Toward antibody-catalyzed hydrolysis of organophosphorus poisons -- Data Supplement - Supplemental Text -- Proceedings of the National Academy of Sciences

Supplementary material for Vayron et al. (2000) Proc. Natl. Acad. Sci. USA 97 (13), 7058-7063.

Supplemental Results
Synthesis, General Remarks.
Reagents were from Aldrich, except for 1a-f, which were provided by Centre d’Etudes du Bouchet. 1b and 1c were further purified by semipreparative HPLC (column: Zorbax SB-CN 250 × 9 mm; elution conditions: isocratic tBuOMe/hexane 60/40, 3.0 ml/min). All chromatography (flash) was performed with Merck Silicagel 60 (0.02-0.04 mm). TLC was performed with fluorescent Merck F254 glass plates. NMR spectra were recorded on a Brucker AC-300 MHz. Chemical shifts (d ) are given in parts per million, and the coupling constant J is expressed in Hertz. IR spectra were recorded on a Perkin-Elmer spectrophotometer 2000-FT/IR, and MS were obtained with a Finnigan-Mat 4600 quadrupole system. Elemental analysis was done by the Institut des Substances Naturelles in Gif sur Yvette, France. High-resolution MS was done by the Centre Régional des Mesures Physiques de l’Ouest in Rennes, France. Methylphosphinic Acid Ethyl Ester (4a).
Ethanol (67 ml, 1.15 mol) was dissolved in 200 ml of cooled (0°C) tetrahydrofuran (THF). Dichloromethylphosphine (50 m, 557 mmol) was then added dropwise, while the temperature was maintained below 4°C. The reaction mixture was allowed to reach room temperature (2 h), and THF was evaporated. Crude 4a was distilled (bp₁₀ = 57°C) yielding 52.9 g (88%) of colorless oil. ¹H NMR (CDCl₃): d 7.24 (dq, ¹J_P-H = 540 Hz, ³J_H-H = 2 Hz, 1H), 4.15 (m, 2H), 1.57 (dd, ²J_P-H = 11 Hz, ³J_H-H = 2 Hz, 3H), 1.37 (t, ³J_H-H = 7 Hz, 3H). ¹³C NMR: d 62.75 (d, ²J_P-C = 6 Hz), 29.8, 16.35, 14.8 (d, ¹J_P-C = 95 Hz). ³¹P NMR: d 34.05 (d, ¹J_P-H = 540 Hz). (3-Amino-1-hydroxypropyl)-methyl-phosphinic Acid Ethyl Ester (5a).
Methylphosphinate 4a (1.0 g, 9.2 mmol), diisopropyl ethylamine (1.7 ml, 9.7 mmol), and (3-oxopropyl)carbamic acid benzyl ester (7) (1.86 g, 9.0 mmol), prepared by pyridinium chlorochromate oxidation of the corresponding aminoalcohol, were dissolved in 10 ml of dry 1,2-dimethoxyethane (DME). The mixture was heated at 50°C for 7 days, DME and the amine were evaporated, and the crude mixture was directly chromatographed on silicagel (CH₂Cl₂/MeOH 9/1 to 5/5), yielding 1.95 g (69%) of (3-benzyloxycarbonylamino-1-hydroxypropyl)methylphosphinic acid ethyl ester as a colorless solid (mp = 135-137°C), mixture of the two diastereomers. ¹H NMR (CDCl₃): d 7.35 (m, 5 + 5H), 5.5 (m, 1 + 1H), 5.1 (s, 2 + 2H), 4.07 (m, 2 + 2H), 3.83 (m, 1 + 1H), 3.57 (m, 1 + 1H), 1.97 (m, 1 + 1H), 1.78 (m, 1 + 1H), 1.50 and 1.48 (d, ²J_P-H = 15 Hz, 3H), 1.30 (t, ³J_H-H = 7 Hz, 3H), 1.29 (t, ³J_H-H = 7Hz, 3H). ³¹P NMR: d 55.5 and 54.7. IR (neat) 3306, 2592, 1704, 1536, 1258, 1179, 1037. MS (CI, NH₃): 316([M + H]⁺), 333 ([M+NH₄]⁺).

(3-Benzyloxycarbonylamino-1-hydroxypropyl)methylphosphinic acid ethyl ester was then dissolved in 5 ml of ethanol (containing ~5% water), 20 mg of Pd/C 5% was added, and the reaction mixture was left overnight under hydrogen. The palladium was filtered off, and the ethanol evaporated yielding 1.1 g (97%) of 5a as a colorless oil and a 50/50 mixture of diastereomers. ¹H NMR (CD₃OD), carbonated amine: d 4.13 and 4.11 (quint, ³J_P-H = ³J_H-H = 7 Hz, 2 + 2H), 3.95 (m, 1 + 1H), 3.01 (m, 2 + 2H), 1.97 (m, 1 + 1H), 1.78 (m, 1 + 1H), 1.53 and 1.51 (d and d, ²J_P-H = 13.5 Hz, 3 + 3H), 1.33 (t, ³J_H-H = 7 Hz, 3H), 1.29 (t, ³J_H-H = 7 Hz, 3H). ¹³C NMR (CD₃OD), carbonated amine d first isomer: 67.3 (d, ¹J_P-C = 115 Hz), 62.6 (d, ²J_P-C = 4.5 Hz), 39.2 (d, ³J_P-C = 12 Hz), 33.2, 16.9, 9.55 (d, ¹J_P-C = 90Hz). Second isomer: 68.6 (d, ¹J_P-C = 115 Hz), 62.3 (d, ²J_P-C = 4.5 Hz), 39.2 (d, ³J_P-C = 12 Hz), 33.2, 16.9, 10.22 (d, ¹J_P-C = 90 Hz). ³¹P NMR (CD₃OD): d 60.6 and 59.2. IR (neat) 3277, 2985, 1302, 1183, 1037, 962. MS (CI, NH₃): 182 ([M+H]⁺).

