Stereoselective Two-Step Biocatalysis in Organic Solvent: Toward All

Article pubs.acs.org/OPRD

Stereoselective Two-Step Biocatalysis in Organic Solvent: Toward All Stereoisomers of a 1,2-Diol at High Product Concentrations Jochen Wachtmeister,† Andre Jakoblinnert,‡ and Dörte Rother*,† †

IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany Piramal Healthcare UK Ltd., Division of Biocatalysis, The Wilton Centre, R345, TS10 4RF Redcar, United Kingdom

‡

S Supporting Information *

ABSTRACT: Biotransformations on larger scale are mostly limited to cases in which alternative chemical routes lack sufficient chemo-, regio-, or stereoselectivity. Here, we expand the applicability of biocatalysis by combining cheap whole cell catalysts with a microaqueous solvent system. Compared to aqueous systems, this permits manifoldly higher concentrations of hydrophobic substrates while maintaining stereoselectivity. We apply these methods to four different two-step reactions of carboligation and oxidoreduction to obtain 1-phenylpropane-1,2-diol (PPD), a versatile building block for pharmaceuticals, starting from inexpensive aldehyde substrates. By a modular combination of two carboligases and two alcohol dehydrogenases, all four stereoisomers of PPD can be produced in a flexible way. After thorough optimization of each two-step reaction, the resulting processes enabled up to 63 g L−1 product concentration (98% yield), space-time-yields up to 144 g L−1 d−1, and a target isomer content of at least 95%. Despite the use of whole cell catalysts, we did not observe any side product formation of note. In addition, we prove that, by using 1,5-pentandiol as a smart cosubstrate, a very advantageous cofactor regeneration system could be applied.

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INTRODUCTION Optically pure 1,2-diols are versatile building blocks for the manufacturing of pharmaceuticals, agrochemicals, and chiral catalysts. Among them, 1-phenylpropane-1,2-diol (PPD) itself serves both as an agent against neurodegenerative disease and as building block for anti-inflammatory drugs and cardiovascular agents.1 There are diverse chemical approaches toward 1,2-diols. These include ketone reduction using hydride transfer agents, Sharpless dihydroxylation of alkenes, epoxide hydrolysis, and asymmetric aldol condensation.2−11 Unfortunately, these routes often share a lack of stereoselectivity in that only mixtures of isomers are obtained.11−15 According to Bommarius and Riebel, this becomes particularly problematic in a pharmaceutical context, where any component, 1% or higher, of a drug demands thorough toxicological investigation.16 Other methods are more selective but only allow access to one of four possible stereoisomers.11,17 As an alternative way of obtaining optically pure 1phenylpropane-1,2-diol, biocatalytic routes generally enable high regio-, chemo-, and stereoselectivity. However, unfortunately, they are often limited by low product concentration and poor space-time-yields (STY).11,18 Here, we present the means to stereoselective access all four isomers of PPD at high product concentration. To do this we have chosen a toolbox approach, combining the appropriate enzymes for carboligation and oxidoreduction (Scheme 1).19 The major advance of our approach is the use of highly selective enzymes or enzyme variants that enables us to access intermediates and products in high chiral purity. As a consequence, the need for an intermediate workup is avoided, and the resulting cascades can be operated in a narrow time frame. For the coupling of benzaldehyde and acetaldehyde we use two different thiamine diphosphate-dependent enzymes © 2016 American Chemical Society

with carboligase activity, Pseudomonas f luorescens benzaldehyde lyase20,21 (BAL, Supporting Information SI-1) and Pseudomonas putida benzoylformate decarboxylase22,23 (BFD; also referred to as BFDC24) variant L461A25 (BFDL461A, Supporting Information SI-1). These are complementary in their stereoselectivity, yielding the (R)- or (S)-configured 2-hydroxy-1phenylpropanone (HPP), respectively. Combined with the use of two different alcohol dehydrogenases (ADHs), namely, Ralstonia sp. ADH26−28 (RADH, Supporting Information SI-1) or Lactobacillus brevis ADH29 (LbADH, Supporting Information SI-1), for the reduction of the 2-hydroxy ketones, these catalyst combinations allow the production of all four stereoisomers of PPD (Scheme 1).19,27 In further advances we (i) switched from aqueous media into microaqueous organic solvents to enable high substrate concentrations of poorly water-soluble compounds and (ii) employed lyophilized whole cells as a cheap and stable catalyst source.19,30,31 The microaqueous organic reaction is set up by adding buffer in a volume of 1 μL per mg of lyophilized whole cell catalyst (resulting in a maximum of 10 vol %) to an organic solvent. Initially this forms a biphasic system, but upon addition of lyophilized cells, the entire buffer is soaked up by the cells. This results in a reaction system comprising only one visible liquid phase thus circumventing the need for liquid−liquid product extraction. The raw product is simply obtained by the removal of the whole cell catalyst by centrifugation and evaporation of the solvent. Using this solvent and catalyst combination, (i) the encapsulated enzymes are shielded from the organic solvent by residual cell wall components, (ii) external cofactor addition becomes unnecessary, (iii) in situ Received: July 6, 2016 Published: September 19, 2016 1744

DOI: 10.1021/acs.oprd.6b00232 Org. Process Res. Dev. 2016, 20, 1744−1753

Organic Process Research & Development

Article

Scheme 1. Synthetic Routes to All Four Stereoisomers of 1-Phenyl-1,2-propanediol by a Combination of Two Carboligases and Two Alcohol Dehydrogenases

a

BAL: benzaldehyde lyase from Pseudomonas f luorescens, BFDL461A: benzoylformate decarboxylase variant L461A from Pseudomonas putida, RADH: alcohol dehydrogenase from Ralstonia sp., LbADH: alcohol dehydrogenase from Lactobacillus brevis.

