One-Pot Synthesis of D-Halotryptophans by Dynamic Stereoinversion

Dec 21, 2018 - Tryptophan (Trp) derivatives constitute important building blocks found in many natural products, peptides and drugs. Accordingly, ther...
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One-Pot Synthesis of D-Halotryptophans by Dynamic Stereoinversion Using a Specific L-Amino Acid Oxidase Christian Schnepel, Isabell Kemker, and Norbert Sewald ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b04944 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on December 24, 2018

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ACS Catalysis

One-Pot Synthesis of D-Halotryptophans by Dynamic Stereoinversion Using a Specific L-Amino Acid Oxidase Christian Schnepel, Isabell Kemker, and Norbert Sewald* Organic and Bioorganic Chemistry, Department of Chemistry, Bielefeld University, PO Box 100131, 33501 Bielefeld, Germany, *[email protected] ABSTRACT: Tryptophan (Trp) derivatives constitute important building blocks found in many natural products, peptides and drugs. Accordingly, there is a high demand for suitable biocatalytic pathways providing selective derivatization of Trp like C–H functionalization or C-oxidation. The specific L-amino acid oxidase RebO from the actinomycete L. aerocolonigenes was harnessed for chemoenzymatic oxidation of substituted Trp derivatives. An array of potential substrates was tested and several Trp derivatives are being accepted in reasonable turnover. The highest selectivity was observed for 7-halotryptophan being converted about 35-fold faster than non-substituted Trp. This selectivity is also useful for establishing a colorimetric halogenase highthroughput assay. RebO was also employed in dynamic stereoinversion in presence of ammonia-borane complex to provide access to non-native D-configured Trp analogues. Optimized reaction conditions yielded ~70% D-amino acid with >98% ee for halotryptophans on analytical scale. Dynamic stereoinversion preceded by enzymatic halogenation in a sequential one-pot reaction cascade provided D-configured 5- or 7-bromotryptophan, resp., with improved conversion of approx. 90%, >92% ee and good isolated yields.

KEYWORDS: tryptophan halogenase; amino acid oxidase; stereoinversion; one-pot synthesis; high-throughput screening; non-canonical amino acid

INTRODUCTION The synthesis of chiral building blocks like amino acids strongly benefits from tremendous progress in biocatalysis within the last decades. Proteinogenic amino acids are being predominantly produced by fermentation processes, e.g. the production of glutamate and lysine proceeds on million-ton scale per year. Many of them find application as bulk materials for chemistry and nutrition or serve as additives in feedstuff, respectively.1 Particularly, the synthesis of non-native amino acids attracted interest in the last decade, as such building blocks have potential in protein research, for example, to study the influence of nonnatural amino acids on biocatalyst properties.2 Non-canonical amino acids can be integrated into peptides by solid-phase synthesis, while so-called xenoproteins are usually synthesized in a recombinant fashion by making use of selective pressure incorporation or the expanded genetic code.3,4 Even though tryptophan (Trp) is a low abundant amino acid, its biological significance is extraordinary and made Trp derivatives widely applied handles.5,6 This particular amino acid acts as a biosynthetic precursor for plenty of natural products.7,8 Thus, many efforts were undertaken to develop selective synthetic strategies yielding functionalized Trp by means of chemo- and biocatalysis. For example, assembly of the chiral Trp scaffold could be accomplished by enantioselective Strecker amino acid synthesis. Schöllkopf’s chiral auxiliary was frequently applied to obtain chiral amino acids (Scheme 1A).9,10 Acylated Trp derivatives can be derived by simple reaction of L-serine with substituted indoles in presence of acetic anhydride. Kinetic

