[en] The title compound 4 has been prepared in four steps from ethylglycinate in 63% overall yield. This amino analog of citric acid has been co-crystallized with the class A beta-lactamase BS3 of Bacillus licheniformis and the structure of the complex fully analyzed by X-ray diffraction. Tris-ethyl aminocitrate 3 and the free tris-acid 4 have been tested against a member beta-lactamase from all distinct subgroups. They are novel inhibitors of class A beta-lactamases, still modest but more potent than citrate and isocitrate.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Beck, Josephine
Sauvage, Eric ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Charlier, Paulette ; Université de Liège - ULiège > Département des sciences de la vie > Cristallographie des macromolécules biologiques
Marchand-Brynaert, Jacqueline
Language :
English
Title :
2-Aminopropane-1,2,3-tricarboxylic acid: Synthesis and co-crystallization with the class A beta-lactamase BS3 of Bacillus licheniformis.
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Preparation of compound 3.Step i: hydrochloride salt of ethyl glycinate (1 g, 7.16 mmol) and triethylamine (1 equiv, 1 mL, 7.16 mmol) were dissolved in dichloromethane (10 mL) and stirred for 6 h at 20 °C. Magnesium sulfate (1.5 g) and 3-chlorobenzaldehyde (0.9 equiv, 730 μL, 6.44 mmol) were then added and the mixture was further stirred overnight. After filtration, the organic solution was washed three times with brine, dried (MgSO4) and concentrated under vacuum to give 1 as yellow oil (1.45 g, quantitative yield). HRMS (ESI): 226.0645 (calculated for C11H12ClNO2+H: 226.0635); 1H NMR (CDCl3, 300 MHz): δ ppm, 1.32 (t, 3H, CH3, J = 7.14 Hz), 4.26 (q, 2H, CH2, J = 7.14 Hz), 4.41 (s, 2H, CH2), 7.33-7.45 (m, 2H, Ar), 7.63 (d, 1H, Ar, J = 7.5 Hz), 7.82 (s, 1H, Ar), 8.25 (s, 1H, NCH). 13C NMR (CDCl3, 125 MHz): δ ppm, 14.36 (CH3), 61.35 (CH2), 62.07 (CH2), 127.00 (CHAr), 128.23 (CHAr), 130.02 (CHAr), 131.31 (CHAr), 134.98 (CAr), 137.47 (CAr), 164.03 (NCH), 170.01 (CO2Et). Step ii (Method C): to the crude imine 1 (580 mg, 2.6 mmol) dissolved in dry THF (12 mL) and cooled at -78 °C, was added under argon atmosphere LDA (commercial solution in THF, 2.1 equiv, 2.7 mL, 5.42 mmol). After 1h at -78 °C, ethyl bromoacetate was added (2.1 equiv, 570 μL, 5.42 mmol) and the mixture was allowed to slowly warm up to 20 °C, overnight, under stirring. After addition of water, the solution was concentrated under vacuum. The oily residue was dissolved in ether and the organic layer was washed successively with aqueous NaHCO3and brine. Drying over MgSO4 and concentration under vacuum furnished 2 as a brown oil (1 g, quantitative yield). 1H NMR (CDCl3, 500 MHz): δ ppm, 1.24 (t, 6H, CH3, J = 7.14 Hz), 1.29 (t, 3H, CH3, J = 7.16 Hz), 3.13 (d, 2H, CH2, J = 15.79 Hz), 3.29 (d, 2H, CH2, J = 15.77 Hz), 4.14 (q, 4H, CH2, J = 7.10 Hz); 4.26 (q, 2H, CH2, J = 7.14 Hz), 7.34 (dd, 1H, Ar, J = 8 Hz and J = 7.6 Hz), 7.40 (ddd, 1H, Ar, J = 8 Hz, J = 2.17 Hz and J = 1.14 Hz), 7.57 (dt, 1H, Ar, J = 7.6 Hz and J = 1.3 Hz), 7.76 (s, 1H, Ar), 8.28 (s, 1H, NCH). 13C NMR (CDCl3, 125 MHz): δ ppm, 14.37 (CH3),40.83 (CH2), 60.79 (CH2), 61.94 (CH2), 67.86 (C), 127.24 (CHAr), 128.19 (CHAr), 130.02 (CHAr), 131.39 (CHAr), 135.00 (CAr), 137.85 (CAr), 159.63 (N{double bond, long}CH), 170.65 (CO2Bn), 171.00 (CO2Me).Step iii: the crude imine 2 (857 mg, 2.15 mmol) was dissolved in acetonitrile (3 mL) and 1 N HCl was added (1.5 equiv, 3.2 mL, 3.22 mmol). After 30 min at room temperature, the solution was concentrated under vacuum, and the aqueous phase was extracted three times with ether. The aqueous phase was then neutralized with NaHCO3 and basified to pH 10 with 1 N NaOH. This was extracted five times with CH2Cl2. The organic layers were collected and washed with brine. After drying (MgSO4) and concentration under vacuum, the residue was purified by column chromatography on silica gel to furnish 3 as yellow oil (296 mg, 50% yield). Rf = 0.5 (CH2Cl2/EtOAc, 1:1); HRMS (ESI): 276.1450 (calculated for C12H21NO6+1: 276.1447); 1H NMR (CDCl3, 250 MHz): δ ppm, 1.30 (t, 9H, J = 7.13 Hz, CH3), 2.56 (s, 2H, NH2), 2.66-2.72 (d, 2H, J = 16.0 Hz, CH2), 2.82-2.88 (d, 2H, J = 16.0 Hz, CH2), 4.15 (q, 4H, J = 7.13 Hz, CH2); 13C NMR (CDCl3, 62.5 MHz): δ ppm, 14.3 (CH3) 43.6 (CH2), 55.8 (C), 61.0 (CH2), 61.9 (CH2), 170.5 (CO2Et), 175.0 (CO2Et).