Scheme 2. Representation of the Two-Step Reaction Combining a Carboligase for C−C-Coupling (Step 1) with an Alcohol Dehydrogenase (ADH) for Hydroxy Ketone Reduction (Step 2)

a

1,5-Pentanediol is used as a smart cosubstrate.33.

Figure 1. Screening of organic solvents for (a) the carboligation catalyzed by BFDL461A-harboring cells and (b) the oxidoreduction by LbADHexpressing cells.

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RESULTS AND DISCUSSION Setup and Optimization: Choice of Organic Solvent and Buffer for the Microaqueous Reaction System. To obtain all PPD stereoisomers, four different catalysts are required and need to be modularly combined, namely, BAL and BFDL461A for carboligation as well as RADH and LbADH for oxidoreduction (Scheme 1). As the optimization of the twostep reaction to (1R,2R)-PPD with BAL and RADH in a microaqueous reaction system has already been published,31 optimization of BFDL461A and LbADH-catalyzed whole cell reactions remained to be investigated. First, we tested the impact of nine different organic solvents on both catalysts in single biotransformations. These solvents represent a selection chosen to cover a wide range of polarity (logP) and functionality, as both of these parameters have been described as being influential on biocatalytic activity.35,36 As shown in Figure 1a, MTBE is by far best solvent for the carboligation reaction with BFDL461A cells (as it was for BAL31). For LbADH-harboring cells differences between solvents are less pronounced (Figure 1b). LbADH shows best reaction rates in cyclohexane, although 2-propanol, MTBE, and n-hexane still

cofactor regeneration is enabled, and (iv) extraordinary high substrate loads can be achieved.31,32 This practical approach had already been optimized for the production of (1R,2R)-PPD to yield up to 55 g L−1 of product at extraordinary space-time yields (327 g L−1 d−1).31 Here we expand the use of these methods to produce all stereoisomers of PPD and in doing so demonstrate the transferability of the microaqueous reaction system to other biocatalysts and biocatalyst combinations. Motivated by the recent finding of so-called “smart cosubstrates”33 which enables savings of up to 90 mol % of cosubstrate, we also investigate the applicability of such diols for the oxidoreduction in our microaqueous system (Scheme 2).33,34 We find that not only is waste generation diminished but also costs are cut. Overall, the combined processes we describe provide advantages such as (i) high product concentrations, (ii) high conversion, (iii) easier product recovery, (iv) low catalyst cost, (v) increased atom economy, (v) utilization of cheap substrates, (vi) no need for external cofactor addition, and (vii) good selectivity. Finally we demonstrate proof that the benefits of microaqueous reactions are not limited to BAL and RADH.31 1745



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Figure 2. (a−c) Screening of different buffer conditions during carboligation of benzaldehyde and acetaldehyde using BFDL461A cells. (a) Screening of different buffer species at 1 M concentration. (b) Screening of different TEA buffer concentrations at pH 10. (c) Screening of different pHs of TEA buffer at 1 M concentration. (d) Comparison of the use of buffer versus deionized water during carboligation using BFDL461A cells and reduction of benzaldehyde employing LbADH cells.

changes in the buffer system tremendously affect the carboligation efficiency of both BAL31 and BFDL461A cells. In summary, the system of choice was a microaqueous reaction system with MTBE as the organic phase and 1 M TEA buffer (pH 10.0). The buffer (1 μL of buffer per mg catalyst) was entirely taken up by the cells, and only one liquid phase was visible. Setup and Optimization: Carboligation Providing (S)HPP. The carboligation reaction mediated by the BAL whole cell catalyst in microaqueous MTBE to provide (R)-HPP has already been demonstrated and optimized by Jakoblinnert and Rother.31 It was found that 500 mM benzaldehyde and 180 mM acetaldehyde gave an ideal initial reaction rate. For both substrates, the initial rate activity dropped at higher concentrations due to inhibition or inactivation of the catalyst. This process provided space-time-yields up to 324 g L−1 d−1 together with product concentrations of more than 67 g L−1.31 Inspired by those results we sought to modify the process to now produce (S)-HPP. In the first instance we tested the effect of aldehyde concentration on the activity of BFDL461A cells. With benzaldehyde we observed Michaelis−Menten-like behavior with a plateau of an astonishing 2 M. However, to ensure complete carboligation on a reasonable time scale (95%, Figure 2c). Screening pH 4 to 8.5, purified wildtype BFD in solely aqueous systems shows an optimum at pH 8.5,23 although no screening for activity at higher pHs was carried out. Finally, in a comparison of a nonbuffered system with a system containing 2.5% (v/v) 1 M TEA buffer (pH 10.0), the optimized microaqueous system led to 47-fold increase in carboligation activity using BFDL461A cells. Conveniently, the optimized solvent conditions were also compatible with LbADH cells, with a 1.3-fold increase in oxidoreduction rate being observed (Figure 2d). These results highlight the importance of a thorough investigation of the aqueous phase when establishing microaqueous systems for biocatalysis. While the activity enhancements for ADHs (LbADH and RADH) are rather small, 1746



