Enzymatic Cascades for the Regio- and Stereoselective Synthesis of Chiral Amines School of Chemistry, University of Manchester, Manchester Institute of Biotechnology, 131 Princess Street, Manchester, M1 7DN, UK Elaine O’Reillyǂ & Nicholas J. Turner E-Mail:
[email protected] [email protected]
Abstract Significant advancements in protein engineering and DNA technology have seen biocatalytic transformations take the place of traditional chemical manipulations in both academia and industry for the preparation of active pharmaceutical ingredients (APIs) and other medicinally relevant compounds. However, despite the large repertoire of commercially available biocatalysts which are readily accessible, enzymes which mediate the formation of C-C bonds and those which enable convergent synthesis remain largely undeveloped. In order to expand the scope of biocatalytic retrosynthesis and enable it to complement traditional chemical retrosynthesis it is essential to develop a ‘toolbox’ of biocatalysts which build molecular complexity. Of particular interest is the development of one-pot enzymatic cascades for the synthesis of functionalised, chiral building blocks without the need for protecting group manipulations or harsh reaction conditions. Highly regio- and stereoselective chemoenzymatic cascades have been developed for the synthesis of a range of chiral amines employing ω-transaminases and monoamine oxidase variants.
Introduction Nature boasts an extraordinary variety of biocatalysts which have the ability to mediate synthetically challenging transformations with exceptional levels of regio- and stereoselectivity. These properties, coupled with advances in protein engineering and DNA technologies mean that enzymes represent important alternatives to more traditional chemical catalysts [1, 2]. Directed evolution has allowed the substrate range, stability and kinetic properties of enzymes to be tailored and means that we no longer rely solely on their natural activity [3]. Addition of enzymes to the catalytic “toolbox”, including a diverse range of commercially available biocatalysts (figure 1), has enabled chemists to redesign synthetic routes to important target structures including bioactive natural products and active pharmaceutical ingredients (APIs), providing more efficient and cost-effective syntheses. A number of chemoenzymatic routes to the blockbuster API, Lipitor, have been developed including ketoreductase-, nitrilase-, halohydrin dehalogenase- and aldolase-mediated transformations and these diverse approaches are testament to the broad synthetic utility of biocatalysts (figure 2) [1, 4]. Chiral amines are prevalent motifs in drug molecules and the development of efficient synthetic routes to optically pure derivatives remains a fundamental goal for the pharmaceutical industry. ωtransaminases (ω-TAs) and variants of monoamine oxidase from Aspergillus niger (MAO-N) represent two important families of biocatalysts for the production of optically pure chiral amines. TAs can mediate the reductive amination of pro-chiral ketones and allow access to the corresponding (S)- or (R)-amines in high optical purity [5-9]. Recognising their potential, Merck and Codexis have engineered an (R)-selective ω-TA which has replaced a rhodium-catalyzed asymmetric enamine ǂ
Current address – School of Science & the Environment, Manchester Metropolitan University, John Dalton East, Oxford Road, Manchester, M1 5GD, UK
hydrogenation in the industrial production of the oral antihyperglycemic drug, Sitagliptin (figure 3) [7]. MAO-N catalyses the oxygen-dependent conversion of amines to imines [10-17] and has been extensively engineered to accommodate a diverse range of architectures, including the API, boceprevir [15] (figure 3) and related motifs. The hydrogen peroxide produced following oxidation has contributed towards the successful evolution of MAO-N as it has enabled the development of a highly effective high-throughput screen which is substrate independent (figure 4).
Figure 1. Examples of commercially available enzymes from the ‘biocatalytic toolbox’.
Figure 2. Chemoenzymatic routes for the synthesis of the side-chain of Atorvastatin; KRED = ketoreductase; HHDH = halohydrin dehalogenase.
Figure 3. APIs Sitagliptin and Boceprevir accessed via a chemoenzymatic route employing and engineered ωTA and MAO-N variant.
Figure 4. A high-throughput colorimetric screen for monoamine oxidase activity employing horseradish peroxidase (HRP) and a HRP substrate.
