Ind. Eng. Chem. Res. 2006, 45, 7101-7109
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Investigation of the Compatibility of Racemization and Kinetic Resolution for the Dynamic Kinetic Resolution of an Allylic Alcohol Chayaporn Roengpithya,† Darrell A. Patterson,†,§ Emma J. Gibbins,† Paul C. Taylor,‡ and Andrew G. Livingston*,† Department of Chemical Engineering and Chemical Technology, Imperial College London, Prince Consort Road, London, SW7 2AZ, United Kingdom, and Department of Chemistry, The UniVersity of Warwick, CoVentry, CV4 7AL, United Kingdom
Allylic alcohols, such as methyl styryl carbinol (MSC), are required as single enantiomers for use as chiral building blocks, flavor additives, and aroma compounds in perfumes. This paper investigates the use of dynamic kinetic resolution (DKR) for this purpose, by determining concentration-time data for all species in the racemization, kinetic resolution (KR), and DKR of MSC in toluene. Racemization was catalyzed by ruthenium p-cymene combined with a number of different bases, while Novozym 435 catalyzed the KR. Results showed that the racemization is the rate-limiting step in MSC DKR because the hydrogenation step of the catalyst cycle is deactivated by the KR products. Furthermore, the racemization bases and ruthenium p-cymene made the KR less enantiospecific by either catalyzing nonenantiospecific allylic acetate formation or interfering with enzyme enantiospecifity. Nevertheless, DKRs using ruthenium p-cymene combined with triethylamine, trioctylamine, or P1-t-Oct gave enantiopure (R)-allylic acetate yields above 68% at 80-97% ee. Introduction Enantiomerically pure secondary alcohols are important synthetic intermediates and chiral auxiliaries for both pharmaceutical and agrochemical industries. In particular, allylic alcohols, such as 4-phenyl-3-buten-2-ol (also known as methyl styryl carbinol), are used in a wide range of applications: as chiral building blocks to produce active pharmaceuticals, food flavor additives, and aroma compounds in perfumes. A major method for producing enantiopure compounds from a racemic substrate is via the classic kinetic resolution (KR), where one enantiomer (i.e., 50%) of a racemic substrate is reacted into a separable compound at a faster rate than the other enantiomer. Typically, KRs are catalyzed by highly enantiospecific enzymes so that the unwanted enantiomer is not reacted at all, or at a very low rate.1 However, like most other resolution methods (e.g., chromatography and diastereomeric resolution), KR can only ever produce a 50% yield since only one of the enantiomers is converted. A dynamic kinetic resolution (DKR) can be used to minimize this waste. In a DKR, the racemic substrate is subjected to both a racemization and a KR. Racemization converts the unwanted substrate enantiomer into its racemate, and the desired enantiomer can then be reacted via KR to the desired product. Therefore, unlike a KR, a DKR can give a pure enantiomeric product yield of over 50%. However, this can only be achieved under strict conditions. For an efficient DKR, (i) the racemization must equilibrate the enantiomers rapidly, maintaining a substrate enantiomeric excess (ee) of near zero; (ii) the KR and racemization procedures should be compatible with each other; and (iii) the KR should be irreversible to ensure high enantioselectivity.2,3 Over the past decade, several groups have been developing new catalysts and procedures both to increase the number of * To whom correspondence should be addressed. Telephone: +44 20 75945582. Fax: +44 20 75945604. E-mail: a.livingston@ imperial.ac.uk. † Imperial College London. § Present address: The Department of Chemical and Materials Engineering, The University of Auckland, Private Bag 92019, Auckland, New Zealand. ‡ The University of Warwick.
compounds to which DKRs can be applied and to enable milder reaction conditions to be used in the DKRs already available.2-5 Overall, the greatest progress has been achieved with secondary alcohols,6-10 where the bulk of the reported work has been conducted by two groups: that of Park and M.-J. Kim in Korea6,11-15 and Ba¨ckvall in Sweden.16-18 DSM Pharma Chemicals in Austria now produce secondary alcohols industrially by DKR.19 Despite this, little published work has been conducted on the DKR of allylic alcohols. There are only two full papers, one dealing with the DKR of allylic alcohols to allylic acetates14 and the other the reverse reaction.20 The application of several new catalysts to allylic alcohols is mentioned in a number of additional papers.6,21 All of these studies report the final concentrations, yield, conversions, and ee data for the reactions conducted. This paper seeks to understand the change in reactant, intermediate, and product concentrations over the entire reaction period, to identify the rate limitations, and to derive reaction control strategies. The KR of the allylic alcohol methyl stryl carbinol (MSC) is shown in Figure 1. Lipases are the most common enzymes used in KRs as they have broad substrate specificity, provide high ee’s, and can be used in organic solvents. Candida antarctica lipase (CAL or CALB) and Pseudomonas cepacia lipase (PCL or PS-C) are used in allylic alcohol systems.2 Figure 2 shows the DKR of (R,S)-MSC as it is currently understood. Of the two processes which comprise the DKR, the racemization has been more refined over the years in order to obtain more stable catalysts (in particular, air-stable catalysts), which function under milder reaction conditions. The racemization of (R,S)-MSC (1) using Ru p-cymene is currently understood to occur via nonselective hydrogen transfer following the metal dihydride mechanism.22 In this mechanism, a ruthenium dihydride species is generated from the Ru p-cymene catalyst precursor as both O-H and R-C-H hydrogen atoms of the alcohol are transferred to the metal.23 Many of the reported racemization catalysts are organometallics incorporating transition metals. There are several catalysts based on ruthenium complexes which have been used to racemize secondary alcohols, including allylic alcohols.6,21,24,25 However, many of
10.1021/ie060394o CCC: $33.50 © 2006 American Chemical Society Published on Web 09/09/2006
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Figure 1. Kinetic resolution of (R,S)-MSC (1) to (R)-allylic acetate (4a) and (S)-MSC (1b).