resolution of racemic Trp catalyzed by an acylase affords the Lconfigured enantiomer.11 A straightforward enzymatic approach developed by Goss et al. utilizes tryptophan synthase from Salmonella enterica to obtain a range of substituted tryptophans.12,13 In the enzyme, a Michael-type addition takes place where indole-C3 attacks a pyridoxal phosphate-linked amino acrylate residue initially formed upon serine dehydration. This simple one-step biotransformation made a large set of halogenated and alkylated Trp derivatives accessible. Recently, Arnold and Micklefield independently embarked on engineering Trp synthase to accept L-threonine as an electrophilic co-substrate resulting in -methyl Trp derivatives.14,15 A major obstacle is that these synthetic pathways require previous formation of substituted indoles, e.g. via Fischer indole synthesis or Pd-catalyzed annulation, respectively, which may render the process inefficient and laborious.16,17 In contrast, a plethora of biotransformations has been discovered capable to perform direct modification of the tryptophan scaffold with high selectivity, while avoiding elaborate directing groups.18 Typical modifications of Trp encompass hydroxylation known from the initial step of serotonin biosynthesis, nitration catalyzed by a cytochrome P450 enzyme or alkylations, respectively.19–22 Particularly, enzymatic halogenation emerged as an important methodology for selective C–H activation (Scheme 1B). The halogenated products allow for a range of subsequent derivatizations, e.g. nucleophilic substitution or Pd-catalyzed cross-couplings. Thus we and other groups sought to employ flavin-dependent Trp halogenases to selectively halogenate the indole moiety, while merely consuming O2, the cofactor FADH2 and a halide salt.23,24

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Scheme 1. (A.) Representative examples describing asymmetric synthesis of substituted Trp derivatives by making use of organo- and biocatalysis. (B.) Overview of enzyme-catalyzed reactions towards substituted L-Trp derivatives providing modification of the indole nucleus or the amino acid backbone (L-AAO: L-Amino acid oxidase; TDC: Tryptophan decarboxylase). A. EtO

X N H

O

OEt

N

Br

OH

N

H 2N

Ph

Backbone modification NH2

X N H Strecker synthesis

NaCN, AcOH

(or vice versa)

B.

H Indole modification O

Schöllkopf method O

O

OH O N H

X N H

CH3

X = Hal, OMe, NO2

NH2

OH NH2

Aminoacylase

TDC O

OH

Trp synthase

X

X N H

O H 2N R

OH OH

Its preparative utility became feasible by CLEA immobilization providing access to bromotryptophan (Br-Trp) on a gram scale.25 The directing influence of substituents present at the indole of Trp on the RebH-catalyzed halogenation was investigated.26 Enzyme engineering by directed evolution to improve halogenase efficiency was shown to provide improved catalyst stability, highthroughput assay design as well as an extended substrate scope.27– 30 Notably, the halogen substituent serves as a reactive handle for Pd-catalyzed cross-couplings. The groups of Goss and O’Connor first embarked on sequential cascades combining enzymatic halogenation and Suzuki-Miyaura cross-coupling also allowing for altered spectral properties of the aryl-substituted tryptophans.31–33 One-pot procedures carried out on preparative scale were later reported by the Lewis group34 and by us.35 Inspired by a seminal study by Gröger et al.,36 Micklefield and coworkers employed compartmentalization to overcome incompatibility between the halogenase and the Pd catalyst thus providing an important milestone in this field.37 Very recently, Goss and coworkers embarked on an intriguing chemogenetic approach that merged enzymatic halogenation with cross-coupling in vivo, albeit without mentioning conversions.38 The use of biocatalysts in reaction cascades, in multistep enzyme reactions or combined with chemocatalysts attracts great attention. Especially when enzymes and metal catalysts are mutually used, compatibility issues have to be overcome. Multienzyme cascades provide novel pathways applicable in the production of valuable chemicals in vitro. Current developments on chemoenzymatic synthesis and their challenges have been reviewed by Flitsch et al.39 and Bornscheuer et al.40

N H N H X = Hal, Me, NH2; also -methyl Trp using L-threonine

X = Hal

O

NH2

Trp halogenase

X

OH

N H Decarboxylation

OH

L-AAO

N H

O N H Oxidative deamination (described herein)

