Cation–π Interactions Contribute to Substrate Recognition in γ‐Butyrobetaine Hydroxylase Catalysis

Abstract γ‐Butyrobetaine hydroxylase (BBOX) is a non‐heme FeII‐ and 2‐oxoglutarate‐dependent oxygenase that catalyzes the stereoselective hydroxylation of an unactivated C−H bond of γ‐butyrobetaine (γBB) in the final step of carnitine biosynthesis. BBOX contains an aromatic cage for the recognition of the positively charged trimethylammonium group of the γBB substrate. Enzyme binding and kinetic analyses on substrate analogues with P and As substituting for N in the trimethylammonium group show that the analogues are good BBOX substrates, which follow the efficiency trend N+>P+>As+. The results reveal that an uncharged carbon analogue of γBB is not a BBOX substrate, thus highlighting the importance of the energetically favorable cation–π interactions in productive substrate recognition.


General methods
All experiments were conducted under the following conditions, unless otherwise stated: Commercially available compounds were used as supplied without purification. Dried solvents were obtained by purification of HPLC grade solvents over activated alumina column using an MBraun SPS800 solvent purification system. Compound purification was done by column chromatography, using silica gel, Merck TM grade (pore size 60 Å; particle size 230-400 mesh, 40-63 µm). Reaction progress was monitored using glass TLC plates (TLC Silica gel 60G, F 254 , Merck, Germany) and observed by UV light and/ or by staining in permanganate. Compound analysis done by NMR ( 1 H, 13 C NMR analyses used), were recorded on a Varion Inova 400 at 400 MHz and 101 MHz respectively, while 31 P NMR analysis were done on either a Bruker DMX300 or Bruker Avance III 400 MHz at 121 MHz and 162 MHz respectively. Reported chemical shifts are in parts per million (ppm), moving from high to low frequencies and referenced to the residual solvent resonance (CDCl 3 or DMSO-d 6 ). Reported coupling constants (J) are noted in hertz (Hz) to the nearest 0.5 Hz. To assign multiplicity of signals the follow standard abbreviations were used: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, br = broad. When possible, 1 H assignments were made using appropriate 2D NMR methods, such as COSY, HSQC and HMBC. High resolution mass spectrometry (MS) analyses were performed using Electrospray Ionization MS using a JEOL AccuToF machine. Mass spectrometry and chromatography analysis were done using a Shimadzu UFLC LC-20AD LC/MS system, equipped with a RPC18 200 × 2 guard column. Typical conditions for an LC-MS run are: 157 bar, mobile phase; 2 minutes 5% MeCN 95% H2O (both with 0.1% formic acid), in 16 minutes decreasing polarity to 100% MeCN, maintaining this polarity for 5 minutes. Subsequently the polarity is increased to 95% H2O for 5 minutes. UV/VIS detection of this machine was done by Ultraviolet Visible Shimadzu SPD-M20A (200-600 nm), while MS analyses have been done using the Thermo scientific LCQ Fleet. When specified, purification of products was carried out by preparative HPLC using a Shimadzu LC 20AT, equipped with a Phenomenex Gemini NX (particle size: 10 μm, pore size: 110 Å, C18) in conjunction with a Shimadzu SPD 20A deuterium lamp at 215 and 254 nm as detector. A typical run was performed as follows: initial grade 10% MeCN in H 2 O (both solvent contain 0.1% TFA), 10 mL/min, at 18 minutes polarity has decreased to 70% MeCN and further to 100% MeCN at 20 minutes which was maintained for 3 minutes. Afterwards the polarity was increased to 10% MeCN in H 2 O over 2 minutes maintaining a constant polarity for 5 minutes (30 minutes total runtime).

Synthesis of (4-(benzyloxy)-4-oxobutyl) trimethylphosphonium bromide (10)
A microwave vial was charged with benzyl 4-bromobutarate ester 9 (500 mg, 1.94 mmol, 1 equivalent) under Ar (g) atmosphere. Subsequently a solution of PMe 3 (4.0 mL, 2 equivalents, 1 M in toluene) was added and the resulting solution was stirred for 60 minutes in a microwave machine applying a maximum of 400 W. The sample was then allowed to cool to r.t. and reaction mixture was diluted with MeOH and transferred into a round bottom flask. The solvent was removed and the crude solid was dissolved in a minimum amount of CH 2 Cl 2 and precrystallized from Et 2 O, affording 10 as a white solid (530 mg, 1.59 mmol, 82%

Synthesis of 4-trimethylphosphoniobutanoic acid bromide (2)
Bromide 10 (250 mg, 0.748 mmol, 1 equivalent) was dissolved in MeOH (20 mL) and treated with Pd/C (125 mg, 10% Pd/C). The suspension was stirred under H 2(g) for 20 hours and subsequently filtered through a pad of Celite. After removal of the solvent, the crude product was recrystallised from CHCl 3

Synthesis of (R)-(3-carboxy-2-hydroxypropyl)trimethylphosphonium trifluoroacetate (5)
To an aqueous solution of NaOH (4.4 mg, 0.110 mmol, 2.5 equivalents in H 2 O) and 1,4-dioxane (5 mL) was added ester 15 (15 mg, 0.044 mmol, 1 equivalent). The solution was stirred for 2 hours at r.t. before acidifying to pH 1 with HCl (3 N, 1 M in H 2 O). The solvent was evaporated (caution, do not heat for an extend period of time, as this induced product degradation) and the crude solid was suspended in a minimal amount of MeOH, filtered and purified by preparative HPLC (t r 6.5 minutes, mobile phase contain 0.1% TFA), affording acid 5 (11 mg, 0.0375 mmol, 85%) as a clear colorless oil.

Synthesis of (R)-(3-carboxy-2-hydroxypropyl)trimethylarsonium iodide (6)
To an aqueous solution of NaOH (5 mL, 4 M in H 2 O) and 1,4-dioxane (5 mL) was added ester 17 (23 mg, 0.061 mmol, 1 equivalent). The solution was stirred for 2 hours before acidifying to pH 3 with HCl (1 M in H 2 O). The solvent was evaporated (caution, do not heat for an extend period of time, as this induced product degradation) and the crude solid was suspended in a minimal amount of MeOH, filtered and purified by preparative HPLC (t r 6.6 minutes), affording acid 6 (15 mg, 0.043 mmol, 71%) as a clear colorless oil.

Synthesis of (S)-(3-carboxy-2-hydroxypropyl)trimethylphosphonium trifluoroacetate (7)
To an aqueous solution of NaOH (4.8 mg, 0.120 mmol, 2.5 equivalents in H 2 O) and 1,4-dioxane (5 mL) was added ester 21 (16 mg, 0.048 mmol, 1 equivalent). The solution was stirred for 2 hours before acidifying to pH 1 with HCl (1 M in H 2 O). The solvent was evaporated (caution, do not heat for an extend period of time, as this induced product degradation) and the crude solid was suspended in a minimal amount of MeOH, filtered and purified by preparative HPLC (t r 6.5 minutes, mobile phase contains 0.1% TFA), affording acid 7 (12 mg, 0.042 mmol, 87%) as a clear colorless oil.
To this was added AsMe 3 (115 μL, 1.08 mmol, 4 equivalents) and the resulting solution was heated to 75 °C. After 24 hours the solution was allowed to cool down to r.t. and the solvent was removed under reduced atmosphere. The resulting crude oil was purified by preparative HPLC (t r 20.

Supporting figures
indicates the formation of the product, likely the corresponding 3-keto derivative of 3.