these ruthenium complexes (including the well-known Shvo complex24 and some of the newer ruthenium cyclopentadienyl complexes21) are unsuitable catalysts for the DKR of allylic alcohols. This is because they isomerize some or all of the allylic alcohols to saturate ketones (e.g., 4-phenyl-2-butanone (3 in Figure 2)) rather than racemizing the alcohol. These undesired side reactions are illustrated on the left-hand side of Figure 2. Lee et al.14 found that by using p-cymene ruthenium(II) combined with a base, this problem could be minimized, enabling relatively high alcohol yields in the racemization step. In combination with the lipase PCL, this system permitted successful DKRs with a wide range of allylic alcohols with conversions (to both the acetate and side products) of 94% to over 99%, with ee’s of 95% to greater than 99% for the acetylated products.14 However, yields of the R-allylic acetate were as low as 84%, which is low compared with those of DKRs of other substrates. Evidently, the complexity of the racemization conditions and formation of side product are the key issues preventing a yield of 100% enantiopure product for these DKRs. This paper investigates the time course racemization, KR, and DKR of the allylic alcohol MSC in toluene using ruthenium p-cymene catalyst, with different bases and the immobilized lipase Novozym 435 (CALB immobilized on acrylic resin). First, the KR was studied in isolation. The effect of enzyme loading on the KR was investigated. Thereafter, the effect of the racemization components on the KR was examined by dosing the KR with different bases and ruthenium p-cymene, to determine how exposure time and concentration of racemization components affect the yield and enantiospecifity. Second, the racemization was studied in isolation. The effect of different bases and different concentrations of ruthenium p-cymene on the racemization was investigated in order to maximize the racemization rate while minimizing the undesired side reactions. The effect of the end products of the KR on the racemization was also examined to determine whether these components deactivated the racemization catalyst. Finally, the racemization and KR were studied together, in a DKR, to reconcile the effect of different bases and concentrations with the results seen in the uncoupled KRs and racemizations. 2. Experimental Section 2.1. Materials. The concentrations and suppliers of the chemicals used in the majority of the experiments are outlined in Table 1. Solvents toluene, hexane, and 2-propanol were purchased from Fisher UK and ethyl acetate was sourced from VWR, UK. All solvents were used as received. 2.2. KR, Racemization, and DKR. All reactions were carried out in a parallel reaction carousel (Radley Discovery Technologies, UK) to ensure uniformity of temperature and stirring speed. For the KRs, 3 mg of Novozym 435 was added to a 25 mL solution containing racemic (R,S)-MSC (33.7 mM) and vinyl acetate acyl donor (48.8 mM) in toluene. The literature suggests that this enzyme can undergo denaturation at high temperature,26,27 and so KR temperature was maintained at 30 ( 1 °C (Figure 1). For the racemizations, 2-4 mg of ruthenium p-cymene catalyst was dissolved in 25 mL of toluene containing one of the bases in Table 1, before (S)-MSC (33.7 mM) was
added. Initial racemization tests were conducted at 30 ( 1 °C. For the DKRs, 3 mg of Novozym 435 was added to 25 mL solution containing 2-4 mg of ruthenium p-cymene catalyst, a base, vinyl acetate, and toluene. (S)-MSC (33.7 mM) was added last. All experiments were repeated at least once and the results reported are based on an average of at least two experiments. Samples were taken at appropriate intervals. For samples containing ruthenium catalyst, the racemization was stopped by adding one small drop of concentrated hydrochloric acid. For HPLC analysis, flash chromatography was employed, using a mixture of 80:20 hexane:ethyl acetate through silica gel (200400 mesh, 60 Å) in glass pipet columns, to remove the ruthenium catalyst from the samples. The solution was then gently evaporated, followed by redissolution of the samples in the HPLC mobile phase, ready for analysis. 2.3. Analytical Techniques. Concentrations were determined using an Agilent 6850 Series gas chromatograph (GC) fitted with a flame ionization detector (FID) and Agilent 7683 autoinjector. Separation was achieved on a HP-1 capillary column (30 m × 0.32 mm i.d. × 0.25 um, AnaChem, UK). The injector and detector temperatures were set at 250 and 300 °C, respectively, and the oven temperature profile was programmed as follows: starting from 100 °C, heating at 10 °C/ min to 140 °C, then heating at 5 °C/min to 200 °C, and finally heating at 20 °C/min to 250 °C (which was held for 5 min). Concentrations and peaks were identified and quantified by external calibration using pure standards. The peak areas of each alcohol-related component (the (R)- and (S)-MSC enantiomers, the (R)- and (S)-allylic acetate product enantiomers, and ketone) were summed and used for the normalization of each component peak area. Thus, component concentrations are presented as the percentage of the total area of identified peaks reported by the component peak. Note that the two ketone intermediates in Figure 2 (2 and 3) were not well separated by this method. Consequently, their concentrations were combined and will henceforth be referred to as “ketone”. The enantiomeric excess (ee) is defined as
ee )
[Cmajorenantiomer] - [Cminorenantiomer] [Cmajorenantiomer] + [Cminorenantiomer]
× 100%
(1)
ee was determined by normal phase high performance liquid chromatography (HPLC) on a Unicam Crystal 200 autosampler HPLC with a Varian 2550 UV-vis detector (set at 254 nm) using a Chiralcel OJ column (4.6 mm × 250 mm × 10 µm, Chiral Technologies Europe, France). A mobile phase of 95:5 hexane:2-propanol at 0.5 mL min-1 (for 45 min) was used to separate the enantiomers of (R,S)-MSC. Peaks were identified by external calibration using enantiopure standards. 3. Results and Discussion 3.1. Kinetic Resolution of (R,S)-MSC. The only detailed study of the DKR of allylic alcohols (by Lee et al.14) uses a relatively high enzyme loading of 150 (g of PCL)(mol of MSC)-1. This may be necessary for this enzyme since other DKRs using PCL also use high loadings.13 In this study Novozym 435 has been used since it is widely used in DKR studies, is immobilized (so is easier to handle), and is readily available. However, Novozym 435 is typically used at a much lower enzyme loading than PCL.6,7,10 Note that Candida (e.g., CALB in Novozym 435) and Pseudomonas (e.g., PCL) are lipases from the same genus but differ in selectivity and activity, as they are supplied by different commercial sources (and produced via different preparation techniques). Pseudomonas
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Figure 2. Reaction scheme for the DKR of (R,S)-MSC (1) to R-allylic acetate (4a). Hcat is the hydrogen associated with the Ru p-cymene catalyst. Table 1. Composition, Basicity Constant (pKb), and Concentrations of Reactants and Catalysts Used in Reaction Testing in the Parallel Reaction Carousela reactant or catalyst (R,S)-MSC R:S ) 40:60 (S)-MSC ee ) 89-95% Ru p-cymene
concentration (mmol L-1)b
MSC molar equivalent
33.7
1
33.7
1
1.35
pKb in solvent Substrate n/a
CAS number
supplier
17488-65-2
University of Warwick, UK
n/a
0.04
University of Warwick, UK
Racemization Catalyst n/a
52462-29-0
Strem, UK
161118-69-0 121-44-8
Fluka, UK Aldrich, UK
102-86-3 2411-36-1 1116-76-3
Fluka, UK Fluka, UK Aldrich, UK Fluka, UK
P1-t-Oct triethylamine (TEA)
7.49 33.7
trihexylamine (THexA) triheptylamine (THepA) trioctylamine (TOA)
33.7 33.7 33.7
Bases (Racemization Co-catalyst) MeCNpK + 26.5 0.22 BH MeCNpK + 18.5 1 BH 10.8 in H2O 1 n/a 1 n/a 1 10.3 in H2O
vinyl acetate (VA)
48.8
Acyl Donor for Kinetic Resolution 1.45 n/a
108-05-4
Kinetic Resolution Catalyst n/a
9001-62-1
Novozym 435
1.2 g/L
Novo Nordisk, USA
Solvent (toluene) volume ) 25 mL. Novozym 435 concentration was 36 g unless stated otherwise.
