Chemoenzymatic Synthesis of Indole-Containing Acyloin Derivatives

Indole-containing acyloins are either key intermediates of many antimicrobial/antiviral natural products or building blocks in the synthesis of biologically active molecules. As such, access to structurally diverse indole-containing acyloins has attracted considerable attention. In this report, we present a pilot study of using biotransformation to provide acyloins that contain various indole substituents. The biotransformation system contains the tryptophan synthase standalone β-subunit variant, PfTrpB6, generated from directed evolution in the literature; a commercially available L-amino acid oxidase (LAAO); and the thiamine-diphosphate (ThDP)-dependent enzyme NzsH, encoded in the biosynthetic gene cluster (nzs) of the bacterial carbazole alkaloid natural product named neocarazostatin A. The utilization of the first two enzymes, the PfTrpB variant and LAAO, is designed to provide structurally diverse indole 3-pyruvate derivatives as donor substrates for NzsH-catalysed biotransformation to provide acyloin derivatives. Our results demonstrate that NzsH displays a considerable substrate profile toward donor substrates for production of acyloins with different indole ring systems, suggesting that NzsH could be further explored as a potential biocatalyst via directed evolution to improve the catalytic efficiency in the future.

Thiamine diphosphate (ThDP)-dependent enzymes are widely found in the biological systems and have been demonstrated to be involved in diverse biotransformation, including C-C, C-N, C-S, and C-O bond cleavage and formation [5]. The general mechanism underlying all these reactions is that the enzyme's active site-mediated dissociation of the C2-H proton from the thiazolium ring of ThDP will generate the C2 anion ylid which is covalently bound to the donor substrate ( Figure 1B). Subsequent decarboxylation results in the ThDP-bound enolate intermediate with nucleophilic reactivity which readily react with electrophilic acceptors, such as aldehydes, ketones or α-ketoacids to form the α-hydroxyl ketone (an acyloin) [6].
In many cases, pyruvate is the donor in the ThDP-dependent reactions. However, some of ThDP-dependent enzymes utilise a range of α-ketoacids to initiate carboligation reactions. For example, the ThDP dependent enzyme, ScyA, was found to couple p-hydroxyphenylpyruvate 6 as the donor and indole-3-pyruvate 7 as the acceptor to afford the β-ketoacid product 8, the key intermediate during the biosynthesis of scytonemin 9, a In many cases, pyruvate is the donor in the ThDP-dependent reactions. However, some of ThDP-dependent enzymes utilise a range of α-ketoacids to initiate carboligation reactions. For example, the ThDP dependent enzyme, ScyA, was found to couple phydroxyphenylpyruvate 6 as the donor and indole-3-pyruvate 7 as the acceptor to afford the β-ketoacid product 8, the key intermediate during the biosynthesis of scytonemin 9, a pigment produced by cyanobacteria [7]. During our biosynthetic studies of the bacterial carbazole alkaloid, neocarazostatin A 10 [8][9][10][11], we found that the ThDP-dependent enzyme NzsH catalyses an unusual carboligation between indole-3-pyruvate 7 as the donor and pyruvate as the acceptor to generate an indole-containing acyloin intermediate 11 [9], followed by further modifications to finally decorate 10 [10,11], consistent with a parallel study in the biosynthetic pathway of the bacterial carbazole analogue, carbazomycin [12]. NzsH only accepts 2-oxobutyrate, an analogue of pyruvate, as the acceptor, unlike other ThDP enzymes that normally display a considerable range of aldehyde and ketones [9]. Phylogenetic analysis demonstrated that NzsH formed a Here, we report a pilot study of new three-enzyme coupled biotransformation to access indole-containing acyloin derivatives with structural diversity. The reaction mixture includes an engineered tryptophane synthase β-subunit, a commercially available L-amino acid oxidase (LAAO), and NzsH. This coupled reaction is initiated from commercially available indole or indole derivatives with L-Ser catalysed by the engineered tryptophane synthetase to provide tryptophane derivatives, followed by addition of LAAO for oxidation reactions to give the corresponding indole-3-pyruvate analogues. Inclusion of NzsH in the reactions finally allow access of a new range of indole-containing acyloin derivatives. This newly introduced functionalised indole-3-pyruvate analogues substantially broaden the donor substrate range of the ThDP-dependent NzsH enzyme, thus holding potential to generate new antiviral/antimicrobial acyloins with different indole ring systems.

