 3.9 mg), and imidazole (0.01 mmol, 0.7 mg) were added. The reaction mixture was stirred at 60 °C for 20 h. After evaporating the solvent, the crude mixture was purified by column chromatography on silica gel (3–7% EtOAc in PE/DCM 3/1 mixture), providing dimethyl (R)-2-hydroxy-2-(1-phenylallyl)malonate 2a (48 mg, 91%) as a white solid. To a suspension of dimethyl (R)-2-hydroxy-2-(1-phenylallyl)malonate 2a (47 mg, 0.17 mmol, ee 26%) in DMSO (0.18 mL) and sodium phosphate buffer (pH 8.0, 0.71 mL), PLE (150 EU) was added, and the mixture was stirred at 35 °C for 3 h. During this period, samples were taken from the reaction mixture for ¹H NMR (20 μL in 600 μL DMSO-d₆) and HPLC (30 μL). HPLC samples were acidified to pH 2 with 1 M aq HCl solution and extracted with diethyl ether. Enantiomeric excess was determined by chiral HPLC analysis of the sample obtained by preparative TLC.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c02973.

• Synthesis of starting compounds; optimization procedures; copies of ¹H and ¹³C spectra; and HPLC chromatograms (PDF)

The authors declare no competing financial interest.