Methoxamine Synthesis in a Biocatalytic 1-Pot 2-Step Cascade

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Methoxamine Synthesis in a Biocatalytic 1-Pot 2 Step Cascade Approach Vanessa Erdmann, Torsten Sehl, Ilona Frindi-Wosch, Robert C. Simon, Wolfgang Kroutil, and Dörte Rother ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b01081 • Publication Date (Web): 02 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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ACS Catalysis

Methoxamine Synthesis in a Biocatalytic 1-Pot 2-Step Cascade Approach Vanessa Erdmann†,⊥, Torsten Sehl†,‡, Ilona Frindi-Wosch†, Robert C. Simon§,||, Wolfgang Kroutil§, Dörte Rother*,†,⊥

†Forschungszentrum

‡HERBRAND

§University

Jülich GmbH, IBG-1: Biotechnology, 52425 Jülich, Germany;

PharmaChemicals GmbH, 77723 Gengenbach, Germany;

of Graz, Department of Chemistry, Heinrichstrasse 28, 8010 Graz, Austria;

||Roche-Diagnostics

⊥

GmbH, 82377 Penzberg, Germany

RWTH Aachen University, Aachen Biology and Biotechnology, 52056 Aachen, Germany.

ABSTRACT

ACS Paragon Plus Environment

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Due to its function as a vasopressor, the vicinal amino alcohol methoxamine is a potential candidate for the treatment of hypotension, incontinence, or has applications in ophthalmology. In this study, a biocatalytic method was developed to produce each of the four stereoisomers of methoxamine in a sequential 1-pot 2-step cascade reaction, starting from readily available pyruvate and 2,5-dimethoxybenzaldehyde without intermediate isolation. All four isomers are accessible with high conversions and very good stereoselectivities through the modular combination of carboligases and transaminases with high stereoselectivities. The development of these cascades was made possible, amongst other factors, by the integration of a recently engineered triple variant of the pyruvate decarboxylase from Acetobacter pasteurianus (ApPDC-E469G-I468A-W543F), which

provided

access

to

the

intermediate

(S)-1-hydroxy-1-(2,5-

dimethoxyphenyl)propan-2-one with a high enantiomeric excess of 98 %. For the amination of these sterically demanding 2-hydroxy ketones, Bacillus megaterium transaminase in particular has proven to be a highly active and selective catalyst. All four methoxamine stereoisomers were achieved with isomeric contents between 94 % and 99 % and total conversions of both steps between 59 % and 80 %. A preparative scale


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(75 mL) of the 1-pot 2-step cascade to (1S,2R)-methoxamine including non-optimized product isolation showed 85 mg HCl salt (46 % isolated yield) with 94 % purity (NMR) and an isomeric content of 98 %.

KEYWORDS

Biocatalysis, 2-step cascades, methoxamine, amino alcohol, transaminase

INTRODUCTION

The vicinal amino alcohol 2-amino-1-(2,5-dimethoxyphenyl)propan-1-ol (methoxamine) belongs to the class of sympathomimetic drugs such as nor(pseudo)ephedrine1,2 and phenylephrine.3 Sold under the trade name Vasoxyl until 2001 (FDA), methoxamine hydrochloride was used as an 1-adrenergic receptor agonist and acted as a vasoconstrictor in the treatment of hypotension.4–8 The stereoisomer (1R,2S)methoxamine is also known for its function in the treatment of faecal incontinence, as a nasal decongestant and in ophthalmology.9 Due to the direct pharmaceutical benefit of


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methoxamine, a cheap, easily applicable and stereoselective – at best environmentally friendly – synthesis method is required to produce this substance.10,11 The racemic amino alcohol is chemically synthesized by a nitroaldol reaction, followed by a separation of the diastereoisomers by flash chromatography and final hydrogenation of the nitro alcohol to an amino alcohol.12 An alternative strategy is the synthesis of methoxamine by the reduction of -amino ketones by hydrosilanes under acidic conditions. Here, the products were formed with high diastereoisomeric excess (de)13, but as racemate. Biocatalytic cascades extent the spectrum of chemical reactions and can sometimes contribute to completely new synthesis strategies. Starting from easily available substrates, the synthesis enables the formation of almost optically pure stereoisomers of methoxamine in a sequential 1-pot 2-step cascade reaction (Scheme 1), thus eliminating the need for subsequent isomer separation. The use of enzymes as non-toxic catalysts – in addition to the unrequired isolation of intermediates and the high atom- and step efficiency – are further advantages of this enzyme cascade approach.14,15


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Scheme 1. Sequential 1-pot 2-step cascade reaction to methoxamine, starting from the easily obtainable substrates 2,5-dimethoxybenzaldehyde and pyruvate.

