Enantioselective One-pot Synthesis of Biaryl-substituted Amines by Combining Palladium and Enzyme Catalysis in Deep Eutectic Solvents

The first application of Deep Eutectic Solvents ( DESs ) in asymmetric bioamination of ketones has been accomplished. The amine transaminases (ATAs) turned out to be particularly stable in DES -buffer mixtures at a percentage of up to 75% (w/w) neoteric solvent. Moreover, this reaction medium was used to perform a chemoenzymatic cascade toward biaryl amines by coupling a Suzuki reaction sequentially with an enantioselective bioamination catalyzed by the recently discovered ATA from Exophiala xenobiotica (EX-  TA). The solubilizing properties of DESs enabled the metal-catalyzed step at 200 mM loading of substrate and the subsequent biotransformation at 25 This paper demonstrates how the unique properties of deep eutectic solvents enable a chemoenzymatic cascade consisting of a Suzuki cross-coupling followed by the unprecedented enzymatic bioamination in these bio-based solvents.


INTRODUCTION
The biaryl moiety has emerged to a broadly used, valuable structural motif not only in the field of chiral ligands for asymmetric catalysis (as underlined by BINOL and BINAP as the presumably most prominent examples bearing such a biaryl structure), 1-3 but also in the field of natural products 4 and pharmaceuticals. 5,6 A commercialized product in this field is Valsartane (1) developed by Ciba-Geigy (now, Novartis), which is used as an angiotensin receptor blocker for treatment of, e.g. high blood pressure ( Figure 1). 6 At the same time, chiral amine structural motifs can be widely found in today's marketed drugs. 4 Indeed, the FDA database reveals that 84% of the approved small molecule drugs bear at least one nitrogen atom. 5 Thus, "merging" these two structural motifs has raised interest in the field of drug development, and the related biaryl-substituted amines have already turned out to represent versatile intermediates for promising drug candidates. For example, the biaryl-amine 2 is used for the synthesis of the cathepsin C inhibitor Odanacatib (3), which was evaluated in phase III for fracture prevention in postmenopausal women with osteoporosis by Merck & Co. 6 This potential for pharmaceutical applications also raised the question about efficient approaches to such chiral biaryl-substituted amine molecules. Retrosynthetically the biaryl unit can be constructed through a palladium-catalyzed Suzuki-cross coupling reaction, 10 a process which has been successfully implemented in eco-friendly media. [11][12][13] On the other hand, the asymmetric reductive amination enables to convert ketones into enantiomerically pure amines. 14 The combination of these two steps represents an elegant and straightforward approach toward these target molecules. The biocatalytic reductive amination can be conducted by amine transaminases. 15 Such an approach has been recently demonstrated for the enantioselective synthesis of biaryl amines by the Bornscheuer group for the first time. This was exemplified for the conversion of a halogenated acetophenone with a boronic acid in a Suzuki reaction. Subsequent conversion of the resulting biaryl ketones in the presence of an amine transaminase led to the formation of amines with up to 84% overall conversion and >99% ee when conducting the two reactions sequentially at 2 mM and 1 mM substrate concentration, respectively. 16 In terms of a high overall process efficiency, the combination of these two steps within a one-pot process would be highly desirable as well as the increase of the substrate loading for achieving an improved volumetric productivity. Combinations of chemo-and biocatalysis have been identified as attractive process options in recent years. This is underlined by many successful examples. [17][18][19][20][21] Our groups have reported a related combination of a Suzuki-cross coupling reaction and subsequent enzymatic reduction in which biaryl-substituted alcohols were formed. [22][23][24] In these studies, Deep Eutectic Solvents (DESs) 25 were used as a well-known environmentally benign solvent class which turned out to be an attractive reaction medium.
In recent times, the pharmaceutical industry has become more receptive to the use of biocatalysis for the manufacture of active pharmaceutical ingredients in a sustainable manner. 26 Although the greenness of this technology is typically assumed, most biotransformations suffer from issues such as water consumption, wastewater production or unfavorable metrics due to the poor solubility of reagents in water. 27 To circumvent this drawback, organic solvents can be supplemented as co-solvents, to the expense of enzyme stability, generally limited in these media. On the other hand, a new awareness has arisen in today´s synthetic organic chemistry to replace toxic/carcinogenic petroleum based volatile organic compounds (VOCs) by new, greener and more sustainable solvents. 28,29 In this context, Abbot introduced DESs, 30 which consist of 2-3 compounds from renewable sources establishing an extensive H-bond network throughout the solvent with a melting point far below those of the individual components. Compared to the related ionic liquids (ILs), DESs are cheaper, easier to make, tunable, highly biodegradable, virtually non-toxic and do not require further purification. As illustrated by the exponential growth of literature, DES technology has been applied to a wide-ranging area of research topics such as organic synthesis, 31,32 metal-catalysis [33][34][35][36] and organocatalysis, 37-40 energy technology, 41,42 material chemistry, 43 or separation processes. 44 In biocatalysis, since the 2008 proof-of-concept, 45 many examples have showcased ad hoc protocols for biotransformations in DESs and DES-buffer mixtures. 46 Interestingly, hitherto there is not any example involving ATAs. Recent advances in protein engineering have enabled the conversion of a variety of sterically demanding ketones by ATAs. For example, the rationally engineered (S)-selective ATA from V. fluvialis catalyzed an ortho-biaryl ketone to the corresponding (S)-biaryl amine. 47 Bornscheuer and coworkers, 16 in parallel to us, 48 have recently generated ATAs from Aspergillus fumigatus (4CHI-TA) and Exophiala xenobiotica (EX-TA) respectively, suitable for producing meta-and para-(R)-biaryl amines. Thus, we became interested in developing an efficient chemoenzymatic one-pot process route to biaryl-substituted amines based on the combination of a Suzuki-cross coupling reaction and an enzymatic transamination in DESs as sustainable reaction media. In the following, we report exactly such a process. It also represents the first example of an application of the enzyme class of amine transaminases in DESs.