handling of L-AAOs is hampered by severe obstacles: In particular, heterologous L-AAO expression proves challenging due to low protein solubility, misfolding and potential toxicity for the expression host. Therefore, these enzymes are less attractive for large scale applications. An L-AAO from Rhodococcus opacus is one of the few well-studied representatives where expression succeeded albeit with low yield and unsatisfying enzyme activity.43 Very recently an elaborate survey reported about a fungal L-AAO expressed in Pichia pastoris followed by its biochemical characterization.44,45 D-AAOs found wide application in biotechnology up to now, e.g. in kinetic resolution of chiral amines as well as for the synthesis of -keto acids. In 2015, Turner et al. embarked on the synthesis of non-natural Phe analogues using a multistep cascade starting from substituted cinnamic acids. These compounds were initially aminated by ammonia lyase and subsequently deracemized with either a deaminase or a D-AAO in presence of a non-selective reducing agent.46 As the current state of the art on L-amino acid oxidases accepting Trp derivatives is very limited, we felt inspired to study an AAO from L. aerocolonigenes (RebO) as the only described member with high specificity towards substituted Trp. RebO (EC 1.4.3.23, Uniprot ID: Q8KHS0), a 52 kDa flavoprotein, was previously characterized to elucidate its role in the biosynthesis of the indole carbazole rebeccamycin.47–49 RebH-catalyzed C7halogenation of Trp is followed by an oxidative deamination induced by RebO resulting in 7-chloroindolyl pyruvate as a biosynthetic intermediate of the rebeccamycin pathway (Scheme 2). Prior studies indicated that RebO prefers L-7chlorotryptophan, but the biotechnological use of this enzyme has not been explored yet.

Amino acid decarboxylation or deamination offer important transformations providing the corresponding tryptamines or keto acids, respectively. Enzymatic deamination catalyzed by aminotransferases, amine dehydrogenases, or amino acid oxidases (AAO), for example, can be employed in the latter case.41 In general, AAOs only require molecular oxygen to catalyze Coxidation yielding an -imino acid intermediate that spontaneously hydrolyzes to give the -keto acid. The catalytic cycle starts with the hydride transfer to FAD. The resulting FADH2 subsequently reacts with O2 to form the flavin hydroperoxide, which finally releases hydrogen peroxide.42 There are L-selective AAOs as well as their D-selective counterparts known. While D-AAOs found broad application in biotechnology,

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ACS Catalysis

Scheme 2. Sequential action of Trp 7-halogenase and TrpAAO RebO in rebeccamycin biosynthesis. CO2H NH2 N H L-tryptophan

CO2H

Trp 7-halogenase

O 2, H + FADH2

Amino acid oxidase

NH2

RebH 2 H 2O FAD

O2

L-7-chlorotryptophan

H N

O

NH

RebO

N H

Cl

CO2H

H 2O 2

N H

Cl 7-chloroindolyl2-iminopropionate

O Further biosynthetic steps

Cl

N H

N O

Cl

HO H3CO HO rebeccamycin

RESULTS AND DISCUSSION The gene rebO was isolated by PCR from L. aerocolonigenes and ligated into the host vector for heterologous expression in E. coli appropriate for chemoenzymatic reactions (Figure S1). The gene of the His6-tagged fusion protein was expressed in E. coli BL21 (DE3) at 16 °C. Folding was supported by the chaperones GroEL/ES and the protein was isolated by metal ion affinity chromatography. SDS-PAGE analysis confirmed successful isolation of RebO (Fig. S2) resulting in approx. 15 mg soluble protein per 1.5 L expression culture sufficient for biocatalytic assays. Taken into consideration that C-oxidation catalyzed by an AAO releases one equivalent of hydrogen peroxide per substrate turnover, the enzymatic activity of RebO can be assayed by coupling to horseradish peroxidase (HRP) (Scheme 3). HRP catalyzes the oxidation of a chromogenic substrate to give a visible dye to be detected photometrically. Among the large set of available chromophores, we chose a water-soluble HRP substrate pair consisting of 4-phenolsulfonic acid (Ph(4-OH)SO3H) and 4aminoantipyrine (4-AAP). Compared to other frequently applied HRP substrates like o-dianisidine these chromogenic substrates allow an enzyme assay in aqueous solution, while being less toxic.50 As a precondition to study the substrate profile of RebO, the extinction coefficient was determined being adjusted to the currently applied reaction conditions (Figure S3). This setup served to determine the activity of RebO towards a panel of Trp analogues, with a focus on halogenated compounds. The halogenated tryptophans were synthesized by using immobilized halogenase that gave the desired L-amino acids in good yield and purity (see Supp. Inform. for details).25 However, as fluorinated aromatic compounds are not accessible using a halogenase, these derivatives were synthesized using Trp synthase as described.13,26 Reaction progress curves revealed that L-6-bromotryptophan (6Br-Trp) is not well accepted as indicated by the negligible increase of absorbance (Scheme 3; Figure S4). Similarly, the 5hydroxy derivative was also not preferred, but substantially higher activities were observed for 5-halotryptophans. Interestingly, among the tested Trp substrates, RebO exhibits a strong preference for C7-isomers as indicated by a tenfold higher activity. Pronounced differences in specific activity demonstrate that RebO is capable to discriminate between the substitution pattern of the indole moiety, while Trp lacking an indole substituent was only poorly converted. It could be presumed from these data that the active site of RebO undergoes substantial interactions with the side chain that make a substituent on the indole moiety necessary for tight substrate binding.