The development of new variants of the pyruvate decarboxylase (PDC) from

Acetobacter pasteurianus16 enabled the production of -hydroxy ketones such as phenylacetylcarbinol (PAC) and derivatives of this compound in (S)-configuration, which has not been possible so far.17,18

Since (R)-PAC is also accessible with high

stereoselectivities from wild type enzymes isolated from nature19, all four 1,2-amino alcohol stereoisomers should theoretically be accessible by a modular combination of ThDP-dependent enzymes and transaminases with stereocomplementary selectivities. The success of this sequential 1-pot 2-step cascade reaction had already been demonstrated in the synthesis of nor(pseudo)ephedrine.19–22 In this study, the product


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platform was expanded to the more spatially demanding substituted amino alcohol methoxamine. A simple superposition of previously combined catalysts led neither to the formation of all four stereoisomers nor to convincing conversions. New enzymes and enzyme variants of the ThDP-dependent enzyme- and transaminase toolboxes had to be screened to build and optimize cascades for all methoxamine stereoisomers with the highest possible conversion and purity. With Bacillus megaterium transaminase a very potent and selective catalyst for the amination of sterically demanding 2-hydroxy ketones is herein described. With regards to economic and ecologic efficiency, the high step efficiency of the 2-step cascade concept described prevents intermediate isolation and enzyme removal, which results in less waste and higher product yields.23,24 And with 3,5-dimethoxybenzaldehyde, pyruvate and isopropylamine, low-cost and partially renewable starting material were used. The challenge of na unfavoured equilibrium of the asymmetric transaminase reaction25 was solved by evaporating the coproduct acetone, shifting the equilibrium to the product side and increasing the overall cascade conversion. Solubility problems with


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benzaldehyde derivatives in aqueous media can be solved by adding water-miscible cosolvent, in this case dimethoxy sulfoxide (DMSO).26,27

RESULTS AND DISCUSSION

Selection of a suitable carboligating enzyme for the first cascade step. The enzymes with the highest selectivities and activities for the formation of (R)- and (S)-1-hydroxy-1(2,5-dimethoxyphenyl)propan-2-one (2,5DMPAC) were selected from the existing toolbox of ThDP-dependent enzymes. For the (R)-selective carboligation step, acetohydroxyacid synthase I from E. coli (EcAHAS-I) was selected for its excellent performance in cascades producing nor(pseudo)ephedrine.19 For the (S)-specific carboligation from 2,5dimethoxybenzaldehyde (2,5DMBA) and acetaldehyde (after decarboxylation from pyruvate occuring on the same active side of the pyruvate decarboxylase), the recently engineered variant ApPDC-E469G-I468A-W543F from Acetobacter pasteurianus was tested. This enzyme variant was optimized for (S)-PAC synthesis by directed evolution.17,18 The exchanges of glutamic acid in position 469 for the smaller amino acid glycine, and isoleucine in position 468 to alanine, opened the so called “(S)-pocket” to


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accept benzaldehyde. The amino acid tryptophan at position 543 was identified to stabilize the unwanted orientation of benzaldehyde, which results in an (R)-configured PAC. An exchance for phenylalanine destabilized the unwanted orientation and thus increased the optical purity for (S)-PAC.17,18 With the new enzyme ApPDC-E469G-I468AW543F, stereoselectivities of >97 % enantiomeric excess (ee) were achieved in the formation of (S)-PAC.17,18 For the synthesis of (S)-2,5DMPAC, ee values of 98 % (S) together with high non-isolated yields of 94 % were reached within 9 h (calculations are presented in the Supporting Information in chapter 4). In comparison, for the formally applied ApPDC-E469G26,28, only 90 ± 2 % non-isolated yields and an ee of 67 % for the (S)-product were achieved within 8.5 h (Figure 1).