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Setup and Optimization: Investigation of Diol Cosubstrates for ADH-Catalyzed Reactions. Traditionally, substrate-coupled cofactor regeneration requires the addition of large molar excesses of alcohols such as 2-propanol as cosubstrates. In an attempt to avoid this, the possibility of implementing smart diol cosubstrates in the microaqueous reaction system was investigated (Scheme 2).33,34 Using benzaldehyde as a model substrate we tested five commercially available diols in MTBE, to explore the reduction catalyzed by cell lyophilizates containing either LbADH or RADH. In the first instance, the five diols were compared to a 2- and 5-fold excess of 2-propanol, the commonly used cosubstrate for LbADH-catalyzed reactions.29 As can be seen in Figure 3, 1,5-

diol substrates, namely, 3-methyl-1,5-pentanediol (91%), 1,6hexanediol (93%), and 1,4-butanediol (97%), gave better than 90% conversion. Conversely, 1,4-pentanediol allowed conversion of no more than 77% in 24 h, while cyclohexanol gave only incomplete conversion regardless of whether it was used in 3-fold (91%) or 5-fold (95%) excess. It should be noted that, although 1,5-pentanediol enabled complete conversion, it did so with a lower initial rate than either 2-propanol or cyclohexanol. Therefore, if atom economy and waste prevention is of higher priority than reaction time, 1,5pentanediol would be the cosubstrate of choice with RADH cells. However, in the present circumstances, 2-propanol is the preferred cosubstrate as it allows full conversion at high rates and is more benign in comparison to cyclohexanol. Two-Step Reactions: Rational Design. Generally, twostep reactions can be operated in either simultaneous (both catalytic steps at the same time) or sequential (catalytic steps carried out successively) reaction mode. While simultaneous reactions may allow higher space-time yields they are also prone to cross reactions due to lack of substrate specificity. Sequential reactions may result in less side-product formation, but it often comes at the expense of space-time yields due to the temporal separation of the reaction steps. The optimal mode will depend on the cross-reactivity of the chosen catalysts and their individual reaction condition requirements.31,32,38 In the two-step reactions with LbADH described here, a simultaneous operation is not feasible as the enzyme maintains up to 36-fold higher activity with the aldehyde substrates than with HPP (Table 1).27 This would lead to formation of benzyl

Figure 3. Screening of different cosubstrates during reduction of benzaldehyde using LbADH cells.

Table 1. Specific Activity of RADH and LbADH towards Different Substrates27

pentanediol and 1,4-butanediol result in better than 99% conversion when used at only 0.5 molar equivalent to the benzaldehyde concentration. The other diols, 3-methyl-1,4pentanediol, 1,4-pentanediol, and 1,6-hexanediol, were not quite so effective at 94, 85, and 77% conversion, respectively. When compared to 5-fold excess of 2-propanol the two best diols also provide up to ∼4-fold increase in the initial reaction rate, meaning there is ∼4-fold higher productivity using only 10% of the molar amount of cosubstrate. Consequently, 1,5pentanediol was used as the cosubstrate for further 2-step reactions employing LbADH cells. It should be emphasized that this result should be relevant for any application utilizing LbADH, an intriguing observation considering its synthetic potential.29 With RADH cells a conversion ≥99% was only achieved within 24 h when using a 3- or 5-fold excess of 2-propanol, or 0.5 molar equivalent of 1,5-pentanediol (Figure 4). Three other

specific activity (U mg−1) LbADH RADH a

benzaldehyde

acetaldehyde

(R)-HPP

(S)-HPP

8.0 ± 0.1 3.0 ± 0.1

109.8 ± 3.1 n.a.a

3.0 ± 0.0 239.9 ± 7.6

3.2 ± 0.1 10.0 ± 0.1

n.a.: no activity detected.

alcohol and ethanol rather than the desired diol. On the other hand, a simultaneous cascade with RADH is more favorable since the enzyme prefers HPP over the aldehydes. Nonetheless, a sequential operation with RADH results in fewer sideproducts and thereby increases the yield of the diol. As a consequence, all two-step reactions were operated in a sequential mode as, in this instance, maximizing yields/product concentrations was of higher priority than maximizing spacetime-yields. Two-Step Reactions: Synthesis of (1S,2S)-PPD Using BFDL461A and LbADH Cells. Accessing the (1S,2S)-PPD isomer requires the combination of BFDL461A for production of the intermediate (S)-HPP and LbADH for its subsequent reduction (Scheme 1). This was the most challenging cascade as the activities and yields in both reactions are relatively low (Table 1, Supporting Information SI-3). Optimization of the cascade revealed that, so long as fresh aldehyde substrates are used to avoid inhibiting/inactivating impurities, a cell load of 100 g L−1 BFDL461A provides a sufficient rate of HPP production (Supporting Information SI-3). Further, due to favorable partitioning, intermittent removal of BFDL461A cells resulted in higher product concentrations (Supporting Information SI-4). Subsequently, 1,5-pentanediol was em-

Figure 4. Screening of different cosubstrates during reduction of benzaldehyde using RADH cells. 1747



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ployed to optimize HPP reduction with LbADH cells (Figure 3). The carboligation using BFDL461A cells yielded 378.9 ± 1.8 mM of (S)-HPP (ee > 98%) within 6.5 h (Figure 5), which

Figure 6. Concentration curves during the two-step reaction of BAL and LbADH cells for the production of (1S,2R)-PPD.