mol-1
lipases are found to be highly selective on “narrow” substrates with limited steric requirements, and thus are often unable to accept bulky compounds. Candida lipases are more selective for substrates intermediate between “bulky” and “narrow”. So substrates which are selectively hydrolyzed by a Pseudomonas type27 usually can also be selectively hydrolyzed by a Candida lipase. To investigate whether a moderate to high ee allylic alcohol KR could still be accomplished using a lower enzyme loading than that employed by Lee et al.,14 KRs at two enzyme loadings were conducted. The data in Table 2 show that this is possible. Entry 1 is the KR with an enzyme loading equivalent to that described by Lee et al.14 Entry 2 is a KR using 24% of the catalyst loading used by Lee et al. (i.e., 36 g (mol of MSC)-1). This suggests that a higher product enantiopurity can be achieved with less Novozym 435 (ee of (R)-allylic acetate: entry 2, 87%; entry 1, 63%) when the substrate concentration available for
Table 2. Effect of Enzyme Concentration on the Kinetic Resolution of (R,S)-MSCa
a
(MSC) for KRs and DKRs. Units for these values are mmol L-1, b
ee (%) entry
Novozym 435 loading (g (mol of MSC)-1)
conversion (%)
S-MSC
R-allylic acetate
1 2
150 36
63 52
100 94
63 87
a Reactants and catalysts: (R,S)-MSC (33.8 mmol L-1), VA (1.5 equiv), Novozym 435 varied as stated, toluene (25 mL).
each enzyme is greater. However, the drawback was a lower nonenantiospecific conversion of the alcohol (entry 2, 52%; entry 1, 63%). These results can be explained as follows. Rotticci et al.28 observed that lower substrate concentration resulted in lower enantioselectivity when using CALB lipases in KRs. It was thought that internal diffusion limitations were the cause of sub-
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Figure 3. Enzymatic KR of (R,S)-MSC in toluene over time. Reactants and catalysts: (R,S)-MSC (33.8 mmol L-1), VA (1.5 equiv), Novozym 435 (36 g (mol MSC)-1), toluene (25 mL). (a) Mole percent of R-allylic acetate: benchmark KR (]), KR with addition of 1 equiv of TOA (O), KR with addition of 1 equiv of TEA (4), and KR with addition of 0.08 equiv of racemization catalyst (Ru cymene) (0). (b) ee % of R-allylic acetate: benchmark KR (]), KR with addition of 1 equiv of TOA (O), KR with addition of 1 equiv of TEA (4), and KR with addition of 0.08 equiv of racemization catalyst (Ru cymene) (0). (c) Mole percent of R-allylic alcohol: benchmark KR (]), KR with addition of 1 equiv of TOA (O), KR with addition of 1 equiv of TEA (4), and KR with addition of 0.08 equiv of racemization catalyst (Ru cymene) (0); mole percent of S-allylic alcohol: benchmark KR ([), KR with addition of 1 equiv of TOA (b), KR with addition of 1 equiv of TEA (2), and KR with addition of 0.08 equiv of racemization catalyst (Ru p-cymene) (9).