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ApPDC-wt

ApPDC-E469G

(S)-pocket blocked

(S)-pocket partially opened

parallel orientation of substrates leads to (R)-configuration

yield (2,5-DMPAC) [%]

legend:

antiparallel orientation of substrates enables (S)-configuration

cofactor: Mg2+: magnesium ion ThDP: thiamine diphosphate

ApPDC-E469G-I468A-W543F

(R)-configuration still possible

(S)-configuration stabilized

W543 and I468 facilitate undesired (R)-configuration

aliphatic substrate: covalently bound to cofactor ThDP

ApPDC-E469G-I468A-W543F optimally stabilizes (S)-configuration aromatic substrate: coordinated parallel or anti-parallel to the aliphatic substrate in the active site

100

100

80

80

60

60

40

40

20

20

0

0 0

2

ApPDC-E469G, yield* Ap PDC-E469G, ee

4 6 reaction time [h]

8

ee [%]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ApPDC-E469G-I468A-W543F, yield* Ap PDC-E469G-I468A-W543F, ee

Figure 1. Synthesis of (S)-2,5DMPAC by ApPDC-E469G (duplicates) and the new variant

ApPDC-E469G-I468A-W543 (quadruplicate) under optimized reaction conditions: 100 mM HEPES buffer, pH 7.5, 2.5 mM MgSO4, 0.1 mM thiamine diphosphate (ThDP), 5 mM


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2,5DMBA, 10 % DMSO, 20 mM pyruvate, 1.5 mg/mL purified enzyme, 30 °C, 700-750 rpm (*non-isolated), HPLC analytics are described in detail in the Supporting Information, chapter 3.

By increasing the substrate concentration to 20 mM, with variant ApPDC-E469G-I468AW543 non-isolated yields of >96 % (26 h) and ee values of 97 % were achieved (20 mM 2,5DMBA, 80 mM pyruvate, 20 % DMSO, 1.5 mg/mL lyophilisate of purified ApPDCE469G-I468A-W543F) (Figure 2). Raising the pyruvate concentration or amount of enzyme also increases the hydroxy ketone yields, but reduces ee values over time (data not shown). This phenomenon has already been observed for the synthesis of PAC by this enzyme.18 PAC concentrations decrease over time, while the regioisomer HPP is formed. A higher background isomerization within E.coli cells (e.g. by keto-enolisomerases of the glycolysis) was found compared to the use of purified enzyme. Thus, the rate for ee decrease was 5 times lower in the presence of purified enzymes compared to whole cells. Consequently, higher optical purity can be achieved if the following factors are taken into account: i) use of purified enzymes ii) optimal reaction times iii) avoidance


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of long standing-times and harsh acidic/basic conditions (especially in the workup flow). As a disadvantage, the use of purified enzymes often increases catalyst costs compared to whole cells or crude cell extract. The benefit of higher (optical) purity must be in proportion to the catalyst costs and the cost for additional effort during further processing. This must be evaluated separately for the requirements of each process strategy. Screening of a suitable transaminase for the second cascade step. Ten transaminases were screened with the focus on the conversion of (S)-2,5DMPAC with three different amine donors. The highest conversion for (R)-selective transamination of 2,5DMPAC was determined for Arthrobacter sp. transaminase (AsTA) (Table 1). Isopropylamine was exposed as the best amine donor, but alanine and 1-phenylethylamine were also suitable for the transamination of (S)-2,5DMPAC. Among the catalysts described as (S)-selective for selected substrates,29–31 the transaminases from Bacillus megaterium (BmTA) and Arthrobacter citreus (AcTA) were identified as potential catalysts for the methoxamine synthesis. Here, the addition of the amine donor isopropylamine allowed a much higher conversions compared to alanine and 1-phenylethylamine. No purities of the end products were measured for these


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preliminary experiments. The optical purities of the methoxamine isomers were determined subsequently in the cascades carried out (see below). Table 1. TA-screening for (S)-2,5DMPAC conversion (5-10 mM) with three different amine donors: 250 mM alanine (including alanine dehydrogenase, 2 mg/mL formate dehydrogenase, and 1 mM NADH to shift the equilibrium to the product side by pyruvate recycling), 50 mM racemic 1-phenylethylamine, 100 mM isopropylamine, each in 100 mM HEPES buffer, pH 7.5, equipped with 0.5-1 mM pyridoxal-5’-phosphate hydrate (PLP). Catalyst formulation was mostly crude cell extract, VfTA with alanine and 1phenylethylamine was purified, lyophilized enzyme, 30 °C, 700-750 rpm; the expected stereoselectivity of each enzyme was assumed on the basis of published data cited in the table and confirmed for the best performing transaminases in the cascade setups as described below. +++ good conversion (>90 %), ++ moderate conversions (>20 %, 98 % (24 h), ic >99 % (only step 2)), lyophilized E. coli whole cells with heterologously expressed AsTA (conv. 94 % (24 h), ic >99 % (only step 2)) can also be used as catalysts to achieve high conversions and perfect isomeric contents (Supporting Information, chapter 5.2, Figure S23). The main product formed was identified as anti-methoxamine by comparison


