The Broad Aryl Acid Specificity of the Amide Bond Synthetase McbA Suggests Potential for the Biocatalytic Synthesis of Amides

Abstract Amide bond formation is one of the most important reactions in pharmaceutical synthetic chemistry. The development of sustainable methods for amide bond formation, including those that are catalyzed by enzymes, is therefore of significant interest. The ATP‐dependent amide bond synthetase (ABS) enzyme McbA, from Marinactinospora thermotolerans, catalyzes the formation of amides as part of the biosynthetic pathway towards the marinacarboline secondary metabolites. The reaction proceeds via an adenylate intermediate, with both adenylation and amidation steps catalyzed within one active site. In this study, McbA was applied to the synthesis of pharmaceutical‐type amides from a range of aryl carboxylic acids with partner amines provided at 1–5 molar equivalents. The structure of McbA revealed the structural determinants of aryl acid substrate tolerance and differences in conformation associated with the two half reactions catalyzed. The catalytic performance of McbA, coupled with the structure, suggest that this and other ABS enzymes may be engineered for applications in the sustainable synthesis of pharmaceutically relevant (chiral) amides.


General information
Reagents were purchased from Sigma-Aldrich, Alfa Aesar, Acros Organics or Fluorochem and used as received unless otherwise stated. Petroleum ether refers to the fraction of petroleum that is collected at 40-60 °C. Reactions requiring anhydrous conditions were carried out using Schlenk techniques (high vacuum, liquid nitrogen trap on a standard inhouse built dual line). Room temperature upper and lower limits are stated as 13-25 °C, but typically 21 °C was recorded. Brine refers to a saturated aqueous solution of NaCl.
Thin layer chromatography (TLC) was carried out using Merck 5554 aluminium backed silica plates (silica gel 60 F254) and spots were visualized using UV light (at 254 nm). Where necessary, plates were stained and heated with one of potassium permanganate, anisaldehyde or vanillin as appropriate. Retention factors (R f ) are reported along with the solvent system used, in parentheses. Flash column chromatography was performed according to the method reported by W. C. Still et al 1 using Merck 60 silica gel (particle size 40-63 μm) and a solvent system as reported in the text.
NMR spectra were obtained in the solvent indicated, using a JEOL ECX400 or JEOL ECS400 spectrometer (400MHz, 101 MHz and 162 MHz for 1 H, 13 1 H and 13 C, respectively). Spectra were typically run at a temperature of 298 K. All 13 C NMR spectra were obtained with 1 H decoupling. NMR spectra were processed using MestReNova software (Version 11.0.2-18153, released October 2016). The spectra given below were saved as .emf or .pdf files in MestReNova and inserted directly into a Microsoft Word Document. For the 1 H NMR spectra the resolution varies from 0.15 to 0.5 Hz; the coupling constants have been quoted to ± 0.5 Hz in all cases for consistency. 1 H and 13 C NMR chemical shifts are quoted to 2 decimal places.
Infrared spectra were obtained using a Bruker ALPHA-Platinum FTIR Spectrometer with a platinum-diamond ATR sampling module. MS spectra were measured using a Bruker Daltronics micrOTOF MS, Agilent series 1200LC with electrospray ionisation (ESI and APCI) or on a Thermo LCQ using electrospray ionisation, with <5 ppm error recorded for all HRMS samples. Mass spectral data is quoted as the m/z and mass to charge ratios (m/z) are reported in Daltons.
Melting points were recorded using a Stuart digital SMP3 machine.
After gel analysis of the PCR, the relevant band was eluted using a PCR Cleanup kit ® (Qiagen) and the gene cloned into the pETYSBLIC-3C vector using a ligation-independent cloning (LIC) procedure which has been described previously. 2 Recombinant plasmid containing the McbA gene were then used to transform E. coli XL10-Gold Ultracompetent cells (Novagen). Small cultures of transformants yielded plasmids using standard miniprep techniques that were submitted for sequencing to confirm the integrity of the gene. Recombinant vectors were then used to transform E. coli BL21 (DE3) cells, using 30 g mL -1 kanamycin as antibiotic marker on Luria-Bertani (LB) agar. One colony produced overnight was then used to inoculate 5 mL of LB broth containing 30 g mL -1 kanamycin, which was then grown for 18 h at 37°C with shaking at 180 r.p.m. This starter culture was then used to inoculate a 500 mL culture of LB broth containing 30 g mL -1 kanamycin and the cultures was grown until the optical density (OD 600 ) had reached 0.6. Expression of the corresponding protein were then induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), after which the culture was incubated at 16 °C in an orbital shaker at 180 r.p.m. for approximately 18 h. Cells were then harvested by centrifugation for 15 min at 4225 g in a Sorvall GS3 rotor in a Sorvall RC5B Plus centrifuge and subsequently resuspended in 50 mL 50 mM KP i buffer pH 7.5 containing 5 % glycerol (w/ v) and 300 mM NaCl. Cells were sonicated for 3 x 30 s bursts at 4 °C with 1 min intervals and the soluble and insoluble fractions separated by centrifugation for 30 min at 26,892 g in a Sorvall SS34 rotor. The clear supernatants were loaded onto a 5 mL His-Trap™ Chelating HP nickel column. After washing with 10 column volumes of buffer 50 mM KP i buffer pH 7.5 containing 5 % glycerol (w/v) and 300 mM NaCl, McbA was eluted with a gradient of 0-500 mM imidazole over 20 column volumes. Column fractions containing the protein, as determined by SDS-PAGE analysis, were pooled and then concentrated using a 30 kDa cut-off Centricon® filter membrane. The concentrated enzymes were then loaded onto a pre-equilibrated S75 Superdex™ 16/60 gel filtration column, and eluted with 120 mL of the buffer, at a flow rate of 1 mL min -1 . Fractions containing pure McbA, as determined by SDS-PAGE analysis were pooled and stored at 4 °C.