However, the fluoro substituent is less well accommodated in the active site compared to bulkier Cl or Br thus leading to lower affinity. The strong preference for C7-halogenated Trp (Figure S5) suggested to exploit these features in a RebO-coupled halogenase assay. In a previous report a high-throughput screening methodology employing Suzuki-Miyaura crosscoupling was established to monitor enzymatic halogenation in directed evolution.30 As only few selective assays for halogenase activity are known, the design of a colorimetric screening could expand the current knowledge on detecting halogenase activity. It was anticipated from prior data that notable RebO-catalyzed conversion associated with H2O2 formation will only occur in presence of halogenated amino acid. In contrast, Trp should not lead to considerable turnover by RebO (Figure 1A). Thus, colorimetric detection of H2O2 would correlate with enzymatic halogenation. A concentration series of L-7-chlorotryptophan (7Cl-Trp) incubated with RebO, HRP and chromogenic substrates gave a linear increase of absorbance at 490 nm proportional to the formation of chlorinated amino acid (Figure S6). As these findings indicated that such an assay is generally feasible, the approach was employed in E. coli lysate with overexpressed halogenase. Lysate mixtures containing a variable ratio of 7-ClTrp to Trp were incubated with the assay components. Despite the presence of both components in lysate medium, a clear linear correlation is observed showing proportionality between absorbance and halogenation product formation (Figure 1B; S7). Noteworthy, slow conversion of Trp did not deteriorate quantitative detection of 7-Cl-Trp even at lower product concentrations owing to the remarkable specificity. Furthermore, RebO-induced formation of H2O2 was unaffected by other contaminants present in lysate. A simple colorimetric screening to quantify halogenation of Trp in a high-throughput manner resulted, which opens up a methodology for enzyme engineering of Trp 7-halogenase variants aiming at improved efficiency or stability, respectively.

Interestingly, the type of substituent is also crucial, as a more polar hydroxyl group was not tolerated. In contrast, L-7halotryptophans turned out as the most promising substrates.

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Scheme 3. RebO-catalyzed oxidation of different substituted Trp derivatives. By coupling with HRP and a chromogenic substrate, the oxidative deamination can be continuously monitored at 490 nm for activity determination. Notably, RebO showed a high preference for C7-halogenated Trp. Activities given herein are based on spectrophotometric assays applied for purified protein.

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A.

Oxidative deamination R

R

RebO

NH OH

H 2N

NH3

H 2O

O substrate

O2

B.

NH

H 2O 2

Ph(4-OH)SO3H + 4-AAP

OH

O

O -keto acid HRP

HN

Ph N N

Peroxidase-coupled assay

O

quinone-imine adduct (chromophore)

NH OH

H 2N O

Trp 1.37 ± 0.4 mU mg–1 Br

OH

O 7-chloro-Trp 46.6 ± 2.0 mU mg–1 Cl

H 2N

OH

O 6-bromo-Trp 99

68.9

7

32

H3N·BH3 (100)

--

>99

66.9

[a]

Enantiomeric excess determined using Marfey’s test (FDAA coupling and RP-HPLC separation); [b] relative ratio of -amino acid to -hydroxy acid determined via RP-HPLC The substrate (0.5 mM) was incubated with 18 µM RebO and 5 mM (10 equiv.) of reducing agent. After incubation at 25 °C for 20 h the enantiomeric ratio was determined by derivatization with Marfey’s reagent forming the respective diastereomers separable via RP-HPLC (Scheme S1).58 The first attempt already reached an enantiomeric excess (ee) of >98% indicating that the enzymatic