99%), and only ∼10 mM benzaldehyde and ∼2 mM (R)benzoin remained. After the second reaction step, 420 ± 15.4 mM (63.8 g L−1) of 1-phenylpropane-1,2-diol had been produced, with ∼95% being the desired (1S,2R)-stereoisomer. Taking into account the dilution during the switch to the second (oxidoreduction) step, this catalyst combination enables astonishing 98% yield. Two-Step Reactions: Production of (1R,2R)-PPD Using BAL and RADH Cells. Previously, the combination of BAL and RADH whole cell catalysts to produce (1R,2R)-PPD was explored under microaqueous conditions with the emphasis on maximizing space-time-yields.31 Here we report the reexamination of the reaction using the newly available alternative cosubstrates and with the focus on optimizing the final product concentration. Our initial concern was that cyclohexanol, which had been used to facilitate the one pot cascade, did not generate full conversion of the hydroxy ketone substrate even when used in a 5-molar excess31 (Figure 4). The first thought was to replace it with either 2-propanol or 1,5-pentanediol. Accordingly both cosubstrates were tested with the carboligation reactions being run in parallel to ensure that the oxidoreduction reactions started from similar carboligation results, i.e., ∼ 428.0 ± 14.3 mM and 441.5 ± 1.1 mM of HPP (ee 99% R), for the 2propanol and 1,5-pentanediol reactions, respectively (Figure 7). When a 5-fold excess of 2-propanol was added (956 μL to 5 mL reaction), an initial drop in HPP concentration was seen, presumably due to the dilution effect (Figure 7a). Overall, the reaction rate of RADH cells with 2-propanol as cosubstrate was high, and the reaction was completed after only 11 h (Figure 7a). A PPD concentration of 366.5 ± 7.0 mM was attained (99% conversion of HPP), almost 99% of which was the desired (1R,2R)-stereoisomer. Clearly the two-step process using 2-propanol provides excellent stereoselectivity. Conversely, when 0.5 molar equivalent of 1,5-pentanediol was employed as the cosubstrate (Figure 7b) even after 30 h ∼ 54.3 ± 11.3 mM of (R)-HPP remained unconverted (i.e, 83% conversion). Strikingly, a slight loss of stereoselectivity was observed as the reaction proceeded (ic ∼ 98% at 11 h and 94.5% after 30 h). Given its (i) shorter reaction time, (ii) higher overall yield and (iii) better stereoselectivity the traditional alcohol cosubstrate, 2-propanol, was the cosubstrate of choice. Finally, when compared to the published use of cyclohexanol (Table 2), 2-propanol yielded a 14% higher final product concentration together with an increase of 10% in overall yield while maintaining excellent stereoselectivity.

Figure 5. Concentration curves during the two-step reaction of BFDL461A and LbADH cells for the production of (1S,2S)-PPD.

corresponds to 82% yield (dilution considered, Materials and Methods). After removal of BFDL461A cells, the addition of LbADH cells, buffer, and cosubstrate led to a slight drop in analyte concentrations due to dilution. During the oxidoreduction step, the HPP was almost completely converted, as only 2.5 ± 1.3 mM (S)-HPP were detected after 16 h. Residual benzaldehyde was entirely reduced to 88.1 ± 2.8 mM benzyl alcohol. Out of 339.8 ± 13.1 mM HPP at the start of oxidoreduction 312.5 ± 9.9 mM (47.5 g L−1) of diol were formed, leaving the mass balance between both compounds unclosed by 8%. Generally, the mass balance of the entire process remains unclosed, which is not surprising giving (i) expectable difference of the analytes regarding partitioning between the organic and the more polar surrounding/interior of the cells, and the (ii) the compartmentalization of the reaction (analytes within the cells are not detected). The content of the desired (1S,2S)-stereoisomer among all PPD built (defined as “isomer content”, Materials and Methods) is 96.7% (ee 99.0%, de 94.3%), which is good to excellent. Two-Step Reactions: Production of (1S,2R)-PPD Using BAL and LbADH Cells. For (1S,2R)-PPD production, we used a combination of BAL cells for the intermediate production of (R)-HPP and LbADH cells for its subsequent reduction (Scheme 1). This is the only cascade of the four described herein, for which the intermediate removal of the C− C-coupling catalyst was necessary so as to circumvent the undesired back reaction of the carboligation products (Supporting Information SI-5). Once again, a sequential operation mode was advised as LbADH has higher activity with both aldehyde substrates than it does with the hydroxy ketone (Table 1). As before, by comparison with 2-propanol, 1,5-pentanediol as “smart co-substrate” was used to enable higher final product concentrations (Figure 3). At first, the carboligation reaction catalyzed by BAL cells gave an increase in (R)-benzoin (Figure 6). This was not a surprise as it is well-known that BAL catalyzes both the self-ligation of benzaldehyde as well the breakdown of benzoin.20,21,39 Upon addition of increasing amounts of acetaldehyde, the equilibrium shifted to favor HPP formation, and the (R)-benzoin concentration decreased. By the end of the carboligation reaction, HPP had reached a concentration of ∼440 mM (ee 1748



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Figure 7. Concentration curves during the two-step reaction of BAL and RADH cells for the production of (1R,2R)-PPD employing (a) 2-propanol or (b) 1,5-pentanediol as cosubstrate.

Table 2. Comparison of Performance Parameters for the Two-Step Reaction towards (1R,2R)-PPD with Different Cosubstrates for Oxidoreduction (1R,2R)-PPD concentration overall yield (dilution considered) STY stereoselectivityb a

cyclohexanola

2-propanol

1,5-pentanediol

321.2 mM (48.8 g L−1) 84% 167 g L−1 d−1 (7 h) ee > 99%, de 99.6%

366.5 mM (55.7 g L−1) 94% 144 g L−1 d−1 (9 h) ic 98.7%

357.8 mM (54.3 g L−1) 80% 44 g L−1 d−1 (30 h) ic 94.5%

From Jakoblinnert and Rother, 2014.31 bic is defined as the fraction of the target stereosiomer in a mixture of multiple stereoisomers.

Figure 8. Concentration curves during the two-step reaction of BFDL461A and RADH cells for the production of (1R,2S)-PPD employing (a) 1,5pentanediol or (b) 2-propanol as cosubstrate.

