Highly Efficient Oxidation of Amines to Aldehydes with Flow‐based Biocatalysis

A new mild and efficient process for the aqueous preparation of aldehydes, which are employed as flavour and fragrance components in food, beverage, cosmetics, as well as in pharmaceuticals, was developed using a continuous‐flow approach based on an immobilised pure transaminase‐packed bed reactor. HEWT, an ω‐transaminase from the haloadapted bacterium Halomonas elongata, has been selected for its excellent stability and substrate scope. Sixteen different amines were rapidly (3–15 min) oxidised to the corresponding aldehydes (90 to 99 %) with only 1 to 5 equivalents of sodium pyruvate. The process was fully automated, allowing for the in‐line recovery of the pure aldehydes (chemical purity >99 % and isolated yields above 80 %), without any further work‐up procedure.


Introduction
Aromatic aldehydes are important intermediates in an umber of synthetic processes and have ap rominent role as flavour and fragrance components.A mongo ther synthetic methods, [1] they can be obtained from the corresponding primary aromatic amines,w hich are readilya vailable substrates. Methods for the oxidation of amines to carbonyl compounds have received significant attention, but these approaches are frequently poorly sustainable, because they produce waste and by-products that are difficult to recycle, require drastic reaction conditions, andoften proceed with poor selectivity. [1a, 2] Biocatalytic processes are interesting alternatives for amine oxidations under mild and benign conditions. For example, coppera mine oxidases (CAOs)h ave been used to catalyse the oxidation of primary aminest oa ldehydes (whileO 2 is simultaneously reduced to H 2 O 2 ). [3] Vanillin has been prepared by oxidation of vanillylamine using an amineo xidase (AO) from Aspergillus niger. [4] Recently,s elective oxidation of amines to aldehydes has been obtained using al accase with TEMPO (2,2,6,6tetramethylpiperidine N-oxide) as mediatora nd O 2 as oxi-dant. [5] Aromatic aldehydes can also be enzymatically prepared using other approaches, such as oxidation of primary alcohols [6] and reduction of carboxylic acids. [7] In this context,w ed eveloped an efficient bio-preparation of nature-identical flavours andf ragrances exploiting the immobilised amine transaminase from the moderate halophilic bacterium Halomonas elongata (HEWT), [8] which is able to tolerate a range of temperature,p H, salts and co-solvents in ac ontinuous flow reactor.T he combination of biocatalysis and flow reactor technologyc an be considered as an enabling methodology intrinsically compatible with the principles of green chemistry. [9] Flow-basedb iocatalysis was recently applied for peptide condensation, [10] hydrolysis and formation of esters and sugars, [11] stereoselective carbonyl reduction, [12] formation of CÀCb onds, [13] production of nucleosides, [14] monosaccharides, [15] and oligosaccharides, [16] and interconversion of carbonyls and amines using transaminases. [17] We recently reported on the application of HEWT in flow for the biosynthesis of amines [18] and we describe here an ecofriendly and scalable process that enhances the oxidising capability of this covalently immobilised enzymef or the production of aldehydes.T he productsa re aromatic aldehydes used as flavours and fragrancesi nf ood, beverage, cosmetics and pharmaceuticals. They have been obtained in excellent yields, with unprecedented reaction times if compared with traditional batch methods. The use of pyruvate as amino acceptori se xtremely favourable and by-product which it generates, the natural amino acid l-alanine, is completely benign and can be easily recovered. Furthermore, this approachc ircumvents potentiali ssues often encountered with whole-cellbiotransformations, such as generation of debris, swelling and permeability.
An ew mild and efficient process for the aqueous preparation of aldehydes, which are employed as flavour andf ragrance components in food, beverage, cosmetics, as well as in pharmaceuticals, was developed using ac ontinuous-flow approach based on an immobilised pure transaminase-packed bed reactor.H EWT,a nw-transaminase from the haloadapted bacterium Halomonas elongata, has been selected for its excellent stabili-ty ands ubstrate scope.S ixteen different aminesw ere rapidly (3-15 min) oxidised to the correspondinga ldehydes (90 to 99 %) with only 1t o5equivalents of sodium pyruvate. The processw as fully automated, allowing for the in-line recovery of the pure aldehydes (chemical purity > 99 %a nd isolated yields above 80 %), without anyf urther work-up procedure.