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with the reference compound, to the analogy and to the GC-TOF-MS analysis (Supporting Information, chapter 3.4, Figure S19). 2-Step cascade for (1S,2S)-methoxamine synthesis (see Scheme 2). The 1-pot 2-step cascade for (1S,2S)-methoxamine synthesis was set up and combines ApPDC-E469GI468A-W543F and the (S)-selective BmTA as catalysts (see Scheme 2). For the first reaction step, non-isolated yields of 98 ± 1 % (5 mM substrate) with ee values of 97 % for the (S)-hydroxy ketone were measured (16 h). For the subsequent transamination step, a substrate conversion of 96 % and an ic value of 98 % (20.5 h) were achieved with purified and lyophilized BmTA and isopropylamine as amine donor. The overall conversion of the cascade reaction was 79 % and with an ic value of 98 % a high stereoselectivity was reached. Furthermore, 9 % of the overall conversion led to the formation of 2,5-dimethoxybenzylamine. Reaction times can be reduced to seven hours, increasing the isopropylamine concentration to 100 mM (97 % conversion, 97 % ic, and 80 % overall conversion; reaction curves for different isopropylamine concentrations are shown in the Supporting Information, chapter 6.1).


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Scheme 2. Sequential 1-pot 2-step cascade reaction to (1S,2R)- and (1S,2S)methoxamine under optimized reaction conditions. Step 1: 100 mM HEPES buffer, pH 7.5, 2.5 mM MgSO4, 0.1 mM ThDP, 5 mM 2,5DMBA, 10 % DMSO, 20 mM pyruvate, purified and lyophilized 1.5 mg/mL ApPDC-E469G-I468A-W543F, 14 h (SR)/16 h (SS); step 2 (SR): 0.2 mM PLP, 1.3 mg/mL purified and lyophilized AsTA and 100 mM isopropylamine (45 h), step 2 (SS): 0.2 mM PLP, 1.1 mg/mL purified and lyophilized

BmTA and 50 mM isopropylamine (36.5 h), open vial, 30 °C, 750 rpm (*non-isolated yield).


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2-Step cascade for (1R,2S)-methoxamine synthesis (see Scheme 3). (1R,2S)methoxamine was accessible in a 1-pot 2-step cascade reaction, combining the (R)selective EcAHAS-I and BmTA (both enzymes purified and lyophilized). Although stereoselectivities of 96 % were achieved in the first step for the (R)-2,5DMPAC synthesis, the greatest challenge in this set-up was the incomplete product formation of (R)-2,5DMPAC yielding only 77 ± 2 %. Therefore, although an almost complete conversion of the (R)-2,5DMPAC intermediate to methoxamine was performed (conv. 99 %), 19 % 2,5-dimethoxybenzylamine was formed from the remaining 2,5DMBA of step 1. After 20 h the isomeric content of (1R,2S)-methoxamine was 94 % (Supporting Information, chapter 6.2). 2-Step cascade for (1R,2R)-methoxamine synthesis (see Scheme 3). For (1R,2R)methoxamine synthesis, E. coli whole cells expressing EcAHAS-I heterologously were tested to synthesize the cascade intermediate (R)-2,5DMPAC with non-isolated yields of 86 ± 1 % and excellent ee values of 98 % (see Scheme 3).

AsTA was applied for the second cascade step. Both formulations – the purified form and the whole cell catalyst (AsTA heterologously expressed in E. coli) – were tested and


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compared. For 2 mg/mL purified enzyme, conversion of 67 % was achieved, while similar high conversion of 67 % were reached in reactions with 10 mg/mL E. coli whole cells containing AsTA. If 20 mg/mL cells were used, the conversion was significantly increased to 86 % (28 h; detailed reaction plots are depicted in the Supporting Information, chapter 6.3). This proves that whole cell catalysis was a valuable alternative to purified enzymes due to its higher conversions. 2,5-Dimethoxybenzylamine was formed as a side product with 3-6 % conversion and another unidentified side product occurred in small amounts. For the 10 mg/mL whole cell sample, the final ic value of 98 % for the (1R,2R)methoxamine stereoisomer was excellent. The main product was identified as synmethoxamine by comparison to the reference compounds, conclusion of analogy and the GC-TOF-MS analysis (Supporting Information, chapter 3.4, Figure S21).