Chemo-enzymatic cascade for the regio- and stereoselective synthesis of 2,5disubstituted pyrrolidines Enzymatic and chemo-enzymatic cascades involving the combination of several enzymatic transformations in a concurrent one-pot process allows complex molecular architectures to be accessed with high optical purity, under ambient conditions and without the need for costly intermediate purification steps or protecting group manipulations [18-20]. Elegant cascades employing both ω-TAs [18-21] and MAO-N variants [10-17] have been reported for the asymmetric synthesis of a range of important amine scaffolds as well as for effective high-throughput screens. However, until recently, a cascade involving a combination of these two important amine synthesis enzymes had not been explored. Considerable efforts have focused on the development of asymmetric routes to both cis- and trans2,5-disubstituted pyrrolidines as these structures represent important scaffolds in pharmaceutical drugs and natural products [22-29]. Accessing the trans-diastereomers via reduction of the corresponding imine in high de is not straightforward and led us to develop a tandam biocatalytic approach to their synthesis. Our strategy (figure 5) encompasses a highly regio- and stereoselective TA mediated reductive amination and spontaneous cyclisation followed by a diastereoselective chemo-enzymatic conversion to the corresponding pyrrolidine using a combination of an (S)-selective MAO-N variant and the non-selective reducing agent NH3.BH3 [30].
Figure 5. Chemoenzymatic approach to the synthesis of chiral 2,5-disubstituted pyrrolidines [30]
We initially examined the reductive amination of non-symmetrical diketone 1a with the commercially available (S)-selective ω-TA, ATA113, employing the lactate dehydrogenase (LDH)/glucose dehydrogenase (GDH) system. Following reductive amination exclusively on the sterically less demanding methyl ketone, spontaneous cyclisation provided the corresponding 2,5-pyrroline 3a in excellent yield and high optical purity (91% yield, > 99% ee) (figure 6a). A novel transaminase (pfATA) from Pseudogulbenkiania ferrooxidans was also explored which shares 95% sequence identity with the transaminase from Chromobacterium violaceum (cv-ATA) (ATCC 12472). pf-ATA also mediated the reductive amination of diketone 1a, however, the 2,5-pyrroline 3a was obtained with a modest ee (75%). Commercially available (R)-selective ATA117 catalysed the regio- and stereoselective mono-amination of 1a, affording pyrroline (R)-3a in 65% yield and >99% ee (figure 6b). ATA113 and pf-ATA were then tested for their ability to catalyse the reductive amination of a series of structurally related diketones 1b-d (table 1). ATA113 provided methyl-substituted pyrrolines 3b-g as the sole regioisomers in excellent conversion and high ee, while substitution of the small methyl for a larger ethyl group resulted in a slightly reduced ee (entry 11 & 13). Pf-ATA provided pyrrolines 3b-g in modest to poor ee and surprisingly, replacement of the methyl substituent by ethyl resulted in a switch in stereoselectivity, affording (R)-3f-g as the predominant enantiomers (entries 12 & 14).
Figure 6. One-pot reductive amination of diketone 1a employing a) ATA113 and b) ATA117
Table 1. ω-TA mediated reductive amination of 3a-g
Entry
Substrate
1
1a
2 3 4 5 6
1a 1b 1b 1c 1c
ω-TA
Conv. [%]
ee [%]
ATA113
> 99
> 99 (S)
PfATA
> 99 > 99 > 99 > 99 > 99
75 (S) > 99 (S) > 78 (S) > 99 (S) 68 (S)
ATA113 PfATA
ATA113 PfATA
7 8 9 10 11 12 13 14
1d 1d 1e 1e 1f 1f 1g 1g
ATA113 PfATA
ATA113 PfATA
ATA113 PfATA
ATA113 PfATA
> 99 > 99 > 99 > 99 60 > 99 > 99 75
> 99 (S) 76 (S) > 99 (S) 78 (S) 96 (S) 76 (R) 94 (S) 46 (R)
Kroutil and co-workers recently developed a chemo-enzymatic route to related 2,6-disubstituted piperidines employing an ω-transaminase followed by diastereoselective hydrogenation using Pd/C [31]. The same strategy is not effective for the synthesis of the five-membered derivatives due to poor diastereoselectivity in the reduction step and so we sought to employ MAO-N for a chemo-enzymatic deracemisation. Two MAO-N variants (D5 and D9) were selected which have previously been evolved to accept structurally related amine frameworks, including pyrrolidines and piperidines. (S)and (R)-3a were exposed to NH3.BH3 in the presence of (S)-selective MAO-N, resulting in the accumulation of (2S,5R)- or (2R,5R)-pyrrolidine 4a in excellent de (>99%) following successive rounds of non-selective reduction/(S)-selective oxidation (figure 7). The approach exploits the exquisite regio- and stereoselectivity displayed by MAO-N which avoids stereorandomisation of the C-2-stereocentre generated by the (S)-selective ω-TA. Using the combination of NH3.BH3 /MAO-N D9 allowed access to 4b-g with excellent de (96-99%) through successive rounds of non-selective reduction/selective oxidation (table 2).