strate depletion and subsequently a drop in enantioselectivity. Novozym 435 is a heterogeneous catalyst, consisting of CALB lipase on and inside a porous support. For the reaction to be catalyzed, substrate MSC and acyl donor vinyl acetate must diffuse from the bulk solution, through the film around the particle, and into the pores to the CALB.29 There is likely to be resistance to internal diffusion within the pores; therefore, the effective concentration seen by the CALB lipase will be lower than that at the surface of the catalyst particle. With use of a higher substrate concentration, this concentration depletion is minimized. Furthermore, for enzymes suspended in a monophasic organic solution, the ratio of enzyme to water available in the reaction medium can affect the enzyme activity, as enzymes require residual bound water to remain catalytically active. As the amount of water in the reaction medium is reduced, the activity becomes unpredictable and weak enzyme distortion can occur.27 These factors may contribute to the lower enzyme loading being more favorable. The lower enzyme loading was used in all further work and the KR at this lower enzyme loading was benchmarked over time in two experiments. These results are shown in Figure 3 (data labeled “benchmark KR”). All further permutations of this reaction are compared to this benchmark. Note that only two sampling times were chosen for enzymatic KR of (R,S)-MSC, as shown in Figure 3, due to analytical
equipment limitations. In a separate initial enzymatic KR of (R,S)-MSC at similar reaction conditions with the addition of two different amine bases (TEA and TOA), the enzymatic KR occurred rapidly, within 30 min (result not shown here). Samples were taken at 30 min intervals for 4 h, with further samplings at the 8th hour and 24th hour. The 45-48 mol % of (R)-allylic acetate was observed at the first sampling and remained constant throughout the reaction. The ee of (R)-allylic acetate was 95% at 30 min and gradually decreased to 42% in a manner similar to that shown in Figure 3b. An independent KR test using Novozym 435 showed a reduction in product formation when using the enzyme recovered from previous reactions. Furthermore, the lipases became detached from the resin after more than 150 h of stirring in toluene. This demonstrates that Novozym 435 is not durable enough to stay immobilized during longer term reactions. To determine the impact of the racemization on the KR in the DKR, the effect of the individual components of the racemization on the yield and enantiospecificity of the benchmark KR was investigated. KRs were dosed with the same concentration of the different bases and Ru cymene catalyst used in the racemization reaction. The results are presented in Figure 3. Figure 3a shows that the overall conversion of the MSC to (R)-allylic acetate was not affected by the presence of base in
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the reaction: the expected 50 mol % conversion to (R)-allylic acetate was achieved by the end of all the KRs. However, the ee of the (R)-allylic acetate (Figure 3b) and the mol % of both the (R)- and (S)-MSC enantiomers progressively decreased over time (Figure 3c). It is likely, therefore, that (S)-MSC is being converted to (S)-allylic acetate; the kinetic resolution becomes nonenantiospecific over time. This effect was also observed when ruthenium p-cymene was present in the KR (Figure 3b,c). With 0.08 equiv of ruthenium p-cymene added, there was a small decrease in the mol % of (R)-allylic acetate formed compared to that of the benchmark KR (Figure 3a). However, (S)-allylic acetate was rapidly formed, making the average ee of R-allylic acetate 30% after 24 h (Figure 3b). One potential explanation for these results is that the combination of a base and ruthenium p-cymene is capable of racemizing the resolution product (allylic acetate). Therefore, a racemization was conducted on enantioenriched allylic acetate (results not presented), which showed that (R)-allylic acetate did not racemize in the presence of ruthenium p-cymene and base. Alternatively, there are a number of possible mutually inclusive mechanisms which could explain the data in Figure 3. First, the base and ruthenium p-cymene could increase the internal diffusion resistance or could block access to the active sites of the CALB by absorbing or adsorbing onto the Novozym 435. By analogy with Rotticci et al.,28 this substrate depletion would cause a drop in enantiospecificity. Second, they could interfere with the enzyme enantiospecifity so that it also catalyzes the acylation of (S)-MSC. The enantiospecificity of lipases, like the CALB in Novozym 435, results from the one enantiomer having a reduced activity toward the enzyme.1 It is possible that either the ruthenium p-cymene and/or the bases interfere with this mechanism, decreasing the enzyme’s enantiospecifity. Furthermore, a number of studies have shown that solvent properties can be affected by the presence of ruthenium p-cymene and by the bases, which could cause a decrease in enzyme enantiospecificity. A review of these properties are given in Carrea et al.30 Third, either TEA, TOA, or ruthenium p-cymene could catalyze a nonenantiospecific side reaction forming allylic acetate. However, regardless of the mechanism, Figure 3 indicates that the best and perhaps easiest method of reducing the amount of (S)-allylic acetate formed by the nonenantiospecific reaction is to minimize the reaction time. Figure 3 shows that the KRs are completed within 10 h, so reaction times less than this should be used in the future. 3.2. Racemization of (S)-MSC. In the vast majority of DKR reactions, racemization is the rate-limiting step. So in this work, ruthenium p-cymene was combined with a number of bases to determine the combination giving the highest racemization rate and lowest unwanted side reactions during the racemization of enantioenriched (S)-MSC. Initially, the ruthenium p-cymene concentration was kept at 0.04 equiv to the substrate and two different classes (and strengths) of organic base were tested. These were as follows: (1) weak amine bases triethylamine (TEA), trihexylamine (THexA), triheptylamine (THepA), and trioctylamine (TOA) at 1 equiv to substrate (reaction based on Lee et al.14); (2) a stronger phosphazene base, P1-t-Oct, at a lower concentration of 0.22 equiv to the substrate, as used in previous work by this group.31 The basicity constants of each base (i.e., the pKb value) are listed in Table 1 where available. The effect of these different bases on the racemization is summarized in Figure 4. The two fastest racemizations at the same ruthenium loading were with TEA and P1-t-Oct. The racemizations with these bases were essentially equivalent over
Figure 4. (a) Racemization of (S)-MSC (33.8 mmol L-1) using Ru cymene catalyst (0.04 equiv) in toluene (25 mL). % ee of (S)-MSC: racemization with 1 equiv of TEA (2), racemization with 1 equiv of TOA (b), racemization with 1 equiv of THexA (0), racemization with 1 equiv of THepA (×), racemization with 0.22 equiv of P1-t-Oct (+), racemization with 0.22 equiv of P1-t-Oct, and 0.08 equiv of Ru cymene (/). (b) Ketone formation in the racemization of (S)-MSC using various bases: 1 equiv of THexA (9), 1 equiv of THepA (×), 1 equiv of TEA (b), 0.22 equiv of P1-t-Oct with 0.04 equiv of Ru (+), and 0.22 equiv of P1-t-Oct and 0.08 equiv Ru cymene (/).
their common time period. Of the amine bases, racemization with TEA gave the fastest racemization rate (Figure 4a); the percentage ee of (S)-MSC decreased from 100% to 17% in 24 h. However, ketone formation during this reaction was also the highest (Figure 4b), making less substrate available to the KR. These results can be explained in terms of the currently accepted catalytic mechanism for ruthenium p-cymene-base racemization. Racemization via hydrogen transfer is understood to occur for ruthenium catalyst complexes such as ruthenium p-cymene.2 It has been demonstrated2,23 that ruthenium pcymene and TEA racemize secondary alcohols by dehydrogenation to a relatively stable ketone intermediate (2 in Figure 2), which is in turn nonenantiospecifically hydrogenated to give the racemic alcohol (1a and 1b in Figure 2). For allylic alcohols, it has also been shown that the isomerization to saturated ketones (reactions to 3 in Figure 2) is also catalyzed by such catalytic systems.14,21 Since TEA is more electronegative (and thus more basic) than THexA and THepA (Table 1), the activation of the ruthenium p-cymene by the base to form the active hydride complex2,23 should be either more complete and/or faster.