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Scheme 3. Sequential 1-pot 2-step cascade reaction to (1R,2S)- and (1R,2R)methoxamine. Step 1 (RS): 1.4 mg/mL EcAHAS-I, 2.5 % DMSO, 20 mM pyruvate, 3.3 h, step 2 (RS) 0.2 mM PLP, 1.1 mg/mL BmTA and 50 mM isopropylamine (20 h). Step 1 (RR): 100 mM HEPES buffer, pH 7.5, 5 mM MgCl2, 0.1 mM ThDP, 0.05 mM FAD, 5 mM 2,5DMBA, 12.5 % DMSO, 25 mM pyruvate, 30 mg/mL E. coli whole cells of EcAHAS-I, 2 h, step 2 (RR measured without speed vac. method at SFC): 0.2 mM PLP, 20 mg/mL

E. coli whole cells with AsTA, 100 mM isopropylamine, open vial, 30 °C, 750 rpm (28 h, *non-isolated yield).


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Preparative scale of (1S,2R)-methoxamine synthesis. A preparative scale (75 mL) of the 1-pot 2-step cascade to (1S,2R)-methoxamine including product purification has been developed. After 48 h, an (S)-2,5DMPAC yield (non-isolated) of 95 ± 1 % with ee values of 97 % was achieved for the (S)-hydroxy ketone with purified and lyophilized ApPDCE469G-I468A-W543F. The second cascade step was initiated by adding 1.3 mg/mL purified and lyophilized AsTA and after 31 h, a conversion of 94 ± 3 % (with respect to 2,5DMPAC formation) and an excellent ic value >99 % was achieved (Supporting Information, chapter 7). The total conversion of both steps was 85 % due to single step conversions and dilution of the reaction mixture by addition of compounds for step 2. The side product 2,5-dimethoxybenzylamine was formed at 12 ± 2 %. This phenomenon of the undesired side reaction has already been described for the 2-step reaction cascade on nor(pseudo)ephedrine.20 Here, the transaminase of the second step has a higher affinity and velocity for the aldehyde substrate of the first carboligation step than for the hydroxyl-ketone intermediate. Furthermore, it could be demonstrated that due to the reversibility of the carboligation reaction, it is not the amino alcohol but the benzylamine by-product that is thermodynamically preferred. However, this side reaction can be


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) decreased by removing the step 1 enzyme, e.g. by ultrafiltration, before starting the second cascade step.20 By alkaline extraction in ethyl acetate and purification by flash chromatography, a 102 mg isolated product was obtained as colorless oil, which corresponds to an isolated yield of 64 % compared to the starting material. The product was precipitated incompletely as an HCl salt (85 mg product, 46 % isolated yield, 94 % purity according to NMR detection, ic 98 %, Supporting Information, chapter 7). Higher yields can be achieved by optimizing the extraction and purification methods.

CONCLUSION

In this project we were able to synthesize all four stereoisomers of methoxamine with good to high conversions and excellent isomeric contents in sequential 1-pot 2-step cascade reactions without isolation and purification of the intermediate 2,5DMPAC. Based on cheap and easily available substrates, a simple procedure was developed to produce almost stereochemically pure vicinal amino alcohol isomers with high step- and atom efficiency. For the synthesis of (1R,2R)-methoxamine, it was shown that the


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synthetic enzyme cascade approach is not only possible for purified enzymes, but also for whole cell catalysts, in order to achieve comparable conversions and product purities. The optical purity of the product formation was measured by SFC analytics by connecting two chiral columns in a row. This method made it possible to separate all four methoxamine isomers without derivatizing the products. The scalability of the optimized cascade arrangement was demonstrated for the (1S,2R)-methoxamine synthesis, gaining a stereoisomeric purity of 98 % in a preparative scale.

EXPERIMENTAL SECTION

Materials. All buffer substances, substrates, and solvents used were ordered from commercial suppliers in the highest available purities. Gene and protein sequences of the used pyruvate decarboxylase from Acetobacter pasteurianus17,18, acetohydroxyacid synthase I45–48 from E. coli, and transaminases from Arthrobacter sp.41 and Bacillus

megaterium34 can be found in the Supporting Information, chapter 1. Cultivations, heterologous expressions, and purifications of the His-tagged enzymes as well as storage of the biocatalysts are also described in this chapter.