Table 3. Overview of Performance Parameters of All Four Two-Step Reactions towards all PPD Isomers (1S,2S)-PPDd

(1S,2R)-PPDe

(1R,2R)-PPDf

(1R,2S)-PPDg

cosubstrate

pentanediol

pentanediol

2-propanol

2-propanol

product concentration (g L−1) overall yield (%)a space-time-yield (g L−1 d−1) catalyst productivity (kg kg−1)b target isomer content (%)c

47.5 79 71.3 (16 h) ∼0.25 96.7

63.8 98 85.2 (18 h) ∼0.51 95.4

55.7 94 121.5 (11 h) ∼0.96 98.7

39.8 82.0 76.4 (12.5 h) ∼0.20 96.8

a

Yield was calculated with respect to the limiting aldehyde substrate, considering the dilution upon addition of acetaldehyde and dilution at switch to oxidoreduction by addition of buffer and cosubstrate. bReferring to 5 mL of total reaction volume. cic is defined as the fraction of a stereoisomer in a mixture of multiple stereoisomers. The exact distribution of isomers can be found in Supporting Information SI-7. d100 mg/mL BFDL461A cells, 500 mM benzaldehyde, 464 mM acetaldehyde in total, 100 μL/mL 1 M TEA (pH 10.0), in MTBE, 5 mL total; addition of 500 mg of LbADH cells, 500 μL of 1 M TEA (pH 10.0), 155 μL of 1,5-pentanediol for oxidoreduction. e25 mg/mL BAL cells, 500 mM benzaldehyde, 630 mM acetaldehyde in total, 25 μL/mL 1 M TEA (pH 10.0), in MTBE, 5 mL total; addition of 500 mg of LbADH cells, 500 μL of 1 M TEA (pH 10.0), 176 μL of 1,5pentanediol for oxidoreduction. f25 mg/mL BAL cells, 500 mM benzaldehyde, 630 mM acetaldehyde in total, 25 μL/mL 1 M TEA (pH 10.0), in MTBE, 5 mL total; addition of 330 mg of RADH cells, 330 μL of 1 M TEA (pH 10.0), 956 μL of 2-propanol for oxidoreduction. g100 mg/mL BFDL461A cells, 500 mM benzaldehyde, 424 mM acetaldehyde in total, 100 μL/mL 1 M TEA (pH 10.0), in MTBE, 5 mL total; addition of 500 mg of RADH cells, 500 μL of 1 M TEA (pH 10.0), 956 μL of 2-propanol for oxidoreduction.

Two-Step Reactions: Production of (1R,2S)-PPD Using BFDL461A and RADH Cells. The two-step reaction combining the carboligation by BFDL461A cells providing

(S)-HPP and its subsequent oxidoreduction by RADH cells to form (1R,2S)-PPD was performed with 1,5-pentanediol and 2propanol as cosubstrates (Figure 8). 1749



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The parallel carboligation reactions both yielded 354.6 ± 6.0 mM and 357.9 ± 1.5 mM of (S)-HPP (ee > 98%). After oxidoreduction with 1,5-pentanediol as cosubstrate (Figure 8a), the reaction reached a PPD concentration of 275.7 ± 15.6 mM (76% overall yield, dilution-corrected). At this point, 14.8 ± 3.7 mM of (S)-HPP remained unchanged. Surprisingly there was a difference of about 17% in mass balance between PPD and HPP. Such a large difference has not been observed within the other two-step reactions. Consistent with the earlier reaction using 1,5-pentanediol, the PPD only contained ∼91% of the target stereoisomer, a number that was considered to be unsatisfactory. When 2-propanol was employed (Figure 8b), the product concentration after oxidoreduction was found to be to be 261.6 ± 5.8 mM, i.e., a dilution-corrected yield of 70%. On the flip side, the reaction reached a plateau in about half the time (12.5 h) and, at 97%, the target stereoisomer content was much more satisfactory. Synthesis of (1S,2S)-PPD on a Preparative (150 mL) Scale. The preparative synthesis of (1R,2R)-PPD with BAL and RADH in a microaqueous setup has already been successfully carried out.31,32 Here, we wanted to demonstrate the general applicability of the microaqueous system with lyophilized whole cells on preparative scale also for other catalysts. To do this we scaled up the production of (1S,2S)PPD, catalyzed by BFDL461A and LbADH cells, to a 150 mL reaction volume (Supporting Information SI-6). The carboligation yielded 385 mM of (S)-HPP (ee 98.3%) within 6.25 h. The subsequent oxidoreduction was performed with 1,5-pentanediol as cosubstrate and reached 333 mM of diol after 23.5 h and complete conversion of HPP. When compared with the smaller scale reaction, the scaled-up version resulted in a 6.6% higher product concentration while maintaining a target stereoisomer content of ∼97% (Table 3). After the reaction was complete the cells were simply filtered off, lysed, and washed with MTBE. The resulting fraction was combined with the crude organic phase from the reaction, dried, concentrated by rotary evaporation, and subjected to flash chromatography. Ultimately 4.56 g product were obtained (41% overall yield) with no change in stereoselectivity occurring during purification. Potentially the downstream processing might be improved by more efficient cell lysis and extraction of the lactone coproduct. To make the process even more economically feasible, the isolated lactone coproduct can serve as a valuable precursor in the manufacture of biodegradable polymers.40 In summary, the desired product (1S,2S)-PPD was successfully produced on a gram scale by simple scale-up of the test reaction. Summary of the Four Tested Cascades. In brief, each of the four enzymes worked well in the combination of whole cell catalyst and microaqueous solvent. Thorough optimization of the individual and combined reactions resulted in outstandingly high product concentrations (average 52 g L−1), excellent reaction, and space-time-yields, all of which exceeded the industrial thresholds of feasibility for biocatalytic fine chemical synthesis.41,42 At the same time very good selectivities (≥95% target isomer content) were maintained (for precise isomer distribution see Supporting Information SI-7). Some minor amounts of byproducts such as phenylacetylcarbinol or 1phenylpropan-1,2-dione were observed during the reaction, but these had vanished by the end of the reaction. None of the byproducts appeared to affect the chiral purity of the product.