Results and Discussion
To generate structurally diverse indole-containing acyloin derivatives, indole-3-pyruvate derivatives with structural diversity to evaluate the substrate plasticity of NzsH is key. However, most of indole-3-pyruvate derivatives are not commercially available. In nature, indole-3-pyruvate is directly descended from L-tryptophane via deamination reactions. There has been a considerable interest in the application of tryptophane synthase to generate structurally diverse tryptophane derivatives due to its importance as a key building block for many bioactive molecules [14]. Tryptophan synthase is a heterodimeric complex consisting of two subunits, TrpA (α-subunit) catalysing the cleavage of indole glycerol phosphate to indole, and TrpB (β-subunit) mediating the coupling between indole and L-Ser to provide L-Tryptophan using pyridoxal phosphate (PLP) as cofactors (Supplementary Scheme S1A). The activities of both subunits are required for wild type tryptophan synthases, although only the reactivity of TrpB, coupling L-Ser and indole is necessary and desirable for L-tryptophan synthesis. Recent studies reported an engineered β-subunit of tryptophan synthase (Pf TrpB) from Pyrococcus furiosus as standalone function that restore the catalytic efficiency and surpass the activity of the native complex (Supplementary Scheme S1B) [15][16][17]. One variant (Pf TrpB 6 ) with six amino acid site mutations displayed the best kinetics towards L-Ser and indole and a wide range of indole derivatives. To this end, we synthesized the gene coding Pf TrpB 6 for overexpression in E. coli. Pf TrpB 6 was expressed as an N-terminal pHis 6 recombinant protein and was purified to near homogenesis by Ni-NTA chromatography, giving estimated molecular weight of~45 kDa as previously reported (Supplementary Figure S1A). Incubation of Pf TrpB 6 with indole, L-Ser and PLP resulted in the accumulation of L-tryptophan 13 as evidenced in our LC-MS and tandem MS analyses ( Figure 2 and Supplementary Figure S2).
To further in situ generate indole-3-pyruvate from L-tryptophan, we decided to use commercially available L-amino acid oxidase (LAAO) from snake venom (Sigma Aldrich catalogue number: A5147) because this enzyme displays broad substrate tolerance toward amino acids and does not require any additional cofactors (Supplementary Scheme S1C). It also can efficiently shift the reaction equilibrium during the coupled reaction as observed in our previous report when the fluorination enzyme, FlA, from Streptomyces cattleya, was investigated for a chlorination reaction [18]. Addition of LAAO into the aforementioned enzyme system led to the formation of indole-3-pyruvate 14 as evidenced in our MS and tandem MS analysis ( Figure 2 and Supplementary Figure S3). We next overexpressed the synthetic construct containing the gene nzsH in E coli BL-21 (DE3). NzsH was expressed as an N-terminal pHis 6 recombinant protein and was purified to near homogenesis by Ni-NTA chromatography, giving estimated molecular weight of~64 kDa as previously reported [9] (Supplementary Figure S1B). Inclusion of NzsH into the coupled reaction of Pf TrpB 6 and LAAO together with TPP and Mg 2+ resulted in the formation of the indolecontaining acyloin scaffold. However, this molecule is not stable and is subjected to facile decarboxylation in the LC-MS analysis [9]. We used NaBH 4 to reduce the β-ketone of the molecule to generate two diastereomeric diols, 15 and 15 , as evidenced in our LC-MS and tandem MS analyses ( Figure 2 and Supplementary Figure S4). as previously reported [9] (Supplementary Figure S1B). Inclusion of NzsH into the coupled reaction of PfTrpB 6 and LAAO together with TPP and Mg 2+ resulted in the formation of the indole-containing acyloin scaffold. However, this molecule is not stable and is subjected to facile decarboxylation in the LC-MS analysis [9]. We used NaBH4 to reduce the β-ketone of the molecule to generate two diastereomeric diols, 15 and 15′, as evidenced in our LC-MS and tandem MS analyses (Figure 2 and Supplementary Figure  S4). 2 4 min Encouraged by the results of this three-enzyme coupling system, we then acquired four commercially available indole derivatives including 4-fluoroindole 16, 5-fluoroindole 17, 4-bromoindole 18 and 6-hydroxyl-indole 19 (Table 1). Our LC-MS and tandem MS analysis demonstrated that all of these indole derivatives can be efficiently transformed into the corresponding indole-3-pyruvate derivatives, 26-29 as well as the corresponding diols, 31-35, (Table 1), in the corresponding coupled chemoenzymatic systems. Encouraged by the results of this three-enzyme coupling system, we then acquired four commercially available indole derivatives including 4-fluoroindole 16, 5-fluoroindole 17, 4-bromoindole 18 and 6-hydroxyl-indole 19 (Table 1). Our LC-MS and tandem MS analysis demonstrated that all of these indole derivatives can be efficiently transformed into the corresponding indole-3-pyruvate derivatives, 26-29 as well as the corresponding diols, 31-35, (Table 1), in the corresponding coupled chemoenzymatic systems.
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A Next, we acquired three commercially available indole derivatives, 2-methyl-indole 36, indoline 37, and indazole 38 that contain modified indole rings ( Figure 4). Consistent with the previous report [15][16][17], the biotransformation catalysed by Pf TrpB 6 gave the corresponding tryptophan derivatives, 39-41, respectively. In all three cases, addition of LAAO provided the corresponding indole-pyruvate derivatives, 42-44, respectively, further demonstrating the considerable substrate promiscuity of LAAO. To our surprise, these indole-pyruvate derivatives can be further utilized as the donor substrates in the NzsH-mediated enzymatic reaction to provide the final indole-containing acyloins, 45-47, respectively, as observed in our LC-MS and tandem MS analyses of the corresponding reduced forms (Supplementary Figure S20-S28). Taken together, our studies demonstrated that NzsH, unlike its activities towards acceptor substrates, displays considerable substrate tolerances toward its donor substrates. However, further studies are required to improve the reactivities of NzsH enzyme toward the unnatural indole-3-pyruvate derivatives. This can be achieved via either identification of new NzsH homologues through comparative genomics or directed evolution to provide an engineered NzsH with the aim of finding a suitable biocatalyst with better kinetics in order to efficiently generate structurally diverse indole-containing acyloin derivatives. Table 1. Structures of indole derivatives and related indole-containing products generated from the step-wise coupled reaction. Addition of NzsH together with ThDP and Mg 2+ provided the corresponding indole-containing acyloin derivatives, 31-35 (Table 1), as evidenced in our LC-MS and tandem MS analyses (Supplementary Figure S5-S19).