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Furthermore, the active groups of the base, ruthenium p-cymene and MSC, must be in close proximity to enable this mechanism, so it is likely that steric hindrance due to bulky groups would slow the rate of activation and/or racemization, limiting access to the active groups of the catalyst. Consequently, the more bulky amine bases (THexA and THepA) should have a slower racemization rate, as observed. A possible explanation for why the racemization with TEA formed more ketone than its larger counterparts is due to the basicity since the TEA is more basic than THexA and ThepA; it has a stronger tendency to become protonated than the other bases, thereby binding a greater fraction of the hydrogen molecules required to hydrogenate the ketone back into MSC. This mechanism is possible since the amine bases were in excess compared to the ruthenium p-cymene. There may also be an increased rate of isomerization to the unsaturated ketone (3 in Figure 2) for the TEA; however, this could not be detected by the GC method used. It is uncertain why the racemization involving the stronger phosphazene base P1-t-Oct gave an equivalent rate of racemization (Figure 4a) and ketone formation (Figure 4b) to TEA. A possible explanation is that although the P1-t-Oct is more basic than TEA (Table 1) (and should therefore give a more complete or faster activation of the ruthenium p-cymene to form the active hydride complex2,23), it is also more bulky. This steric hindrance may inhibit the catalyst activation and/or racemization, resulting in a compromised racemization rate, which happened to be equivalent to the racemization rate with the weaker yet smaller base, TEA. The effect of ruthenium p-cymene concentration on the racemization reaction profile was also investigated. When the ruthenium p-cymene concentration is doubled when using P1t-Oct as base, the racemization rate also approximately doubled (Figure 4a). However, the amount of ketone formed also doubled. In terms of the currently accepted reaction mechanism (Figure 2), this could mean the following: (1) the increased ruthenium p-cymene concentration favored the dehydrogenation of the MSC to unsaturated ketone (2 in Figure 2) compared with the hydrogenation back to alcohol, and/or (2) the increase in ruthenium p-cymene concentration favored the isomerization reaction to saturated ketone (3 in Figure 2). All of these results show that the racemization in isolation, i.e., when not combined with KR in a DKR, is a viable reaction, provided some ketone formation can be tolerated. However, the ultimate objective of this work was to investigate DKRs. Therefore, to determine whether the racemization is affected by any of the products of the KR, a racemization was conducted using the actual end product mixture from a (R,S)-MSC KR. This racemization, rather than a DKR, was conducted to quantify this effect since the change in (S)-MSC could be more easily attributed to the racemization reaction where there is no need to account for ee changes due to the enzyme reaction. The end product mixture contained enantioenriched (R,S)-MSC (Novozym 435 does not significantly catalyze S-enantiomer acetylation), as well as the KR products ((R)-allylic acetate and acetaldehyde) and residual reactants (such as vinyl acetate) and any side reaction products (such as acetic acid from the hydrolysis of vinyl acetate6), at concentrations equivalent to those at the halfway point of a full yield DKR. The KR (which used (R,S)-MSC, 333.4 mM; VA, 1.5 equiv.; Novozym 435, 150 g mol (MSC)-1) was run to 50% conversion. The enzymes were filtered off and then the remaining components were diluted to give the following concentrations: 17 mM (S)-MSC, 0.3 mM (R)-MSC, 26 mM (R)-allylic acetate, 1 mM
(S)-allylic acetate, 0 mM, ketone. This mixture was racemized using 0.04 equiv of Ru cymene and the two types of bases used before: (1) weak amine bases TEA, THexA, ThepA, and TOA at 1 equiv to substrate and (2) stronger P1-t-Oct at 0.22 equiv to substrate. The results are presented in Figure 5. In Figure 5a, the decrease in (S)-MSC is greater than the spread in the data for different bases. The amount of (S)-MSC compared to that of (R)-MSC is therefore decreasing, similar to a racemization. However, the rate of decrease in Figure 5a is at least 7.8 to 15 times slower than the rate without any of the KR end products present as in Figure 4a. This is because racemization does not occur in this reaction. Instead, (S)-MSC forms ketone, thereby decreasing its concentration relative to (R)-MSC, resulting in a decrease in the ee, as in a racemization. Since no (R)-MSC is formed, the rate of ee decrease is slower than that in a racemization. This reaction to ketone is more clearly illustrated by comparing the mole balance of alcohol-related reactants and products for two different racemizations: first, a racemization using enantioenriched (S)-MSC (33.75 mM) (Figure 5b) and, second, a racemization of the end products from the KR (Figure 5c). Both reactions used 0.04 equiv of Ru p-cymene and 1 equiv of THexA. Figure 5b shows the expected mole balance over time for the first reaction. Racemization occurs, as shown by the molar percent of (R)-MSC enantiomer which reached 50% yield in 24 h, with minimal ketone being formed. However, for the second reaction (Figure 5c), there was no clear indication that the (S)-MSC racemized to form (R)-MSC since the mole percent of (S)-MSC remained constant. Similar concentration profiles of (R)- and (S)-MSC and ketone were observed for the reactions with THexA and ThepA; therefore, only the concentration profile of THexA is shown in Figure 5b. For TEA, TOA, and P1-t-Oct, only short term (5 h) mole data are available (Figure 4), so in this case, the racemization had not reached completion. Thus, a comparison cannot be made with racemization using THexA. Figure 5c therefore suggests that the (S)-MSC simply reacted to form ketone, which could mean that the ketone was not being hydrogenated to (R,S)-MSC (as in Figure 2). It is possible that the hydrogenation reaction step was deactivated by components in the KR end product mixture. These include unreacted vinyl acetate and/or acetaldehyde. Acetaldehyde is an oxidant10,18 and can therefore oxidize (remove hydrogens from) the active dihydride form of Ru cymene, thereby deactivating the catalyst. Furthermore, the solvents and reagents were used as received, so had some residual water content. Thus, vinyl acetate (which was added at 0.5 equiv in excess) could have undergone hydrolysis to form acetic acid. It has been shown that acetic acid completely deactivates ruthenium organometallic based racemization.6 The results in Figure 5 reflect the amount of deactivation that would occur in the racemization system at the halfway point of a DKR reaction, i.e., at the point when only half the required yield has been generated. Therefore, it shows that the DKR of this allylic alcohol system could be improved if the racemization deactivation and ketone formation could be minimized. To determine if these effects were due to the decoupling of the two racemization and KR systems, full DKRs based on these systems were conducted. 