In comparison to Kihumbu et al.19 who also accessed all four PPD-stereoisomer in high optical purity, our approach provides a 12- to 80-fold increase in final product concentrations. More recently Wu et al.18 were able to obtain (1R,2R)- and (1R,2S)PPD with very good stereoselectivity, but our approach reported here resulted in 5- to 8-fold higher PPD concentrations. That is not to say the process cannot be further improved. We have identified the limited catalyst productivity (Table 3)43 as a point for further optimization and are currently working to increase the expression levels of recombinant enzymes within the whole cell catalysts. In addition we a working toward the implementation of additional catalyst recycling strategies.32

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CONCLUSION

In this manuscript, we applied a 2-step biocatalytic approach using lyophilized whole cells in microaqueous reaction system for the synthesis of all four stereoisomers of 1-phenylpropane1,2-diol at outstandingly high product concentrations. Up to 63.8 g L−1 product at up to 98% of yield could be achieved. All space-time-yields accomplished were above 70 g L−1 d−1 at good to excellent stereoselectivities. We demonstrated the scalability of the presented microaqueous reaction approach on 150 mL scale, yielding more than 4.5 g of (1S,2S)-PPD with a purity >95%. In addition, we tested novel cosubstrates for cofactor regeneration to increase atom efficiency of the enzyme cascade. We could prove that the industrially relevant alcohol dehydrogenase from Lactobacillus brevis accepts 1,5-pentanediol as a cosubstrate with a higher initial reaction rate than the abundantly used 2-propanol. With 1,5-pentanediol, up to 90 mol % of cosubstrate can be saved, allowing a reduction in cost and waste. Furthermore, 1,5-pentanediol is considered neither dangerous (volatile, flammable, or explosive) nor hazardous (harmful or toxic).

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MATERIALS AND METHODS Chemicals. All chemicals were obtained in high chemical grade (purity ≥95%) from Sigma-Aldrich, Fluka, and Carl Roth. (R)-HPP was synthesized as a reference for analytics according to Jakoblinnert and Rother.31 (1R,2R)-PPD was produced and purified according to Wachtmeister et al.32 Benzaldehyde was distilled under reduced pressure to remove benzoic acid before application. Acetaldehyde was ordered in smallest quantities available (5 mL) and only opened up to five times (stored under argon at 4 °C) before disposal in order to avoid any catalyst inactivation by oxidized or polymerized substrate. Preparation of a Whole Cell Catalyst. The plasmid coding for BFD variant L461A was obtained as described in the publication of Gocke et al.25 Cells expressing benzaldehyde lyase or benzoylformate decarboxylase variant L461A were cultivated in high density fermentation as described elsewhere.44,45 Plasmids coding for LbADH and RADH were produced as described by Kulig et al.27 and cultivation proceeded in shaking flask culture at 20 °C for 48 h in autoinduction medium.46,47 Cell pellets were harvested by centrifugation and stored frozen at −80 °C. Lyophilization of pellets proceeded for 2−4 days at −45 °C and 1 mbar pressure (Martin Christ Gefriertrocknungsanlagen GmbH). The lyophilizate was ground to crude powder and stored at −20 °C. The powder was later used in different volumetric loads but always rehydrated by addition of 1 μL buffer per mg of powder. For 1750



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reactions had a total volume of 5 mL and were incubated horizontally shaking (1400 rpm) at 30 °C. The fed-batch addition of acetaldehyde in BAL-catalyzed reactions proceeded in pulses of 90 mM at 25, 45, 75, 110, and 180 min, according to published reaction optimizations.31 BFDL461A-catalyzed reactions were fed according to balancing the amount of free acetaldehyde (by subtracting the amount of HPP built from the acetaldehyde applied) and subsequent addition of acetaldehyde needed to restore a concentration of 90 mM. When the concentration of HPP reached a plateau, the vials were placed into 50 mL reaction tubes and centrifuged (1780 g, 10 min, 22 °C). The supernatant was transferred into new 8 mL vials containing the corresponding ADH catalyst (165 mg RADH cells for (R)-HPP-reduction, 500 mg RADH cells for (S)-HPPreduction, or 500 mg of LbADH for both (R)- and (S)-HPP reduction). For RADH-catalyzed reactions using 1,5-pentanediol, cosubstrate was added in half the molar amount of initially present benzaldehyde (500 mM). For LbADH-catalyzed reactions with 1,5-pentanediol, half the molar amount of benzaldehyde (present at start) and residual acetaldehyde within the reaction (calculated from the amount of HPP built and acetaldehyde added) was added. To finally start the oxidoreduction, 1 M TEA buffer (pH 10.0) was added in amounts of 1 μL of buffer per 1 mg of catalyst. Carboligation reactions were followed by HPLC, while oxidoreduction was additionally followed by GC. Preparative Synthesis of (1S,2S)-PPD on a 150 mL Scale. 15 g of lyophilized BFDL461A cells were given into a 250 mL round-bottom flask. 134 mL of a 560 mM benzaldehyde solution in MTBE were added together with 15 mL of 1 M TEA buffer (pH 10) and 760 μL of acetaldehyde. The reaction was incubated stirring (200 rpm, overhead blade stirrer) at 30 °C and fed with acetaldehyde as described above. After carboligation the cells were allowed to precipitate, and the supernatant was transferred into a new 250 mL round-bottom flask already containing 15 g of lyophilized LbADH cells. The residual pellet was washed with 10 mL of MTBE that were then filtered through a funnel with cotton batting and added to the ADH cells, together with 4.67 mL of 1,5-pentanediol and 15 mL of 1 M TEA buffer (pH 10). After 24.5 h the reaction was terminated by precipitation of the catalyst and filtration of the clear supernatant through Celite (Merck, size 0.02−0.1 mM) and cotton batting. Residual turbid liquid was transferred into plastic tubes, centrifuged (4 °C, 1780 g, 10 min), and the supernatant was added to the formerly received filtrate. Residual catalyst cells were resuspended in 60 mL of MTBE and lysed by sonication (4 min). The resulting liquid was clarified as described above by filtration and added to the previously obtained filtrate. Pooled fractions were dried with magnesium sulfate and concentrated by rotary evaporation giving 9.673 g of raw product to be subjected to flash chromatography. Chromatography was performed using 500 g of silica gel and a 6:4 mixture of petrol ether and ethyl acetate as mobile phase. Resulting fractions were roughly screened for PPD and δ-valerolactone using GC and pooled according to purity. Product identity and exact purity were analyzed by 1Hand 13C NMR analysis (600 MHz, CDCl3, Supporting Information SI-6) using 1,3,5-trioxan as a reference. HPLC Analysis. Benzaldehyde, benzyl alcohol, HPP, and benzoin were analyzed by chiral HPLC, using a Dionex Gina 50 autosampler, a Dionex UVD170U detector (Thermo Fisher Scientific, USA) with a Gynkotek high-precision pump model 480, and a Gynkotek Degasys DG 1310 (Thermo Fisher