Indole Derivatives Tryptophan Derivatives Indole-3-Pyruvate Derivatives Acyloin Derivatives
ules 2023, 28, x FOR PEER REVIEW 5 of 10 e 1. Structures of indole derivatives and related indole-containing products generated from the step-wise coupled reaction. tion of NzsH together with ThDP and Mg 2+ provided the corresponding indole-containing acyloin derivatives, 31-35 (Table 1)

HO
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A

HO
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A Table 1. Structures of indole derivatives and related indole-containing products generated from the step-wise coupled reaction. Addition of NzsH together with ThDP and Mg 2+ provided the corresponding indole-containing acyloin derivatives, 31-35 (Table 1)

HO
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A Table 1. Structures of indole derivatives and related indole-containing products generated from the step-wise coupled reaction. Addition of NzsH together with ThDP and Mg 2+ provided the corresponding indole-containing acyloin derivatives, 31-35 (Table 1)

HO
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A    We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A  We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A

HO
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A

HO
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A  We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A Table 1. Structures of indole derivatives and related indole-containing products generated from the step-wise coupled reaction. Addition of NzsH together with ThDP and Mg 2+ provided the corresponding indole-containing acyloin derivatives, 31-35 (Table 1)

HO
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A

HO
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A

HO
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A  We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A  We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A

HO
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A

HO
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A Table 1. Structures of indole derivatives and related indole-containing products generated from the step-wise coupled reaction. Addition of NzsH together with ThDP and Mg 2+ provided the corresponding indole-containing acyloin derivatives, 31-35 (Table 1)

HO
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A Table 1. Structures of indole derivatives and related indole-containing products generated from the step-wise coupled reaction. Addition of NzsH together with ThDP and Mg 2+ provided the corresponding indole-containing acyloin derivatives, 31-35 (Table 1)