Results and Discussion
Pure HEWT (imm-HEWT) was immobilised on an epoxy-resin as reported by Planchestainer et al. [18] andt he supported biocatalyst (5 mg gram resin À1 )w as then used in ap acked-bed flow reactor.T he system was firstly tunedb yo ptimisingt he preparation of benzaldehyde starting from the corresponding benzylamine (Scheme 1).
To maximiset he solubility of the amine, 10 %o fD MSO was used as co-solvent in the phosphate buffer (50 mm,p H8.0). The reaction was performed under optimised conditions at 37 8Ca nd atmosphericp ressure with justo ne equivalent of pyruvate, as the equilibrium for this reaction is extremely favourable;c omplete substrate oxidation (molar conversion > 99 %) was obtained with only 3minutes of residence time (flow rate 0.3 mL min À1 ).
Notably, the use of the same immobilised enzymei nb atch gave af ull oxidation in about 2hours.
The optimised conditions were applied to the bioconversion of different benzylamines into the corresponding flavour aldehydes (Table 1).
Specific reaction rates in the batch and continuous-flow systems werec alculatedu sing the equations reported in the Experimental Section; the time taken (conversion rate) for the reaction to reach maximum conversion,whether in batch or continuous-flow,w as calculated and normalised to the amount of catalystused for both set-ups. [11a] Benzylamine-derivatives (entries 1-8) were oxidised into the corresponding aroma-compounds with high molar conversion; in all cases, ag reater than 4-fold rate increasew as observed if reactions were conducted under flow conditions, as conversions ! 90 %w ere reached within ar esidence time between 3 and 10 minutes (flow rate 0.3 mL min À1 and 0.1 mL min À1 ,r espectively), at 37 8Cand atmosphericpressure.
The process was implemented with the addition of an inline acidifications tep followed by extraction with EtOAc. The two phases werec ontinuously separated using aZ aiput liquid/ liquid separator and the desired aldehydes were recovered in the organic phase, significantly acceleratingt he overall workup, as no furtherpurification is required (Scheme 2).
This protocol wass uccessfully appliedt os ubstrates 1a-1h. Aldehydes obtained from substrates 1i and 1j (entries 9a nd 10) provedi nitially difficult to recover as they were retained by the packing material, despite variousa nd extensive washing steps.
Al iquid-liquid-phase reactions ystem was therefore set up, in which toluene flowed into the system upstream of the packed column( Scheme 3). On acidification, downstream of the process, the products 2i and 2j were extracted in-line and recovered by membranes eparation as pure compounds.R emarkably,t he presenceo ft oluene had no effect on the catalytic efficiency of the immobilisede nzyme which was extensively used over several weeks.
As econd set of amines (1k-1p)w as investigated using the same methodologies( either in am onophasic environmento r the biphasic one) to prove the versatility of the system with different aromatic substrates. (Table 2).
However, the oxidation of cinnamylamine (1o,e ntry 15) to cinnamaldehyde (2o,c innamon aroma)a nd hydrocinnamylamine (1p,e ntry 16) to hydrocinnamaldehyde (2p,h oney aroma), appeared more challenging. The batch reaction with an equimolar concentration of amino donor resultedi np oor conversion after 24 hours (50 and 52 %), without any significant increase over al onger incubation time, likely owing to an unfavourable equilibrium. Under flow conditions, with one equivalent of pyruvate, the conversions achievedw ere 50 % and 25 %r espectively,d espite increasing the residence time to 30 min. To displace the equilibrium, the concentration of pyruvate was increased to 2a nd 5equivalents with respect to the aldehydes 1o and 1p,y ielding 95 %o fc innamaldehyde and  [c] 1.41 [c] 10 300 > 99 0.33 10 > 99 [c] 1.41 [c] [a] Reactions were performed in the presence of 10 mM substrates and pyruvate, 0.1 mM PLP,1 0% DMSO was used as co-solventa t3 78C. Isolated yields are reported in the Experimental Section.
[b] Conversion rates are normalised to the amounto fe nzymeu sed in the reaction and calculated as reported in Ref. [11a]. [ c] Liquid-liquid-phase flow stream (see procedure summarised in Scheme 3), in this case DMSO was not added to the buffer. ChemCatChem 2017, 9,3843 -3848 www.chemcatchem.org This result underlines the fact that process control strategies (in our case, the optimisation of stoichiometric ratio of the substrates) help to maximise the productivity of HEWT by accelerating the reaction, while shifting the equilibrium to the product'sside.