DNA and amino sequences of enzymes see Supporting Information SI-1. Optimizing the Solvent System: Choosing Solvent and Buffer for the Microaqueous Reaction System. Organic solvent screening for BFDL461A and LbADH cells and the screening of different buffer species and buffer concentration for BFDL461A cells was performed as described elsewhere. 31 Screening of the optimal buffer pH for biotransformations with BFDL461A cells was done using 1 M TEA buffer. Here, 25 mg of lyophilized cells were given into 1.5 mL glass vials and mixed with a substrate solution containing 500 mM benzaldehyde and 180 mM acetaldehyde in MTBE. Twenty-five μL of buffer of varying pH were added to give 1 mL of reaction volume in total, incubated shaking (1400 rpm) at 30 °C. Samples were analyzed by HPLC to quantify the increase of (S)-HPP, and linear regression was performed to calculate initial reaction rates. Finally, optimized buffer conditions were compared to the use of water for 25 mg of BFDL461A or LbADH cells, respectively. With BFDL461A cells, 975 μL of substrate mixture containing 500 mM benzaldehyde and 90 mM acetaldehyde were used, and the reaction was started by addition of 25 μL of aqueous phase. With LbADH, the 975 μL of substrate mixture contained 200 mM benzaldehyde and 1 M 2-propanol in MTBE. All reactions were incubated shaking at 1000 rpm at 30 °C and sampled regularly. Product increase was quantified by HPLC analysis, and initial reaction rates were derived from the linear initial increase of product. Optimizing the Carboligation Step toward (S)-HPP. The screening of initial reaction rates at different concentrations of benzaldehyde was performed on 1 mL scale in 1.5 mL glass vials filled with 25 mg of lyophilized cells and 25 μL of 1 M TEA buffer (pH 9.5) using MTBE as main solvent. To start the reaction, 180 mM acetaldehyde was applied using an ice-cold Hamilton syringe. To screen for ideal acetaldehyde concentrations, reactions were set up as described above, but with different amounts of acetaldehyde at a constant concentration of 500 mM benzaldehyde. All reactions were incubated shaking at 1400 rpm at 30 °C. Samples were analyzed by HPLC, and linear regression was performed to calculate initial reaction rates. Investigation of Diol Cosubstrates for ADH-Catalyzed Reactions. To screen different cosubstrates for both ADH whole cell catalysts, the reduction of benzaldehyde to benzyl alcohol served as a model reaction. 100 mg of lyophilized cells were given into 1.5 mL glass vials. A benzaldehyde stock in MTBE was given into each reaction reaching a final concentration of 396 mM, before the addition of 198 mM of diol cosubstrate or 2-, 3-, or 5-fold excess of 2-propanol or cyclohexanol over benzaldehyde, respectively. 1,6-Hexanediol was solubilized in dimethylformamide to avoid the addition as a solid. All reactions were filled up to 900 μL with MTBE and started by addition of 100 μL of 1 M TEA buffer (pH 10.0). Reactions were incubated shaking (1000 rpm) at 30 °C and followed by HPLC. Setup of Two-Step Reactions. Lyophilized catalyst for carboligation (125 mg BAL cells or 500 mg BFDL461A cells) was given into 8 mL glass vials, and a volume of benzaldehyde stock in MTBE was added to give a final concentration of 500 mM. Acetaldehyde was added using an ice-cold glass syringe, giving a concentration of 90 mM (BFDL461A cells) or 180 mM (BAL cells), respectively. 1 M TEA buffer (pH 10.0) was added in an amount of 1 μL buffer per 1 mg catalyst. Resulting 1751



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Scientific, USA). The Chiralpack IC column (4.6 × 250 mm, 5 mm particle size; Daicel Chemical Ind., Ltd,) was operated with an analytical-grade mobile phase of 70% (v/v) n-heptane and 30% (v/v) 2-propanol at 1 mL min−1 flow (25 °C). Samples were diluted in n-heptane, centrifuged, and diluted in an nheptane solution containing toluene as internal standard. Retention times were 4.1 min for benzyl alcohol, 5.2 min for benzaldehyde, 6.2 min for (R)-HPP, 6.8 min for (S)-HPP, 6.9 min for (R)-benzoin, and 7.2 min for (S)-benzoin. GC Analysis. Acetoin, 1-phenylpropane-1,2-dion, and PPD were analyzed by a chiral CP-Chirasil-DEX CB column (Varian; 25 m × 0.25 mm × 0.25 mm). The program started at 70 °C, held for 5 min, followed by a gradient of 30 °C min−1 to 140 °C, and finally held for 32 min. Detection proceeded by a flame ionization detector using hydrogen as carrier gas. Samples were diluted in acetonitrile and centrifuged. The supernatant was subjected to GC analysis. Retention times were 4.6 for (R)-acetoin, 5.2 min for (S)-acetoin, and 29.4, 30.2, 32.6, and 33.5 min for (1S,2S)-, (1R-2R), (1S,2R), and (1R,2S)PPD, respectively. Calculations. Conversion refers to the amount of substrate depleted. Yield refers to the amount of product gained (according to GC analysis) from the amount of the limiting substrate.48 Calculation of yields considered the dilution by addition of acetaldehyde during carboligation and the addition of buffer and cosubstrate upon switch to oxidoreduction. Since acetaldehyde could not be directly quantified via HPLC, the acetaldehyde feed for carboligation was calculated by subtracting the amount of HPP built from the amount of acetaldehyde fed, in order to obtain the theoretic amount of free acetaldehyde within the reaction. To compare two-step reactions more easily, the content of the target isomer (isomer content, ic) of a reaction is given as the percent fraction of the target isomer It in a mixture of all isomers Ii detected (whether enantiomer or diastereoisomer):

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We like to thank Zaira Maugeri and Torsten Sehl for advice and support. Further we thank Frank Hollmann, Selin Kara and Sandy Schmidt for fruitful discussions regarding diol cosubstrates. We also thank Michael McLeish very much for proofreading.