General procedure for the synthesis of the -carboline ester derivates 20 and 21. 2
Acetone (66 L, 0.90 mmol) or acetophenone (54 L, 0.458 mmol) and iodine (115 mg, 0.458 mmol) were added to 2 mL of DMSO and the resulting solution was heated at 90 °C for 1 h. Tryptophan methyl ester (116 mg, 0.458 mmol) was added and the solution was stirred at same temperature for 2-3 h till completion of reaction (monitored by TLC, EtOAc:hexane 1:2). The reaction mixture was then cooled to room temperature followed by addition of water (25 mL) and extracted with EtOAc (2 x 25 mL). The extract was washed with 10% Na 2 S 2 O 3 , dried over Na 2 SO 4 , filtered and the solvent evaporated under reduced pressure. The residue was purified by chromatography on a silica gel using acetone: DCM (0.1: 9.9) as eluent to give the desired product (20, 21) as a yellow solid. 20 (72% yield isolated), 21 (56% yield isolated). Compounds 20 and 21 exhibited physical and spectral properties in accordance with those reported. 3

Synthesis of racemic 22 by reduction of the ketone-derived 20
NaBH 4 (34 mg, 0.90 mmol) was added carefully to a solution of -carboline derivative 20 (185 mg, 0.69 mmol) in a mixture of methanol and CHCl 2 (10 mL, 1:1) in an ice bath. The reaction was stirred at 0 °C for 1 h and then at room temperature for 2-3 h until completion of the reaction (monitored by TLC, EtOAc, 5% v.v -1 MeOH). The reaction was quenched with water (5 mL) and extracted with CHCl 2 (2 x 20 mL). The organic phases were dried over Na 2 SO 4 , filtered and the solvent evaporated under reduced pressure obtaining, after purification by flask chromatography, the desired product rac-22 as pale yellow solid (87% isolated yield). Compound rac-22 4 exhibited physical and spectral properties in accordance with those reported.

Synthesis of 23 by Pictet-Spengler condensation and KMnO 4 oxidation 5
Tryptophan methyl ester (0.5 g; 2.1 mmol), and propionaldehyde (1.2 equiv., 180 L, 2.5 mmol) were dissolved in DCM (20 mL) and trifluoro acetic acid (1.3 equiv., 190 L, 2.5 mmol) was added at room temperature. The reaction was stirred overnight and was then quenched with saturated sodium bi-carbonate and extracted with DCM (2 x 20 mL). The organic phases were dried over Na 2 SO 4 sat , filtered and the solvent evaporated under reduced pressure. The crude product was used in the next step without further purification. 6 KMnO 4 (2 equiv., 660 mg, 4.2 mmol) was added carefully to a cooled stirring solution of the previous crude reaction product in DMF (10 mL) and then the resulting mixture was stirred for 24 h at room temperature. The suspension was filtered over Celite and concentrated under vacuum, obtaining after purification by flask chromatography the desired product 23 as a pale yellow solid (67 % yield isolated). Compound 23 6 exhibited physical and spectral properties in accordance with those reported.

General procedure for the synthesis of acids 6, 8-11. 7
A solution of ester derivated--carboline 20-24 (113-165 mg, 0.5 mmol) in a mixture of NaOH 2M and methanol (10 mL, 2:1) was stirred at 80 °C for 1-2 h until completion of reaction. After completion, the methanol was eliminated under reduced pressure and then 2M HCl was added, adjusting the pH to 5-6. At this point, a yellow solid precipitated and after cooling the flask in an ice bath the solid was filtered, and then washed with cold water (5 mL) and diethyl ether (2 x 5 mL), obtaining the desired products as pale yellow solids (47-74% isolated yield). Compounds 6 8 , 8 9 , 9 8 , 10 10 and 11 11 exhibited physical and spectral properties in accordance with those reported.