3.3. DKR of (R,S)-MSC. DKRs were initially conducted with the weaker amine bases since these bases are more readily available and less expensive than the P1-t-Oct. The DKR reaction time was kept down to 4 h to avoid the drop in product ee by nonenantiospecific reactions, as seen in the KR studies. The time course reaction data, presented as the percentage of
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Figure 5. (a) Racemization of the end product from the enzymatic kinetic resolution of (R,S)-MSC. % ee of (S)-MSC: racemization with 1 equiv of TEA (2), racemization with 1 equiv of TOA (b), racemization with 1 equiv of THexA (0), and racemization with 0.22 equiv of P1-t-Oct (+). For (b) and (c), the racemization with 1 equiv of THexA and THepA produced a similar trend to that of TOA and has been omitted here for clarity. (b) Racemization of enantioenriched (S)-MSC (33.4 mmol L-1) using 0.04 equiv of Ru cymene catalyst and 1 equiv of THexA in 25 mL of toluene. Mole percent: (R)-MSC (]), (S)-MSC (9), and ketone (×). (c) Racemization of the end product mixture from the enzymatic kinetic resolution of (R,S)-MSC end using 0.04 equiv of Ru cymene catalyst and 1 equiv of THexA in 25 mL of toluene. Mole percent: (R)-MSC (]), (S)-MSC (9), ketone (×), and allylic acetate (+).
Figure 6. DKR of (R,S)-MSC in toluene over 4 h at a low substrate concentration. Reactants and catalysts: (R,S)-MSC (33.8 mmol L-1), VA (1.5 equiv), Ru cymene (0.04 equiv), Novozym 435 (36 g.(mol MSC)-1), toluene (25 mL). (a) DKR with 1 equiv of TOA. (b) DKR with 1 equiv of TEA. Primary Y-axis: mol % (R)-MSC (]), (S)-MSC (9), (R)-allylic acetate (4), (S)-allylic acetate (×), and ketone ([). Secondary Y-axis: % ee (R)-allylic acetate (b) and (S)-MSC (+).
the total moles of MSC, ketone, and allylic acetate, is given in Figure 6. Each point is the average of the data from two separate reactions. The DKR reactions in Figure 6, panels a and b, both show that the yield limitation of 50% intrinsic to KRs can be exceeded by a DKR under these conditions since an enrichment to more than 70 mol % (R)-allylic acetate has been achieved by each reaction. The time course data also indicate why each of the DKRs failed to achieve a higher yield. In each case, the base has had a different impact on the overall reaction.
In the DKR using TOA as the base (Figure 6a), the (R)-MSC is reacted away almost completely over the 4 h reaction period, and so the (S)-MSC ee increases to 100%. It appears that the KR is not significantly inhibited and is functioning as expected. The (R)-allylic acetate ee decreases only marginally (reflecting the marginal (S)-MSC mol % increase) over the 4 h reaction period, indicating that the nonenantiospecific reactions observed in the KR (section 3.1) have indeed been minimized by running the DKR over this shortened time
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Figure 7. DKR of (R,S)-MSC in toluene over 24 h at high substrate concentration, 337 mmol L-1, Ru cymene (0.04 equiv), Novozym 435 (148 g (mol of MSC)-1), VA (1.5 equiv) with different bases. (a) DKR with 1 equiv of TEA. (b) DKR with 0.22 equiv of P1-t-Oct. Primary Y-axis: mol % (R)-MSC (]), (S)-MSC (9), (R)-allylic acetate (4), (S)-allylic acetate (×), ketone ([). Secondary Y-axis: % ee of (R)-allylic acetate (b) and (S)-MSC (+).
period. However, the (S)-MSC mol % only decreases over the first 1.5 h of the reaction, leveling out at the same time that the (R)-allylic acetate stops being formed. The mol % of ketone remains approximately constant over the entire reaction period. So the racemization has stopped functioning after 1.5 h, preventing the formation of further ketone and especially (R)MSC. Therefore, there is no further substrate for the KR after 1.5 h, so no further (R)-allylic acetate product can be formed. It is probable, therefore, that the racemization catalyst has been deactivated by the same reaction components which deactivated the racemization in the reaction of KR end products in section 3.2 (Figure 5). In the DKR using TEA (Figure 6b), the reaction appears to be faster compared with the DKR using TOA, as expected, since the rate of racemization (the rate-limiting step) with TEA is faster than that with TOA (see section 3.2). The reaction is so fast that it is complete within 1.5 h. The drop in (R)-allylic acetate ee (and rise in mol % (S)-allylic acetate) thereafter is probably due to the nonenantiospecific reaction of MSC to allylic acetate, catalyzed by the ruthenium catalyst and TEA, as discussed in section 3.1. The decrease in mol % of (S)-MSC can also probably be attributed to this reaction rather than the racemization since the (S)-MSC ee remains approximately constant and there is no increase in (R)-MSC mol % after 1.5 h. This indicates that the racemization has also been deactivated. However, this also means that the KR was not active after 1.5 h; if it was, we would expect that the (S)-MSC ee would increase toward 100% over the reaction period as in the DKR with TOA (Figure 6a). Due to the limited amount of MSC-related material that was available during this project, a low substrate concentration had to be used for most of the work. Consequently, the overall reaction concentration was 10 times lower compared to those in other studies.14 If the racemization and KR can be assumed to be pseudo first order in concentration (a common assumption adopted by many DKR researchers32,33), then the racemization and KR rate should have been lower by a factor of 10. To determine if this had a significant impact on the findings of this research, DKRs were also conducted at the higher concentrations: with (R,S)-MSC concentrations of 337 mmol L-1. The enzyme loading was also increased to the 150 g (mol of MSC) -1 used by other researchers, to enable direct comparison of results. Furthermore, in light of the results of the KR studies dosed with base and ruthenium p-cymene, it is thought that Park and co-workers14 probably used an excess amount of enzyme at 150 g (mol of MSC)-1 to