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Batch reaction to (1S,2R)- and (1S,2S)-methoxamine. For the first carboligation step, 5 mM of 2,5DMBA, 10 % DMSO, and 20 mM pyruvate were dissolved in a 100 mM HEPES buffer, pH 7.5, with 2.5 mM MgSO4 and 0.1 mM thiamine diphosphate (ThDP). Purified, lyophilized enzyme of ApPDC-E469G-I468A-W543F (1.5 mg/mL) was directly weighed into the reaction vessel and incubated at 30 °C and 750 rpm for 9-16 h. When the 2,5DMBA concentration was increased to 20 mM, the pyruvate concentration was also raised to 80 mM with 20 % DMSO. After almost complete conversion of 2,5DMBA, 100 mM isopropylamine, 0.2-1.0 mM pyridoxal-5’-phosphate hydrate (PLP), and 1.3-1.9 mg/mL purified, lyophilized enzyme of AsTA were added and the pH adjusted to 7.5 by 1M HCl to synthesize (1S,2R)-methoxamine. For (1S,2S)-methoxamine synthesis, the parameters of the first carboligation step were the same. For the second transamination step, 20-100 mM isopropylamine, 0.2 mM PLP, and 1.1 mg purified, lyophilized enzyme of BmTA were added and the pH adjusted to 7.5 by 1M HCl. The reaction vessel was incubated without a lid, in order to shift the reaction equilibrium of the second transamination step by acetone evaporation (30 °C, 750 rpm, 20.5 h).


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ACS Catalysis

Batch reaction to (1R,2R)- and (1R,2S)-methoxamine. For the carboligation step of the (1R,2S)-methoxamine cascade, 5 mM 2,5DMBA, 2.5 % DMSO, and 20 mM pyruvate were dissolved in a 100 mM HEPES buffer, pH 7.5, with 5 mM MgCl2, 0.1 mM ThDP, and 0.05 mM flavine adenine dinucleotide disodium salt hydrate (FAD). Purified, lyophilized

EcAHAS-I (1.4 mg/mL) was directly weighed into the reaction vessel (30 °C and 750 rpm, 3.3 h). For (1R,2S)-methoxamine synthesis, 50 mM isopropylamine, 0.2 mM PLP, and 1.1 mg/mL lyophilisate of purified BmTA were added and the pH adjusted to 7.5 by 1M HCl. For acetone evaporation, the reaction vessels were incubated without a lid (30 °C, 750 rpm, 20 h). For set-ups for (1R,2R)-methoxamine synthesis with whole cells, 30 mg/mL E. coli whole cells containing EcAHAS-I were mixed with 5 mM 2,5DMBA, 12.5 % DMSO, and 25 mM pyruvate (2 h, 30 °C, 750 rpm). For the second cascade step, 0.2 mM PLP, 2 mg/mL lyophilisate of purified AsTA, or 10-20 mg/mL E. coli whole cell with heterologously expressed AsTA,100 mM isopropylamine were added and the pH adjusted to 7.5 by 1M HCl (28 h, 30 °C, 750 rpm, open vial for acetone evaporation).


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Preparative scale of (1S,2R)-methoxamine. The carboligation reaction was initiated by adding 40 mM pyruvate to a 100 mM HEPES buffer, pH 7.5, 2.5 mM MgSO4, 0.1 mM ThDP, 10 mM 2,5DMBA, 20 % DMSO, 0.75 mg/mL purified and lyophilized ApPDCE469G-I468A-W543F (reaction volume 75 mL, 30 °C, 160 rpm). After 22 h, 0.5 mM PLP, 100 mM isopropylamine, 1.3 mg/mL purified and lyophilized AsTA were added and the pH adjusted to 7.5 by 1M HCl to initiate the transamination step of the cascade reaction (incubation with opened reaction vessel, 30 °C, 160 rpm, 31 h). The product purification is described in the Supporting Information, chapter 7.3. Analytical methods. A chiral IE column from Chiralpak was used for non-isolated yield measurements of the carboligation step (2,5DMPAC). In addition, 20 µL of the reaction sample was diluted in 180-380 µL acetonitrile and measured using a gradient HPLC method (Agilent Technologies, 1260 Infinity) starting at 80:20 H2O: acetonitrile (flow: 0.7 mL/min, 5 µL injection, 20 °C oven temperature, 200 nm, 24 min, (S)-2,5DMPAC: 18.118.2 min, (R)-2,5DMPAC: 19.1 min, for further information see the Supporting Information, chapter 3.1).