Preparation of compound 4. Amine 3 (100 mg) was treated with 6 N HCl (10 mL) at reflux for 1 night. After extraction with CH2Cl2, the aqueous phase was concentrated under vacuum and the residue was dried under high vacuum to furnish 4 as a white solid (75 mg, 90% yield). HRMS (ESI): 192.0503 (calculated for C6H9NO6+1: 192.0508); 1H NMR (D2O, 250 MHz): δ ppm, 2.98 (s, 4H); 13C NMR (D2O, 62.5 MHz): δ ppm, 39.9 (CH2), 59.3 (C), 173.7 (CO2H).
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Determination of biochemical activity. The enzymes were produced and purified as previously described.18-22 The enzymes (1-100 nM) were incubated with the tested compounds (100 μM, otherwise mentioned) and the chromogenic substrate nitrocefine (100 μM) in a phosphate buffer (50 mM, pH 7.2). The hydrolysis rate of this substrate was followed by spectrophotometry at 482 nm. The residual activity was obtained by comparison with the variation of the absorbance of a reference (sample without inhibitor) and indicated in Table 1. Results are expressed as % of initial activities; variations of results are within ±5%. Plot V/VI versus inhibitor concentration (ratios of hydrolysis in the absence and in the presence of inhibitors) gave the inhibition constant indicated in Table 2. All experiments were performed three times.
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BS3 β-lactamase purification, crystallization, and data collection. The expression, purification and initial crystallization conditions of the BS3 enzyme (BS3-citrate) were described previously.3,24 Typically, monoclinic crystals were obtained using the hanging drop vapor diffusion method with drops containing 5 μL of a protein solution (at a concentration of 40 mg/mL in 50 mM NaCl, 10 mM Tris buffer, pH 7.2) and 5 μL of 10% PEG 6000 in 100 mM sodium citrate buffer (pH 3.4), equilibrated against 1 mL of the latter solution at 20 °C. The BS3-isocitrate crystals were obtained in the same conditions, by replacing the citrate buffer by a 100 mM sodium isocitrate buffer at the same pH. The BS3-aminocitrate crystals were grown in drops containing 5 μL of a protein solution (at a concentration of 38 mg/mL in 50 mM NaCl, 10 mM Tris buffer, pH 7.2), 4 μL of 8% PEG 6000 in 100 mM sodium aminocitrate buffer (pH 3.4) plus 1 μL of 0.1 M urea additive, equilibrated against 1 mL of a 20% PEG 6000 solution, at 20 °C. X-ray diffraction experiments were carried out under cryogenic conditions (100 K) after transferring the crystals into a reservoir solution supplemented with 50% glycerol. The diffraction data for the BS3-isocitrate crystal were measured at ESRF (Grenoble, France) on the FIP-BM30a beamline (λ = 1.0 Å) using a MarResearch 165 mm CCD detector. Data for the BS3-aminocitrate crystal were collected with a Rigaku RU-200 rotating anode generator operating at 40 kV and 100 mA and a MarResearch Mar345 Imaging Plate (λ = 1.5418 Å). Intensities were indexed and integrated using MOSFLM version 6.01. The scaling of the intensity data was accomplished with SCALA of the CCP4 program suite and all corresponding statistics are available as Supplementary data. The atomic coordinates are available at the Protein Data Bank under the codes 1I2S (citrate), 1W7F (isocitrate) and 3B3X (aminocitrate).
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In the class C β-lactamase CMY2 of Klebsiella pneumoniae (β-lactamase structurally close to P99 enzyme), the citrate molecule seems to make a 180° rotation in the active site, in comparison with its position in the BS3 catalytic cleft (see Supplementary data). The atomic coordinates are available at the Protein Data Bank under the code 1ZC2.
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