Conclusions
An ew biocatalytic method for the synthesis of aldehydes with extensive applicationsa sc omponents of flavours and fragrances was developed. This is the first example of at ransaminase exploitedi naflow chemistry reactor under highly favourable oxidisingc onditions for the preparation of aromatic aldehydes, showingexcellent adaptability and stabilityduring the processes. The use of af low-based approacha llowed for dramatic accelerations of the reactions, with isolated yields above 80 % and very short residence times (3-15 min) of the substrates. This system required, in the majority of cases, only one equivalent of pyruvate as the amino acceptor,w hichl eads to the formation of l-alaninea sb y-product. As uccessful implementation was achieved with an in-line extraction step, which permitted the recovery of the desired pure aldehydes in the organic stream and l-alanine in the aqueous one, with an extremely simplified work-up procedure and almost no manipulation. As ar esult of the highl ocal concentration of the (bio)catalyst and the enhanced heata nd mass transfer, [19] the combination between biocatalysis and flow chemistry reactors not only leads to significant reductions of reactiont imes and increased productivity,b ut it can be also considered as ustainable technology for the productiono fa ldehydes commonly used in food, cosmetic, and pharmaceuticalindustry.

Flow reactions with immobilised HEWT
Continuous flow biotransformations were performed using aR 2+ /R4 Vapourtec flow reactor equipped with an Omnifit glass column (0.3421 mm i.d 100 mm length) filled with 0.7 go fi mm-HEWT (5 mg g À1 ). A2 0mm sodium pyruvate in phosphate buffer (50 mm, pH 8.0) containing 0.1 mm pyridoxal phosphate, and 20 mm amino donor solution with 10 %o fD MSO were prepared. The two solutions were mixed in aT -piece and the resulting flow stream was directed into the column packed with the biocatalyst (packed bed reactor volume:1 .0 mL). The flow rate was varied and optimised. An in-line acidification was performed by using an inlet of 1 n HCl aqueous solution (flow rate:0 .1 mL min À1 )t hat was mixed to the exiting reaction flow stream using aT -junction. The resulting aqueous phase was extracted in-line using as tream of EtOAc (flow rate: 0.2 mL min À1 )a nd aZ aiput liquid/liquid separator.B oth the organic and aqueous phase were analysed by HPLC using the above reported conditions. The amount of substrate and product was evaluated by exploiting ap reviously prepared calibration curve. For the optimisation procedure, the reactions have been performed by injecting 250 mLo fe ach starting solutions (volume of EtOAc used for the in-line extraction:1mL). To isolate the product, 10 mL of each starting solution has been used (volume of EtOAc used for the in-line extraction:4 0mL). The organic phase, containing the aldehyde, was evaporated to yield the desired product.
Specific reaction rates in batch and continuous-flow systems were calculated using Equations 1a nd 2: where [n p ]i st he amount of product (expressed in mmol), t is the reaction time (expressed in min), and m B [g] is the amount of biocatalyst employed.
where [P] is the product concentration flowing out of the reactor (expressed in mmol mL À1 ), fi st he flow rate (expressed in mL min À1 ), and m B [g] is the amount of biocatalyst loaded in the column.
Comparison of the rates of the same reaction in ab atch or flowmode was made at similar degrees of conversion.
Flow reactions in liquid-liquid-phase systems with immobilised HEWT 20, 40 or 100 mm pyruvate in phosphate buffer (50 mm,p H8.0) containing 0.1 mm PLP,a nd 20 mm amino donor solutions were prepared. The two solutions were mixed in aT -piece. As econd junction for additional supplement of toluene at the same flow rate was installed before the packed enzyme column. The resulting segmented flow stream was directed to the imm-HEWT.T he flow rate was varied and optimised. After an in-line acidification step, as previously reported, the exiting flow stream was separated by a Zaiput liquid/liquid separator.T he organic and aqueous phases were analysed by HPLC, exploiting ac alibration curve (see conditions above), and the toluene containing the desired product was evaporated to yield the aldehydes.