RESULTS AND DISCUSSION
In the recent report we revisited the synthesis of biaryl alcohols by means of a Suzuki crosscoupling and subsequent bioreduction of the transiently formed ketones with KREDs. 24 With regard to previous research, the use of DESs enabled us to tackle the solubility hurdles and reach concentrations of 200 mM for the coupling step and 75 mM for the subsequent bioreduction.
From a synthetic goal point of view, a first key challenge was to determine if EX-TA is active in these bio-based solvents. Accordingly, the bioamination of the biaryl ketone 4a was investigated as a benchmark reaction with the variant EX-STA (amino acid exchange T273S).
This variant leads to the highest conversions of biaryl ketones. 48 The biotransformation was conducted under the previously optimized reaction setup, namely based on the use of alanine as amino donor and the LDH/GDH recycling system, and supplemented with choline chloride (ChCl)/glycerol (Gly) (1:2). For the sake of comparison, other co-solvents such as DMSO, THF, and i-PrOH were also tested (5% and 15% v/v). As deduced from As the next step, we sought to get more insight in the unveiled stability of ATAs in DES-buffer mixtures by extending the study to enzymes from a commercial kit (Codexis), 50 and also the Sselective TAs from Chromobacterium violaceum (Cv) 51 and (S)-Arthrobacter (ArS) 52 and the R-selective ATAs from (R)-Arthrobacter (ArR) 53 and its evolved variant ArRmut11. 54 For this study, phenylacetone (6) was selected as a substrate, which had been efficiently converted by these ATAs in conventional aqueous medium. 55 Four choline chloride-based eutectic mixtures, namely 1ChCl/2Gly, 1ChCl/2H2O, 1ChCl/1Sorb (Sorb = sorbitol) and 1ChCl/2Urea were screened at variable water content (Table 2). In a typical experiment aimed at evaluating the enzymatic performance, 6 (30 mM) was incubated in a mixture of DES and potassium phosphate buffer (KPi) 100 mM (1 mM PLP and 1 M iPrNH2) at pH 7.0, 30 ºC and 250 rpm during 24 h.
An initial conclusion extracted from Table 2 is that the DES-buffer mixtures resulted in highly suitable reaction media for the ATAs at 25% or 50% (w/w) DES. On the one hand, the commercial enzymes led to very high conversions in the four tested media (  Moving back to the chiral biaryl amines and keeping in mind both the reported Suzuki crosscoupling reaction in DESs and the unveiled good tolerance of EX-STA toward these solvents, we envisaged to set up a cascade combining metal catalysis and biocatalysis in such reaction media. Accordingly, we focused on the first step of the cascade, namely the Suzuki cross-coupling reaction. Equimolar amounts of p-Br-acetophenone (8) and phenylboronic acid (9) were reacted at 40 mM in a mixture of water and different co-solvents at room temperature. As depicted in Figure 2, the measured conversions toward 4'-phenylacetophenone (4b) were very high (≥85%) at 50% of i-PrOH, THF and 1ChCl/2Gly, while the reaction did not work at the same percentage of DMSO. As stated above, the negative impact of THF and i-PrOH on the catalytic performance of the EX-STA precluded the use of these solvents in the chemoenzymatic cascade. As a result, 1ChCl/2Gly emerged as the only co-solvent addressing the requirements of the two-step process.
Based on the knowledge gained in our previous work, the reaction was accomplished at 200 mM substrate concentration in a DES:water 4:1 mixture and 100 ºC.
Once having assessed both steps of the cascade separately and, taking into account that the metal-catalyzed reaction occurs first, the focus was on studying the potential inhibitory effects of the remaining reagents from the first step on the biocatalytic system. Thus, the bioamination of 4a catalyzed by EX-STA under the optimized setup (Table 1, Table 1 displays that a percent higher than 15% impacts negatively on the enzyme activity. Accordingly, the reaction mixture containing the biaryl ketone was diluted to 50 mM, resulting in a final 20% of DES for the biotransformation.
As expected, the conversion for 4b increased up to 30%. Further dilution to 25 mM, which results in 10% of DES, led to an optimized conversion of 45% (Table 3, entry 3). It should be noted that this conversion was identical to that reported in the bioamination of 4b in aqueous the wild-type enzyme, called EX-wt, which had exhibited the highest conversion in the single bioamination of 4b (83%). 23 However, upon the optimal cascade setup described above (25 mM in the biotransformation) the conversion dropped to 35% (  This unprecedented enzymatic activity of ATAs is an excellent proof of concept of the practical value of biorenewable solvents for synthetic chemists. Although immense advances are being made in areas such as protein engineering, it is no less true that a much simpler technique like medium engineering can be a valuable solution for optimizing a given biotransformation.

Supporting Information.
Additional information obtained from this study regarding the characterization of biaryl amines (NMR spectra), inhibition studies and analytical data. This material is available free of charge via the Internet at http://pubs.acs.org.