HO
We also monitored the biotransformation of the formation of fluorinated indolecontaining acyloins in 19 F NMR. As shown in Figure 3A Figure 3D,H), respectively.
In conclusion, we developed a coupled biotransformation system including a genetically modified β-subunit of tryptophan synthase (Pf TrpB 6 ) from Pyrococcus furiosus, and a commercially available L-amino acid oxidase (LAAO) from snake venom and the ThDP-dependent NzsH enzyme from the biosynthetic pathway of neocarazostatins to access structurally diverse (aza)indole-containing acyloin derivatives. The combination of Pf TrpB 6 and LAAO allowed generation of (aza)indole-3-pyruvate derivatives, starting from commercially available (aza)indole derivatives, which were subsequently accessed by NzsH-mediated reaction. Our results demonstrated that NzsH displays considerably good substrate tolerance, suggesting that this three-enzyme coupled system holds potential as a means of new biotransformation to produce indole-containing acyloins, important precursors of many biologically relevant molecules.   [19]. They display distinct physiochemical properties, aqueous solubility and total polar surface area due to the presence of extra nitrogen atom within the six membered ring in comparison to indoles. As such, azaindole-containing building blocks have attracted medicinal chemists to derive a number of pharmacological agents [20]. Among these, 7-azaindole 20 has appeared in a plethora of biologically active molecules [21], such as potent anticancer natural products, variolins [22] and their synthetic derivatives, meriolins [23]. To this end, we tested commercially available 7-azaindole 20 as the starting material in our biotransformation system. Again, the formation of aza-tryptophan 25, azaindole-3-pyruvate 30 and azaindole-containing acyloin 35 were observed in the corresponding PfTrpB 6 , PfTrpB 6 + LAAO and PfTrpB 6 + LAAO +NzsH systems, respectively, as evidenced in our LC-MS and MS tandem analyses (Table 1 and Supplementary Figure S9,S14.S19). Taken together, NzsH display considerable substrate tolerance toward modified benzene rings of indole moieties when indole-3-pyruvate derivatives were used as substrates.
Next, we acquired three commercially available indole derivatives, 2-methyl-indole 36, indoline 37, and indazole 38 that contain modified indole rings ( Figure 4). Consistent with the previous report [15][16][17], the biotransformation catalysed by PfTrpB 6 gave the corresponding tryptophan derivatives, 39-41, respectively. In all three cases, addition of LAAO provided the corresponding indole-pyruvate derivatives, 42-44, respectively, further demonstrating the considerable substrate promiscuity of LAAO. To our surprise, these indole-pyruvate derivatives can be further utilized as the donor substrates in the NzsH-mediated enzymatic reaction to provide the final indole-containing acyloins, 45-47, respectively, as observed in our LC-MS and tandem MS analyses of the corresponding reduced forms (Supplementary Figure S20-S28). Taken together, our studies demonstrated that NzsH, unlike its activities towards acceptor substrates, displays considerable substrate tolerances toward its donor substrates. However, further studies are required to improve the reactivities of NzsH enzyme toward the unnatural indole-3pyruvate derivatives. This can be achieved via either identification of new NzsH homologues through comparative genomics or directed evolution to provide an  [19]. They display distinct physiochemical properties, aqueous solubility and total polar surface area due to the presence of extra nitrogen atom within the six membered ring in comparison to indoles. As such, azaindole-containing building blocks have attracted medicinal chemists to derive a number of pharmacological agents [20]. Among these, 7-azaindole 20 has appeared in a plethora of biologically active molecules [21], such as potent anticancer natural products, variolins [22] and their synthetic derivatives, meriolins [23]. To this end, we tested commercially available 7-azaindole 20 as the starting material in our biotransformation system. Again, the formation of aza-tryptophan 25, azaindole-3-pyruvate 30 and azaindole-containing acyloin 35 were observed in the corresponding Pf TrpB 6 , Pf TrpB 6 + LAAO and Pf TrpB 6 + LAAO +NzsH systems, respectively, as evidenced in our LC-MS and MS tandem analyses (Table 1 and Supplementary Figures S9, S14 and S19). Taken together, NzsH display considerable substrate tolerance toward modified benzene rings of indole moieties when indole-3-pyruvate derivatives were used as substrates.  In conclusion, we developed a coupled biotransformation system including a genetically modified β-subunit of tryptophan synthase (PfTrpB 6 ) from Pyrococcus furiosus, and a commercially available L-amino acid oxidase (LAAO) from snake venom and the ThDP-dependent NzsH enzyme from the biosynthetic pathway of neocarazostatins to access structurally diverse (aza)indole-containing acyloin derivatives. The combination of PfTrpB 6 and LAAO allowed generation of (aza)indole-3-pyruvate derivatives, starting from commercially available (aza)indole derivatives, which were subsequently accessed