system successfully produced D-configured halotryptophan (Figure 2B; Table 1, entry 1). RP-HPLC analysis of the reaction mixture showed a substantial decrease of amino acid accompanied by an increase of another, more abundant compound (Figure 2C), corresponding to the brominated -hydroxy acid 3-bromoindolyllactic acid with m/z 283.99 (Figure 2D). Based on these data, the -keto acid formed from spontaneous imine hydrolysis is being reduced by the ammonia-borane complex to give the hydroxy acid. Changing the reducing agent was not successful confirming that ammonia-borane complex is most efficient, while NaBH4 did not give any D-enantiomer (Table 1, entry 3). The L-amino acid was still the dominant species, but neither -keto nor -hydroxy acid were formed. In light of previous publications about the deracemization of D/L-proline using NaBH4 this result was unexpected.57,59 In addition to its low stability in water, NaBH4 severely suppresses L-AAO activity as obvious from the lack of notable enzyme activity. One reason might be an interference of the reductant with oxidation of flavin cofactor as previously postulated by Soda et al.59 The reduction of Schiff’s bases formed between NH2 groups of the enzyme and the keto acid was also discussed as a plausible inactivation mechanism.57 NaCNBH3 instead slightly induces formation of D-7-Br-Trp (6.5% ratio) correspond. to 87% ee(L)) with mediocre amino acid ratio (Table 1, entry 4) albeit unsatisfactory for stereoinversion. Probably this occurred as a consequence of its substantially lower reactivity, hence turning back to ammonia-borane complex. Ongoing optimization studies revealed that the undesired hydrolysis / carbonyl reduction pathway can be suppressed by further increasing the concentration of H3N·BH3 (Figure S8A). In analytical scale assays the target D-amino acid was obtained in approx. 70% yield (HPLC) and >99% ee using a large excess of H3N·BH3 (100 equiv.). Addition of ammonium that might favor the equilibrium towards the amino acid did not improve the yield of amino acid under these conditions. Interestingly, the excess of H3N·BH3 did not have any severe influence on the enantiomeric excess. Even with only a small excess of reductant, >90% ee of Damino acid are obtained (Figure S8B). In contrast, reducing RebO loading to 4 µM gave the D-enantiomer in 40% ee (Table 1, entry 5), while over 99% of target enantiomer resulted applying a biocatalyst concentration of 12 and 32 µM, respectively (Table 1, entry 6, 7), indicating a minimum threshold concentration of biocatalyst required for efficient dynamic stereoinversion.

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A.

B.

C.

D.

Figure 2. (A.) Two-step biocatalytic synthesis of D-configured tryptophan derivatives by combining biocatalytic halogenation or Trp synthase with RebO-induced dynamic stereoinversion. L-selective oxidation by RebO and subsequent chemical reduction into the racemate induce stepwise accumulation of the D-enantiomer. (B.) HPLC diagram (C18-RP column, 1.9 µm particle size) upon Marfey’s derivatization using FDAA indicates quantitative inversion into the desired D-enantiomer (see Table 1, entry 6-7). (C.) The ratio of amino acid to -hydroxy acid was determined by RP-HPLC (C18-RP column, 3.0 µm particle size) indicating that a large excess of H3N·BH3 was required to avoid the hydrolysis/reduction pathway (Table 1, entry 1-2). (D.) ESI-LC-MS proved the presence of both BrTrp (tr = 3.6 min, m/z = [M+H]+ calc. 283.01) and its hydroxy acid derivative, bromoindolyl-lactic acid, as a side product at tr = 4.1 min (m/z = [M+H]+ calc. 283.99).

Encouraged by these results, the stereoinversion approach was transferred to other Trp derivatives, in order to deracemize substrates with lower affinity towards RebO. Particular emphasis was laid on halotryptophans, as RebO provides a strategy to obtain the corresponding D-enantiomers by means of a convenient chemoenzymatic cascade. Facing the problem that the corresponding D-configured products were not available as reference compounds, diastereomer forming derivatization with Marfey’s reagent prior to LC-MS analysis was used for chiral analysis. Stereoinversion of L-5-Br-Trp was of particular interest in this context, because D-configured 5-halotryptophans are not accessible when attempting enzymatic halogenation of D-Trp. The screening confirmed that all 7-halotryptophan derivatives are obtained in >95% ee. Moreover, -hydroxy acid was only present in low levels ranging from 17-25%. Hence, this side product has no severe detrimental effect on the overall conversion (Figure 3, Table S1).

also possible in reasonable yield (HPLC) under these conditions. The lowered quantity of -hydroxy acid also indicates that weaker substrates of RebO are less prone to the hydrolysis / carbonyl reduction pathway.