Figure 7. MAO-N mediated synthesis of (2S,5R)-4a and (2R,5R)-4a
Table 2. MAO/NH3.BH3 mediated asymmetric synthesis of (2S,5R)-4a-g
Entry
Substrate
1 2 3 4 5 6 7 8 9 10 11 12 13 14
3a 3a 3b 3b 3c 3c 3d 3d 3e 3e 3f 3f 3g 3g
MAO-N variant D5 D9 D5 D9 D5 D9 D5 D9 D5 D9 D5 D9 D5 D9
de [%] (2S,5R) > 99 96 88 98 > 99 > 99 > 99 > 99 68 > 99 64 > 99 56 96
The compatibility of the ω-TA and MAO-N under the same reaction conditions has also allowed the development of a one-pot cascade (figure 8) for the synthesis of target (2S,5R)-4a in 82% isolated yield and >99% de. We have also extended the one-pot TA/MAO-N approach for the preparation of (2S,5R)-4b, (2S,5R)-4d and (2S,5R)-4e in > 99% conversion and > 99% de starting from the corresponding diketones (table 3), demonstrating the generality of this one-pot approach.
Figure 8. One-pot asymmetric synthesis of (2S,5R)-4a on preparative-scale
Table 3. ATA113/MAO-N one-pot cascade for the synthesis of (2S,5R)-4a-4b, and (2S,5R)-4d-e Ketone
ω-TA
MAO-N
Conv. [%]
de [%]
1aǂ
ATA113
D5
> 99
> 99 (2S,5R)-4a
1bǂǂ 1dǂǂ 1eǂǂ
ATA113 ATA113 ATA113
D9 D9 D9
> 99 > 99 > 99
> 99 (2S,5R)-4b > 99 (2S,5R)-4d > 99 (2S,5R)-4e
ǂ
25mM substrate concentration; ǂǂ5mM substrate concentration
Monoamine oxidase/ω-Transaminase Cascade for the Deracemisation and Dealkylation of Amines While the deracemisation of cyclic amines using MAO-N variants has proven to be an effective means of accessing optically pure derivatives, employing this approach for the deracemisation of acyclic derivatives is limited due to competing hydrolysis of the imine in aqueous conditions. We have exploited a MAO-N/ω-TA cascade which relies on this competing hydrolysis for the effective deracemisation of a panel of acyclic amines 5a-h (figure 9, table 4) [32].
Figure 9. MAO-N/ω-TA cascade for the overall dealkylation of amines 5a-h.
Optically pure MBA analogues have been employed as chiral auxiliaries [33], for chiral derivatisation [34] and for the synthesis of peptides [35, 36]. The effective kinetic resolution of MBA-derivatives using ω-TAs has been reported, although employing this method for accessing optically pure amines results in a maximum 50% yield. A two-step approach has been successfully used for the deracemisation of amines by exploiting enantio-complementary TAs to achieve an initial resolution followed by an asymmetric synthesis [37]. Our approach takes advantage of a MAO-N mediated enantioselective oxidation of the (S)-amine to the corresponding ketone followed by reductive amination employing an (R)-selective TA. Exploiting this method, optically pure amines 5a-d can be accessed in high conversion and >99% ee. Further optimisation is ongoing with the aim of reducing the quantity of expensive alanine donor employed including the possibility of recycling the methyl-, ethyl- and isopropylamine by-products formed during the dealkylation as an alternative amine donor source. The same method can also be used for the regioselective dealkylation of amines. Oxidation of N-alkyl amines 6a-c using MAO-N provided the corresponding aldehyde which was quantitatively converted to 8 by the action of a commercially available (R)-selective TA (ATA117) (figure 10).
Table 4. Deracemisation of racemic amines 1a-h using a MAO-N/ω-TA cascade process Rac-1
Conversion[a]
5a 5b 5c 5d 5e 5f 5g 5h
99% 87% 91% 90% 99% 90% 90% 81%
ee (R)-enantiomer >99% >99% >99% >99% >99% >99% >99% >99%
[a] Conversion is based on conversion of the ketone
Figure 10. MAO-N/ω-TA cascade for the overall dealkylation of amines 6a-c
Conclusions The first examples of the use of transaminases in combination with monoamine oxidase variants have been developed. The compatibility of these important amine synthesis biocatalysts enables the regioand stereoselective synthesis of chiral 2,5-disubstituted pyrrolidine building blocks in excellent conversion (>99 %) and de (96-99 %). The same combination also enables the deracemisation and dealkylation of a range of methylbenzylamine derivatives. This technology in combination with the expanding ‘toolbox’ of transaminase and monoamine oxidase variants means that this approach will have broad utility for the construction of chiral amine frameworks.
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