ensure that even if some of the enzyme was compromised by the ruthenium and base, there would still be sufficient enzyme remaining for the DKR reaction to continue. However, as mentioned earlier, immobilized enzymes are expensive, and such a high loading could not be justified throughout the whole of this study. Figure 7a shows the reaction profile for the DKR with TEA. The DKR reaction profile shows similar trends to the DKRs at lower concentration (Figure 6b). The mol % (R)-allylic acetate for this DKR is not much more than that at lower concentration. Thus, product yield is not significantly improved at higher concentrations. Again, the KR was rapid, but despite this, the (R)-MSC present was still not completely converted during the reaction period. This again indicates that the KR has either been inhibited or deactivated as previously discussed. Therefore, the higher loading of enzyme did not make a significant difference. The longer reaction time used in this run clearly shows the impact of the nonenantiospecific reaction to allylic acetate on the mole balance: over the 24 h, the ee of (R)-allylic acetate drops to almost 70% through increased production of (S)-allylic acetate despite more (R)-allylic acetate being formed. Furthermore, the ee of (S)-MSC remains constant throughout the reaction period, indicating that the racemization has also been deactivated. The same trends were found for the equivalent DKR using 0.22 equiv of P1-t-Oct as base, though the nonenantiospecific reaction to allylic acetate was not as substantial. 4. Conclusions Time course reaction data were investigated for the racemization, KR, and DKR of the allylic alcohol MSC in toluene using the immobilized lipase Novozym 435 and ruthenium pcymene catalyst with different bases. When the KR was studied in isolation, it was found that although the overall yield of (R)allylic acetate was not affected by the presence of base and ruthenium p-cymene, the overall KR became less enantiospecific. It is thought that these compounds catalyze nonenantiospecific side reactions, forming allylic acetate, or interfere with enzyme enantiospecificity. To minimize these undesired side reactions, the KR should be run over the shortest time possible. Second, the racemization was studied in isolation. TEA and P1-t-Oct with ruthenium p-cymene racemized (S)-MSC faster than THexA and THeptA, but yielded a greater mol % of ketone byproduct. It is thought this is because TEA and P1-t-Oct are more electronegative (and thus more basic) than THexA and THepA (Table 1), making the formation of the active catalyst
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(a hydride complex) either faster or more complete. Furthermore, since the base, ruthenium p-cymene, and MSC will be in close proximity during the catalytic cycle, it is likely that steric hindrance by the bulky groups in THexA and THepA would slow the rate of activation and/or racemization rate. It was found that the hydrogenation step of the catalyst cycle is deactivated by the presence of the components of the KR. These inhibitory and deactivating factors were present in the DKR also and were responsible for limiting the yield of single enantiomer (R)-allylic acetate to approximately 70%. Increasing the concentrations of all the components did not significantly improve this. To further increase the enantiospecific yield, some method of isolating the racemization and KR catalysts from the inhibiting and deactivating components of the other reaction must be used. Acknowledgment This work was supported by the ESPRC (Grant GR/S13637/ 01) and Imperial College London. The authors thank Dr. Jake Irwin and Jennifer Muir at the University of Warwick for providing the chemicals and for their assistance. Literature Cited (1) Ema, T.; Yamaguchi, K.; Wakasa, Y.; Yabe, A.; Okada, R.; Fukumoto, M.; Yano, F.; Korenaga, T.; Utaka, M.; Sakai, T. Transitionstate models are useful for versatile biocatalysts: kinetics and thermodynamics of enantioselective acylations of secondary alcohols catalyzed by lipase and subtilisin. J. Mol. Catal. B: Enzymol. 2003, 22 (3-4), 181192. (2) Pa`mies, O.; Ba¨ckvall, J. E. Combination of Enzymes and Metal Catalysts. A Powerful Approach in Asymmetric Catalysis. Chem. ReV. 2003, 103 (8), 3247-3261. (3) Huerta, F. F.; Minidis, A. B.; Ba¨ckvall, J. E. Racemisation in asymmetric synthesis. Dynamic kinetic resolution and related processes in enzyme and metal catalysis. Chem. Soc. ReV. 2001, 30 (6), 321-331. (4) Azerad, R.; Buisson, D. Dynamic resolution and stereoinversion of secondary alcohols by chemo-enzymatic processes. Curr. Opin. Biotechnol. 2000, 11 (6), 565-571. (5) El Gihani, M. T.; Williams, J. M. J. Dynamic kinetic resolution. Curr. Opin. Chem. Biol. 1999, 3 (1), 11-15. (6) Choi, J. H.; Choi, Y. K.; Kim, Y. H.; Park, E. S.; Kim, E. J.; Kim, M. J.; Park, J. Aminocyclopentadienyl Ruthenium complexes as racemization catalysts for dynamic kinetic resolution of secondary alcohols at ambient temperature. J. Org. Chem. 2004, 69 (6), 1972-1977. (7) Dijksman, A.; Elzinga, J. M.; Li, Y. X.; Arends, I. W. C. E.; Sheldon, R. A. Efficient ruthenium-catalyzed racemization of secondary alcohols: application to dynamic kinetic resolution. Tetrahedron: Asymm. 2002, 13 (8), 879-884. (8) Dinh, P. M.; Howarth, J. A.; Hudnott, A. R.; Williams, J. M. J.; Harris, W. Catalytic racemisation of alcohols: Applications to enzymatic resolution reactions. Tetrahedron Lett. 1996, 37 (42), 7623-7626. (9) Riermeier, T. H.; Gross, P.; Monsees, A.; Hoff, M.; Trauthwein, H. Dynamic kinetic resolution of secondary alcohols with a readily available ruthenium-based racemization catalyst. Tetrahedron Lett. 2005, 46 (19), 3403-3406. (10) Verzijl, G. K. M.; de Vries, J. G.; Broxterman, Q. B. Removal of the acyl donor residue allows the use of simple alkyl esters as acyl donors for the dynamic kinetic resolution of secondary alcohols. Tetrahedron: Asymm. 2005, 16 (9), 1603-1610. (11) Kim, M. J.; Chung, Y. I.; Choi, Y. K.; Lee, H. K.; Kim, D.; Park, J. (S)-Selective Dynamic Kinetic Resolution of Secondary Alcohols by the Combination of Subtilisin and an Aminocyclopentadienylruthenium Complex as the Catalysts. J. Am. Chem. Soc. 2003, 125 (38), 11494-11495. (12) Koh, J. H.; Jeong, H. M.; Park, J. Efficient catalytic racemization of secondary alcohols. Tetrahedron Lett. 1998, 39 (31), 5545-5548.