General Chemicals, Reagents, and Analytical Methods
All starting materials and reagents were bought from commercial sources and used as received. All biochemical reactions apart from where noted were carried out in triplicate. Before every set of measurements, triplicate control reactions were performed to ensure that the assay were functioning correctly. All flash column chromatography was carried out using silica purchased from Sigma Aldrich using the solvent system noted. 19 F NMR spectra were recorded at 298 K on Bruker Avance III 400 using CFCl 3 as an external reference. Chemical shifts are reported in parts per million (ppm) and coupling constants (J) are reported in Hertz (Hz).
Enzymatic assays were analyzed on a Bruker MaXis II ESI-Q-TOF-MS connected to an Agilent 1290 Infinity II UHPLC fitted with a Phenomenex Kinetex XB-C18 (2.

General Methods of Protein Expression and Purification
The synthetic constructs encoding Pf TrpB 6 and NzsH proteins were purchased from Genscript Ltd. and were individually transformed into E. coli BL21 (DE3). Single colonies from each transformation were grown overnight in LB media (5 mL) containing kanamycin (50 µg/mL) and chloramphenicol (25 µg/mL). The overnight culture was transferred to fresh LB medium (500 mL) supplemented with kanamycin (50 µg/mL) and cultivated at 37 • C until the cell density reached an OD 600 of 0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce protein expression. Cells were grown for 16-20 h at 16 • C and then harvested by centrifugation at 4 • C. The cells pellets were resuspended in ice-cold lysis buffer (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0), and further disrupted by Ultrasonic Homogenizer JY92-IIN. Then, the supernatant of cell debris was loaded onto Ni-NTA-affinity column. Bound proteins were eluted with the same Tris-HCl buffer containing different concentrations of imidazole. The desired elution fractions were combined and concentrated using a Centrifugal Filter Unit (Millipore). The final yields of Pf TrpB 6 and NzsH were estimated to be 10 mg/100 mL culture and 5 mg/100 mL culture, respectively.

Biochemical Reactions
A sample of Pf TrpB 6 (20 µM) was incubated with indole or indole derivatives (1 mM), L-Ser (1 mM) and PLP (1 mM) in phosphate buffer (50 mM, pH 7.5) to the final volume of 100 µL at 28 • C for 3 h and then quenched by addition of 100 µL of acetonitrile. The mixture was centrifuged at 13,000 rpm for 10 min to remove protein precipitates.
For the production of indole-3-pyruvate derivatives, a sample of Pf TrpB 6 (20 µM) was incubated with indole or indole derivatives (1 mM), L-Ser (1 mM) and PLP (1 mM) in phosphate buffer (50 mM, pH 7.5) to a final volume of 50 µL at 28 • C for 3 h. To this mixture were added LAAO (20 µM) to a final volume of 100 µL at 28 • C for another 1 h. The reaction mix was then quenched by addition of 100 µL of acetonitrile. The mixture was centrifuged at 13,000 rpm for 10 min to remove protein precipitates.
For the production of indole-containing acyloin derivatives, a sample of Pf TrpB 6 (20 µM) was incubated with indole or indole derivatives (1 mM), L-Ser (1 mM) and PLP (1 mM) in phosphate buffer (50 mM, pH 7.5) to a final volume of 100 µL at 28 • C for 3 h. To this mixture were added LAAO (20 µM) to a volume of 100 µL at 28 • C for another 1 h. Inclusion of NzsH (25 µM), TPP (1 mM) and Mg 2+ (1 mM) into the mixture was perform to the final volume of 100 µL at 28 • C for 3 h. Because of the instability of acyloins, overdose NaBH 4 was treated to the three-enzyme systems. Finally, the reaction mix was quenched by addition of 100 µL of acetonitrile. The mixture was centrifuged at 13,000 rpm for 10 min to remove protein precipitates.
All the supernatants from the above reaction systems were then analyzed by Bruker MaXis II QTOF in tandem with an Agilent 1290 Infinity UHPLC. Samples were separated on a Phenomenex Kinetex XB-C18 (2.6 µM, 100 × 2.1 mm) column with a mobile phase of 5% ACN + 0.1% formic acid to 100% ACN + 0.1% formic acid in 15 min.