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For conversion measurements of 2,5DMPAC in the transamination step, 20 µL samples were diluted 1:10 or 1:20 in acetonitrile with 0.1 % trifluoroacetic acid (TFA) and 0.1 % DEA. The samples were measured using HPLC (Agilent Technologies, 1260 Infinity) with an achiral RP-18 column from LiChrospher (70 % H2O and 30 % acetonitrile, 0.1 % DEA, 0.1 % TFA, 1 mL/min, 200-280 nm, 25 min, 2,5DMPAC: 6.4 min, methoxamine: 3.3 min, Supporting Information, chapter 3.2). The isomeric content of the methoxamine gained was determined via SFC. Here, 50 µL samples were taken, frozen with liquid nitrogen, and the water removed using an Eppendorf concentrator (30 °C, 1 h). The pellet was dissolved in 50 µL of eluent (MeOH + 0.5 % DEA), centrifuged for 3 min at 13000 rpm (RT), and the samples measured via SFC (Agilent Technologies, 1260/1290 Infinity, in series-connected chiral IA and chiral AD-H-columns from Chiralpak, 85:15 CO2:MeOH + 0.5 % DEA, 1 mL/min, 30 °C oven temperature, 200 nm, 30 min, (1S,2R)-methoxamine, (14.7-15.0 min)). The sample preparation is described in detail in the Supporting Information, chapter 3.3. Before the above-described method was developed, the reaction samples (20 µL) were diluted directly in 40 µL MeOH + 0.5 % DEA, centrifuged (3 min, RT, 13000 rpm), and


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measured via SFC (Agilent Technologies, 1260/1290 Infinity, in series-connected chiral IA and chiral AD-H columns from Chiralpak, 85:15 CO2:MeOH + 0.5 % DEA, 1 mL/min, 30 °C oven temperature, 200 nm, 27 min).

Equations for conversion, yield, ee, de and ic determination Equation 1

Conversion [%] = ([substratet] / ([productt=0]+[substratet=0]))*100

Equation 2

Non-isolated yield [%] = ([productt] / [substratet=0])*100

Equation 3

ee [%] = ((E1- E2)/(E1+E2))*100

Equation 4

de [%] = ((D1-D2)/(D1+D2))*100

Equation 5

ic [%] = (I1 / (I1+I2+I3+I4))*100

ee: enantiomeric excess, E: enantiomer, de: diastereomeric excess, D: diastereomer, ic: isomeric content, I: isomer

Product identification. In order to identify the single methoxamine isomers, reference compounds of the four methoxamine isomers were produced as described in the Supporting Information, chapter 2. Through chemical synthesis it was possible to produce


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a 50:50 mixture of all four methoxamine isomers, after which the diastereomers were separated by flash chromatography. The identity of the reference compounds and the isolated product was verified by NMR (Supporting Information, chapter 2 and 7). The identity of the non-isolated products was verified by GC-TOF-MS (Supporting Information, chapter 3.4). The absolute configuration of the isomers was matched by the conclusion of analogy due to the chosen enzyme combination of each cascade that determines the configuration of each methoxamine isomer.

ASSOCIATED CONTENT

The following files are available free of charge.

Supporting Information (PDF): Cultivation and target protein expression, chemical reference compound syntheses, analytical methods, equations for conversion and optical purity calculations, method optimization and more detailed information on data, described in this manuscript.


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AUTHOR INFORMATION

Corresponding Author * Email for Dörte Rother: [email protected]

Author Contributions

VE planned and carried out most of the experiments. TS brought many helpful ideas and discussions to this topic. IF supported the cultivation and purification of the used ThDP-dependent enzymes and transaminases. RS and WK contributed their know-how in the production of methoxamine reference compounds. In addition WK supplied the TA from

Bacillus megaterium, Arthrobacter citreus, Paracoccus denitrificans and

Arthrobacter sp. and hosted VE for an internship. DR was the supervisor and creative director of this project.

Notes


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This manuscript and the Supporting Information are part of the PhD thesis of Vanessa Erdmann (RWTH Aachen University). The authors declare no competing financial interest.

ABBREVIATIONS

ApPDC pyruvate decarboxylase from Acetobacter pasteurianus; AsTA Arthrobacter sp. transaminase, BmTA Bacillus megaterium transaminase, EcAHAS-I acetohydroxyacid synthase I from E. coli; de diastereoisomeric excess; DEA diethylamine; 25DMBA 2,5dimethoxybenzaldehyde; 25DMPAC 1-hydroxy-1-(2,5-dimethoxyphenyl)propan-2-one;

ee enantiomeric excess; FAD flavine adenine dinucleotide; ic isomeric content; PAC phenylacetylcarbinol (1-hydroxy-1-phenylpropan-2-one); PLP pyridoxal-5’-phosphate; SFC supercritical fluid chromatography; ThDP thiamine diphosphate, TFA trifluoroacetic acid.