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ABBREVIATIONS ADH, alcohol dehydrogenase; AMP, 2-amino-2-methyl-1propanol; BAL, Pseudomonas f luorescens benzaldehyde lyase; BFDL461A, Pseudomonas putida benzoylformate decarboxylase variant L461A; de, diastereomeric excess; ee, enantiomeric excess; HPP, 2-hydroxy-1-phenyl-propanone; ic, target isomer content (see Materials and Methods); LbADH, Lactobacillus. brevis alcohol dehydrogenase; NADPH, nicotinamide adenine dinucleotide phosphate; PPD, 1-phenyl-1,2-propanediol; RADH, Ralstonia sp. alcohol dehydrogenase; STY, space-timeyield; ThDP, thiamine diphosphate

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(1) Sehl, T.; Kulig, J.; Westphal, R.; Rother, D. In Industrial Biocatalysis; Grunwald, P., Ed.; Pan Stanford Publishing Pte. Ltd.: Singapore, 2014; Vol. 1, pp 887−930. (2) Johnson, T. H.; Klein, K. C. J. Org. Chem. 1979, 44, 461−462. (3) McMahon, R. J.; Wiegers, K. E.; Smith, S. G. J. Org. Chem. 1981, 46, 99−101. (4) Noyori, R.; Tomino, I.; Tanimoto, Y. J. Am. Chem. Soc. 1979, 101, 3129−3131. (5) Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino, G. A.; Hartung, J.; Jeong, K. S.; Kwong, H. L.; Morikawa, K.; Wang, Z. M. J. Org. Chem. 1992, 57, 2768−2771. (6) Yamada, S.; Koga, K. Sel. Org. Transform. 1970, 1, 1−33. (7) Wang, Z.; Cui, Y. T.; Xu, Z. B.; Qu, J. J. Org. Chem. 2008, 73, 2270−2274. (8) Tokunaga, M.; Larrow, J. F.; Kakiuchi, F.; Jacobsen, E. N. Science 1997, 277, 936−938. (9) Guillena, G.; Hita, M. D.; Najera, C. Tetrahedron: Asymmetry 2006, 17, 1027−1031. (10) Notz, W.; List, B. J. Am. Chem. Soc. 2000, 122, 7386−7387. (11) Kulig, J. Stereoselective synthesis of vicinal diols with enzymatic cascade reactions. Doctoral thesis, Heinrich-Heine-Universität Düsseldorf, 2013. (12) Campos, C. H.; Torres, C. C.; Leyton, A.; Belmar, J.; Mella, C.; Osorio-Vargas, P.; Ruiz, D.; Fierro, J. L. G.; Reyes, P. Appl. Catal., A 2015, 498, 76−87. (13) Husain, S. M.; Stillger, T.; Dünkelmann, P.; Lödige, M.; Walter, L.; Breitling, E.; Pohl, M.; Burchner, M.; Krossing, I.; Müller, M.; Romano, D.; Molinari, F. Adv. Synth. Catal. 2011, 353, 2359−2362. (14) Ikariya, T.; Blacker, A. J. Acc. Chem. Res. 2007, 40, 1300−1308. (15) Torres, C. C.; Campos, C. H.; Fierro, J. L. G.; Reyes, P.; Ruiz, D. J. Mol. Catal. A: Chem. 2014, 392, 321−328. (16) Bommarius, A. S.; Riebel, B. R. In Biocatalysis: Fundamentals and Applications; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2004; p 379. (17) Periasamy, M. Pure Appl. Chem. 1996, 68, 663−666. (18) Wu, S.; Chen, Y.; Xu, Y.; Li, A.; Xu, Q.; Glieder, A.; Li, Z. ACS Catal. 2014, 4, 409−420. (19) Kihumbu, D.; Stillger, T.; Hummel, W.; Liese, A. Tetrahedron: Asymmetry 2002, 13, 1069−1072. (20) Demir, A. S.; Pohl, M.; Janzen, E.; Müller, M. J. Chem. Soc. 2001, 7, 633−635. (21) González, B.; Vicuña, R. J. Bacteriol. 1989, 171, 2401−2405. (22) Hegeman, G. D. J. Bacteriol. 1966, 91, 1140−1154.

ic = It /∑ Ii

Precise amounts of each isomer in each two-step reaction are given in SI-7.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.oprd.6b00232. DNA and amino acid sequences of all enzymes used, initial rate measurement of BFDL461A cell-catalyzed reactions, reaction optimization results for carboligation with BFDL461A cells, optimization of two-step reaction toward (1S,2S)-PPD, optimization of two-step reaction toward (1S,2R)-PPD, concentration curves, and purification results for preparative synthesis of (1S,2S)-PPD, isomer distribution for all two-step reactions (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

This project was financially supported by Deutsche Forschungsgemeinschaft (DFG) in framework of graduate school “biocatalysis using non-conventional media (BioNoCo)”. 1752



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Stereoselective Two-Step Biocatalysis in Organic Solvent: Toward All

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