Although RebO displayed negligible activity towards 5-hydroxy and 6-Br-Trp, these compounds gave appreciable accumulation of the respective D-enantiomer with approximately 20% inversion ratio. Remarkably, D-5-halotryptophan was also formed with perfect ee. Noteworthy, stereoinversion of poor substrate Trp was

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Figure 3. RebO-induced stereoinversion starting from different LTrp derivatives as substrates. The percentaged of each enantiomer is plotted for different substrates, while the relative ratio (italic) of amino acid to hydroxy acid is mentioned above. Each substrate forms a notable quantity of the respective D-enantiomer confirming the successful stereoinversion of 5- and 7halotryptophan with minor formation of undesired -hydroxy acid. For chiral analysis reaction mixtures were analyzed via Marfey’s derivatization combined with LC-MS for quantification of enantiomeric ratios. Reaction conditions: 1.0 mM substrate, 18 µM RebO, 100 U mL–1 catalase, 0.1 M H3N·BH3, 10 mM Na2HPO4 (pH 8.0), 30 mM NaCl, 20 h at 25 °C. Considering the high enantiomeric ratio of D-amino acid (>95%) along with low side product formation, these findings underscore the overall value of RebO to obtain a variety of halogenated D-Trp analogues hardly accessible otherwise. In this light, RebO-based dynamic stereoinversion was envisioned to be combined with enzymatic halogenation in a one-pot approach without isolation of the halogenated amino acid intermediate (Figure 4A). L-5-Br-Trp was prepared using combiCLEAs comprising the halogenase together with auxiliary enzymes for cofactor regeneration as previously described.25 CLEA immobilization is methodology that is well-established in enzymatic halogenation and was also successfully adopted by others.37 CLEAs provide smooth upscaling of halogenation, convenient separation and high stability. The crude filtrate can be comfortably stored over months and used in portions if desired. Quantitative conversion of 1 mM substrate was achieved in 3-5 days at 25 °C. After CLEA removal from the suspension, the components for dynamic stereoinversion were added to the filtrate. 75 mL reaction buffer containing 1 mM L-5-Br-Trp (approx. 21 mg L-amino acid) was employed for dynamic stereoinversion using RebO. By subsequent usage of RebO as a crude lysate from 0.33 L expression culture, laborious

and yield-limiting enzyme purification was avoided. 100 equiv. H3N·BH3 and catalase were added to the mixture, while glycerol and FAD were found to favor the biotransformation probably due to enhancing enzyme stability. 42% of D-enantiomer was formed after 3 h at 25 °C (Figure 4B). Prolonged incubation resulted in nearly full conversion into D-5-Br-Trp after 24 h. Notably, the ratio of amino acid to hydroxy acid was considerably improved in preparative scale synthesis reaching 91% of amino acid in case of 5-Br-Trp and 89% for 7-Br-Trp compared to approx. 70% in analytical scale reactions. Thus, subsequent desalting via reversed-phase silica yielded the target amino acid and minor quantity of -hydroxy acid as contaminant. HPLC purification gave D-5-Br-Trp in a total yield of 49% over three steps based on starting material L-tryptophan and 92.0% ee (Figure 4C). In light of an enzyme cascade consisting of three reaction steps the overall yield of isolated product is rather satisfying. Moreover, LC-MS analysis of FDAA derivatization assigned elution peaks from the reference and the final product to Marfey’s adduct of Br-Trp indicating good enantiopurity of the isolated product (Figure 4D). Combined with NMR analysis D-5-Br-Trp was unambiguously identified. Analogously, D-7-Br-Trp was obtained with >98% ee and a yield of 44% on a preparative scale further proving that RebO can be broadly applied to prepare D-configured halotryptophans using this one-pot cascade.

CONCLUSION The dynamic stereoinversion of substituted tryptophan derivatives is possible using a specific L-AAO providing access to Denantiomers. RebO exhibits an extraordinary specificity yet recognizing slight variations of the substituted indole attached to the amino acid side chain. Substrate screenings revealed that a set of non-natural Trp derivates was oxidized by RebO into the corresponding -keto acid. Similar to previous observations by

A.

B.

C.

D.