General procedure for the synthesis of the final -carboline amide derivatives (6, 8-11)a-d Protocol A)
The ester derivative 20-24 (50 mg, 0.2 mmol) was treated with an excess of the respective amine a-d (20 equiv.) under neat conditions (the tryptamine b was dissolved in DMSO) at 90 °C overnight. After completion of reaction (monitored by TLC) the reaction was cooled to room temperature and extracted with EtOAc (3 x 5 mL) and water (5 mL). The organic extract was dried over MgSO 4 and concentrated under vacuum. The crude reaction product was purified by flash chromatography affording the final amide derivatives. 12 The acid derivative 6, 8-11 (25 mg, 0.1 mmol), amine a-d (0.11 mmol, 1.1 equiv.), pyridine (0.5 mmol, 5 equiv.) were added to dry DMF (1 mL). The mixture was cooled to -10 °C and T3P solution (50 wt% in EtOAc, 0.2 mmol, 2 equiv.) was added carefully. The solution was stirred at -10 °C for 10 min and then 1 h at room temperature. The reaction was then cooled to 0 °C, quenched with water (4 mL) and stirred for 5 min. HCl 0.2 N (2 mL) was then added and the reaction was extracted with EtOAc (3 x 10 mL). The organic phases were collected and washed with HCl 0.2 N, NaOH 0.2 N and brine, dried over Na 2 SO 4 sat. , filtered and evaporated under reduced pressure. The crude reaction product was then purified by flash chromatography, affording the final amide derivatives. Standards 12a, 13a, 14a, 15a, 16a, 17a,  18a and 19a The acid derivative 18 (100 mg, 0.58 mmol, 1eq.), 2-Phenylethylamine a (81 µL, 0.64 mmol, 1.1 equiv.), and Et 3 N (404 µL, 2.9 mmol, 5 equiv.), were added to DMF (10 mL). T3P solution (50 wt% in EtOAc, 345 µL, 1.2 mmol, 2 equiv.) was added carefully. The solution was stirred at room temperature for 24 h. The reaction was then quenched with water (5 mL) and stirred for 5 min. HCl 0.2 N (10 mL) was then added and the reaction was extracted with EtOAc (3 x 20 mL). The organic phases were collected and washed with HCl 0.2 N, NaOH 0.2 N and brine, dried over MgSO 4 sat., filtered and evaporated under reduced pressure. The amide products were then re-crystallised with Et 2 O to afford the final amide derivatives.
The scale up of 17a was carried out by adding a further 1 mg mL -1 McbA and 1 eq. ATP to the reaction after 24h. The reaction was then incubated at 37 °C for a further 24h.
Method I: 40% of B to 100% B in 5 min, 10 min at 100% B. 35 °C, flow 1 mL min -1 . Method II: 25% of B to 30% B in 5 min, 30% of B to 100% B in 1 min, 10 min at 100% B. 35 °C, flow 1 mL min -1 . Retention Times for acids and amide products are given in Table S1.  Figure S3. Chiral HPLC Analysis of product 6d using a Chiralpak ID4 HPLC column using 20% isopropanol: 80% hexane as eluant with a flow rate of 1 mL min -1 . A: Racemic standard 6d; Synthesis of a standard product of (S)-configuration from 6 and commercially sourced (S)-d, followed by chiral HPLC analysis confirmed the retention times of (S)-and (R)-6d as approximately 35.0 and 40.0 min respectively. B; product of reaction catalysed by McbA with 96% e.e. These crystals were washed in a cryoprotectant solution of 15% ethylene glycol in the mother the flash-cooled in liquid nitrogen prior to analysis at the Diamond Light Source.

Data collection, structure solution and refinement
Data were collected on beamline i03 at the Diamond Light Source and were processed and integrated using XDS 19 and scaled using SCALA 20 within the Xia2 21 processing system. Data collection statistics can be found in Table S2. The structure was solved with MOLREP 22 using the structure of protein acetyltransferase RpPAT from Rhodopseudomonas palustris (28% sequence identity, PDB code 4GXQ) 23 as the molecular replacement model. The structures were refined using iterative cycles of the programmes COOT 24 and REFMAC5. 25 After building of the protein backbone, side chains and water molecules, residual density was present in the omit map all five active sites. This could be modelled in four subunits, A-D, each in the 'adenylation' conformation as AMP and -carboline acid 6. In subunit E, in the 'amidation' conformation, density for 6 was again clearly visible but the AMP peak lacked density for the phosphate group. Coordinate and library files for 6 were prepared using ACEDRG. 26 Refinement statistics for all structures can be found in Table S1. Coordinates and structure factor files have been deposited in the Protein Data Bank (PDB) with the accession code 6H1B.