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ACKNOWLEDGMENT

This project was financially supported by the Helmholtz Synthetic Biology Initiative and the Helmholtz Young Investigators Group “Synthetic enzyme cascades”. We thank our cooperation partners for kindly providing the following enzymes: TA from Vibrio fluvialis and variants thereof - Prof. Uwe Bornscheuer (University of Greifswald, Germany); TA from Chromobacterium violaceum - Prof. Helen C. Hailes (University College London, UK); TA from Aspergillus terreus – Enzymicals AG; carboligase EcAHAS-I – Prof. David M. Chipman (Ben Gurion University of the Negev, Israel) and Prof. Kai Tittmann (GeorgAugust-University, Göttingen, Germany). We would also like to thank Jochem Gätgens, IBG-1, Forschungszentrum Jülich, for his support in GC-TOF-MS analysis.

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Figure 1. Synthesis of (S)-2,5DMPAC by ApPDC-E469G (duplicates) and the new variant ApPDC-E469GI468A-W543 (quadruplicate) under optimized reaction conditions: 100 mM HEPES buffer, pH 7.5, 2.5 mM MgSO4, 0.1 mM thiamine diphosphate (ThDP), 5 mM 2,5DMBA, 10 % DMSO, 20 mM pyruvate, 1.5 mg/mL enzyme, 30 °C, 700-750 rpm (*non-isolated), HPLC analytics are described in detail in the Supporting Information, chapter 3. 98x95mm (600 x 600 DPI)


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Figure 2. Sequential 1-pot 2-step cascade reaction from 2,5DMBA and pyruvate to (1S,2R)-methoxamine. Reaction conditions: step 1: 100 mM HEPES, pH 7.5, 2.5 mM MgSO4, 0.1 mM ThDP, 20 mM 2,5DMBA, 20 % DMSO, 80 mM pyruvate, 1.5 mg/mL purified and lyophilized ApPDC-E469G-I468A-W543F, 30 °C, 750 rpm, 26 h, step 2: pH 9, 1 mM PLP, 1.9 mg/mL purified and lyophilized AsTA, 100 mM isopropylamine, 30 °C, 750 rpm, 51 h, open vial. 208x172mm (300 x 300 DPI)


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1. Sequential 1-pot 2-step cascade reaction to methoxamine, starting from the easily obtainable substrates 2,5-dimethoxybenzaldehyde and pyruvate. 148x42mm (300 x 300 DPI)


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ACS Catalysis

Scheme 2. Sequential 1-pot 2-step cascade reaction to (1S,2R)- and (1S,2S)-methoxamine under optimized reaction conditions. Step 1: 100 mM HEPES buffer, pH 7.5, 2.5 mM MgSO4, 0.1 mM ThDP, 5 mM 2,5DMBA, 10 % DMSO, 20 mM pyruvate, purified and lyophilized 1.5 mg/mL ApPDC-E469G-I468A-W543F, 14 h (SR)/16 h (SS); step 2 (SR): 0.2 mM PLP, 1.3 mg/mL purified and lyophilized AsTA and 100 mM isopropylamine (45 h), step 2 (SS): 0.2 mM PLP, 1.1 mg/mL purified and lyophilized BmTA and 50 mM isopropylamine (36.5 h), open vial, 30 °C, 750 rpm (*non-isolated yield). 180x91mm (600 x 600 DPI)


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Scheme 3. Sequential 1-pot 2-step cascade reaction to (1R,2S)- and (1R,2R)-methoxamine. Step 1 (RS): 1.4 mg/mL EcAHAS-I, 2.5 % DMSO, 20 mM pyruvate, 3.3 h, step 2 (RS) 0.2 mM PLP, 1.1 mg/mL BmTA and 50 mM isopropylamine (20 h). Step 1 (RR): 100 mM HEPES buffer, pH 7.5, 5 mM MgCl2, 0.1 mM ThDP, 0.05 mM FAD, 5 mM 2,5DMBA, 12.5 % DMSO, 25 mM pyruvate, 30 mg/mL E. coli whole cells of EcAHAS-I, 2 h, step 2 (RR measured without speed vac. method at SFC): 0.2 mM PLP, 20 mg/mL E. coli whole cells with AsTA, 100 mM isopropylamine, open vial, 30 °C, 750 rpm (28 h, *non-isolated yield). 179x90mm (300 x 300 DPI)


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ACS Catalysis


Methoxamine Synthesis in a Biocatalytic 1-Pot 2-Step Cascade

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