Figure 4. Dynamic stereoinversion of L-5-bromotryptophan using a one-pot approach on preparative scale. (A.) PyrH-catalyzed halogenation of the reaction mixture yielding C5-brominated L-amino acid was directly employed in stereoinversion without intermediary purification. (B.) RP-HPLC diagram of the reaction progress reveals progressive formation of D-enantiomer as analyzed via Marfey’s derivatization. (C.) HPLC results (Marfey’s test) illustrate that D-5-Br-Trp was isolated in good enantiomeric purity. (D.) LC-ESI-MS data of Marfey’s derivatives confirm that both elution signals in C. correspond to diastereomers formed with FDAA.

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Nishizawa et al. an overall preference for halogenated L-Trp derivatives was observed.47 It can be presumed from these results that a distinct motif within the active site is responsible for this discrimination. This feature was exploited to establish a colorimetric halogenase assay based on RebO in combination with a peroxidase. The remarkable preference for a halogen motif present in the Trp scaffold was harnessed to quantify turnover by Trp 7-halogenase through H2O2-coupled dye formation suitable for analysis in a microtiter plate. This tool adds to the small set of halogenase assays currently available by offering a simple and rapid colorimetric high-throughput screening to monitor enzymatic halogenation in crude lysate. We anticipate that the transfer to a directed evolution campaign will be viable, for example in order to engineer catalytic efficiency or lifetime of Trp halogenases. As RebO additionally distinguishes between regioisomers of halotryptophans, detection of altered regioselectivity in Trp halogenation should be feasible with this assay. In conjunction with the recently published fluorogenic cross-coupling assay,30 both screenings significantly support protein engineering to exploit halogenases as robust biocatalysts. Dynamic stereoinversion using RebO with a concomitant reducing agent gives access to substituted D-Trp derivatives with excellent enantioselectivity when starting from the respective Lenantiomer. Viability of the synthetic methodology is exemplified for a representative set of halotryptophans. Combining enzymatic halogenation and stereoinversion to a one-pot biocatalytic cascade proves efficient to obtain the respective D-enantiomers. Notably, on preparative scale the undesired hydrolysis / carbonyl reduction pathway forming -hydroxy acids becomes less relevant that further enhances the synthetic value of the cascade. Regarding the importance of enantiomerically pure building blocks in synthetic chemistry, this biotechnological approach is of particular interest in the fields of peptide synthesis and drug development, as a wide set of D-configured Trp analogues will become accessible. To the best of our knowledge, this is the first example of combining biocatalytic halogenation with dynamic stereoinversion to synthetically access non-natural D-Trp derivatives. The biocatalytic route showcases how Trp halogenases as tools for selective C-H functionalization can be combined with another biocatalyst in an enzyme cascade. In future, immense efforts on enzyme and reaction engineering will guide further development of nature’s elaborate halogenation arsenal towards fine chemical synthesis by making use of multistep enzyme-based processes.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Supporting figures, tables, analytical data, spectra and detailed experimental procedures (PDF).

AUTHOR INFORMATION Corresponding Author

*Prof. Dr. Norbert Sewald, Organic and Bioorganic Chemistry, Department of Chemistry, Bielefeld University, PO Box 100131, 33501 Bielefeld, Germany, [email protected]. Author Contributions

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C. S. and N. S. conceived the research project. N.S. supervised the project. C. S. designed and carried out experiments presented herein, performed molecular biology, enzyme assays as well as optimization studies and interpreted the results. I. K. carried out synthesis of 5-Cl- and 7-F-Trp. C. S. and N. S. wrote the manuscript. All authors have given approval to the final version of the manuscript.

ABBREVIATIONS AAO, amino acid oxidase; 4-AAP, 4-aminoantipyrine; Br-Trp, bromotryptophan; Cl-Trp, chlorotryptophan; DKR, dynamic kinetic resolution; FDAA, 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide; HPLC, high-performance liquid chromatography, LC-MS, liquid chromatography-mass spectrometry; Ph(4-OH)SO3H, 4phenolsulfonic acid; Trp, L-tryptophan.

ACKNOWLEDGMENT The authors gratefully acknowledge experimental support by Anke Nieß and Monja Jochmann.

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TOC Graphics Halogenase CO2H

CO2H X

NH2 N H

L-tryptophan

NH2 N H

CO2H X

CO2H

NH 2

X

NH2

N H

L-Amino

acid oxidase

N H

H3 N⋅BH3

One-pot cascade ee (D) 90-99%

CO2H X

NH N H

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