(13) Koh, J. H.; Jung, H. M.; Kim, M. J.; Park, J. Enzymatic resolution of secondary alcohols coupled with ruthenium-catalyzed racemization without hydrogen mediator. Tetrahedron Lett. 1999, 40 (34), 6281-6284. (14) Lee, D.; Huh, E. A.; Kim, M. J.; Jung, H. M.; Koh, J. H.; Park, J. Dynamic Kinetic Resolution of Allylic Alcohols Mediated by Rutheniumand Lipase-Based Catalysts . Org. Lett. 2000, 2 (15), 2377-2379. (15) Choi, J. H.; Kim, Y. H.; Nam, S. H.; Shin, S. T.; Kim, M. J.; Park, J. Aminocyclopentadienyl ruthenium chloride: catalytic racemization and dynamic kinetic resolution of alcohols at ambient temperature. Angew. Chem., Int. Ed. 2002, 41 (13), 2373-2376. (16) Csjernyik, G.; Bogar, K.; Ba¨ckvall, J. E. New efficient ruthenium catalysts for racemization of alcohols at room temperature. Tetrahedron Lett. 2004, 45 (36), 6799-6802. (17) Martı´n-Matute, B.; Edin, M.; Boga´r, K.; Ba¨ckvall, J. E. Highly Compatible Metal and Enzyme Catalysts for Efficient Dynamic Kinetic Resolution of Alcohols at Ambient Temperature. Angew. Chem., Int. Ed. 2004, 43, 6535-6539. (18) Persson, A. B.; Larsson, A. L.; Ray, M. L.; Ba¨ckvall, J. E. Ruthenium- and Enzyme-Catalyzed Dynamic Kinetic Resolution of Secondary Alcohols . J. Am. Chem. Soc. 1999, 121 (8), 1645-1650. (19) DSM intermedia publication home page. http://www.dsm.com/ en_US/ downloads/dpc/intermedia_08.pdf (accessed November 2004). (20) Choi, Y. K.; Suh, J. H.; Lee, D.; Lim, I. T.; Jung, J. Y.; Kim, M. J. Dynamic Kinetic Resolution of Acyclic Allylic Acetates Using Lipase and Palladium. J. Org. Chem. 1999, 64 (22), 8423-8424. (21) Martı´n-Matute, B.; Bogar, K.; Edin, M.; Kaynak, F. B.; Ba¨ckvall, J. E. Highly Efficient Redox Isomerisation of Allylic Alcohols at Ambient Temperature Catalyzed by Novel Ruthenium Cyclopentadienyl ComplexesNew Insight into the Mechanism. Chem.sEur. J. 2005, 11 (20), 58325842. (22) Pa`mies, O.; Ba¨ckvall, J.-E. Studies on the Mechanism of MetalCatalyzed Hydrogen Transfer from Alcohols to Ketones. Chem.sEur. J. 2001, 7 (23), 5052-5058. (23) Aranyos, A.; Csjernyik, G.; Szabo´, K. J.; Ba¨ckvall, J. E. Evidence for a ruthenium dihydride species as the active catalyst in the RuCl2(PPh3)catalyzed hydrogen transfer reaction in the presence of base. Chem. Commun. 1999, 4, 351-352. (24) Ba¨ckvall, J. E.; Andreasson, U. Ruthenium-catalyzed isomerization of allylic alcohols to saturated ketones. Tetrahedron Lett. 1993, 34 (34), 5459-5462. (25) Shvo, Y.; Czarkie, D.; Rahamim, Y. A new group of ruthenium complexes: structure and catalysis. J. Am. Chem. Soc. 1986, 108 (23), 7400-7402. (26) Kumer, A.; Gross, R. A. Candida antartica Lipase B Catalyzed Polycaprolactone Synthesis: Effects of Organic Media and Temperature. Biomacromolecules 2000, 1 (1), 133-138. (27) Faber, K. Biotransformations in Organic Chemistry; SpringerVerlag: Berlin, 1992. (28) Rotticci, D.; Norin, T.; Hult, K. Mass Transport Limitations Reduce the Effective Stereospecificity in Enzyme-Catalyzed Kinetic Resolution. Org. Lett. 2000, 2 (10), 1373-1376. (29) Levenspiel, O. Chemical Reaction Engineering, 3rd ed.; John Wiley & Sons: New York, 1999. (30) Carrea, G.; Ottolina, G.; Riva, S. Role of solvents in the control of enzyme selectivity in organic media. Trends Biotechnol. 1995, 13 (2), 6370. (31) Gibbins, E.; Irwin, J. L.; Livingston, A. G.; Muir, J.; Patterson, D. A.; Roengpithya, C.; Taylor, P. C. An Improved Protocol for the Synthesis and Nanofiltration of Kim and Park’s Aminocyclopentadienyl Ruthenium Chloride Racemisation Catalyst. Synlett 2005, 19, 2993-2995. (32) Kitamura, M.; Suga, S.; Oka, H.; Noyori, R. Quantitative Analysis of the Chiral Amplification in the Amino Alcohol-Promoted Asymmetric Alkylation of Aldehydes with Dialkylzincs. J. Am. Chem. Soc. 1998, 120 (38), 9800-9809. (33) Andraos, J. Quantification and Optimization of Dynamic Kinetic Resolution. J. Phys. Chem. A 2003, 107 (13), 2374-2387.
ReceiVed for reView March 29, 2006 ReVised manuscript receiVed July 25, 2006 Accepted August 11, 2006 IE060394O