(1-Hydroxy-3-isopropylamino-propyl)-methyl-phosphinic Acid Ethyl Ester 6a.
Methylphosphinite 5a (90 mg, 5.0 mmol) was dissolved in 5 ml of acetone containing 5% MeOH. MgSO₄ (50 mg) was then added. After 4 h at room temperature, MgSO₄ was filtered off, 10 ml of dry methanol was added together with 10 mg of Pd/C 5%, and the reaction mixture was left overnight under hydrogen. The crude reaction mixture was filtered over celite, the solvents were removed under vacuum, and 102 mg (92%) of pure 6a was recovered as a white powder (mp = 157-158°C) after a single precipitation with pentane (50/50 mixture of diastereomers). ¹H NMR (CD₃OD), carbonated amine: d 4.14 and 4.12 (2 quint, ³J_P-H = ³J_H-H = 7 Hz, 2 + 2H), 4.05 (m, 2 + 2H), 3.30 (hept, ³J_H-H = 6.5 Hz, 1 + 1H), 3.16 (m, 2 + 2H), 2.16 (m, 1 + 1H), 1.98 (m, 1 + 1H), 1.58, and 1.55 (d and d, J²_P-H = 14 Hz, 3 + 3H) 1.34 (t, ³J_H-H = 7 Hz, 3 + 3H) 1.30 (d, ³J_H-H = 6.5 Hz, 6 + 6H). ¹³C NMR (CD₃OD) d 68.3 and 66.2 (d, ¹J_P-C = 115 Hz, 1 + 1C), 63.2 and 62.8 (d, ²J_P-C = 7 Hz, 1 + 1C), 51.45 (2C), 43.6 and 43.5 (d, ³J_P-C = 6 Hz), 28.75 (2C), 19.9 (4C), 16.9 (2C), 10.15 and 9.5 (d, ¹J_P-C = 89 Hz, 1 + 1C). ³¹P NMR (CD₃OD): d 60.79 and 59.61. MS (CI, NH₃): 224 ([M + H]⁺). Hapten Ha: {[(3-Aminopropanoyl)-isopropylamino]-hydroxypropyl}-methylphosphinic Acid Ethyl Ester.
3-Benzyloxycarbonylamino-propionic acid [prepared by Jones oxidation of (3-hydroxypropyl)carbamic acid benzyl ester] (80 mg, 0.36 mmol) was dissolved in dried CH₂Cl₂. Thionyl chloride (23 µl, 0.54 mmol) was added dropwise at 0°C. The reaction mixture was heated at 50°C for 1 h, the solvents and unreacted thionyl chloride were evaporated in vacuo, and the crude reaction mixture was dissolved in dry THF. Amine 6a (62 mg, 0.28 mmol) and triethylamine (1 ml) were successively added at 0°C. The reaction mixture was allowed to stand at room temperature for 2 h, the solvents were then evaporated in vacuo, and the crude product was chromatographed on silicagel (CH₂Cl₂/acetone 90/10) yielding 59 mg (50%) of the corresponding amide: {[(3-benzyloxycarbonylamino-propanoyl)-isopropylamino]-hydroxypropyl}-methylphosphinic acid ethyl ester (two diastereomers) as a clear oil. ¹H NMR (CDCl₃): d 7.35 (m, 5 + 5H), 5.56 (broad s, 1 + 1H), 5.09 (s, 2 + 2H), 4.11 and 4.08 (2 quint, ³J_P-H = ³J_H-H = 7 Hz, 2 + 2H), 4.03 (m, 1 + 1H), 3.66 (m, 2 + 2H), 3.49 (m, 2 + 2H), 2.60 (m, 2 + 2H), 2.06 (m, 1 + 1H), 1.78 (m, 1 + 1H), 1.53 and 1.47 (d and d, ³J_P-H = 14 Hz, 3 + 3H) 1.35 and 1.32 (t, ³J_H-H = 7 Hz, 3 + 3H) 1.25 and 1.19 (d, ³J_H-H = 6.5 Hz, 6 + 6H).³¹P NMR (CDCl₃): d 55.14 and 54.41. MS (CI, NH₃): 429 ([M + H]⁺) 446 ([M + NH₄]⁺).

The amide (28 mg, 0.065 mmol) was dissolved in technical ethanol, 5 mg of Pd/C 5% was added, and the reaction mixture was kept under hydrogen for one night. The palladium was filtered off, and hapten Ha (18 mg, 94% yield) recovered as a pale yellow oil (50/50 mixture of diastereomers). ¹H NMR (CD₃OD): d 4.18 (m, 3 + 3H), 4.03 (m, 1 + 1H), 3.87 (m, 1 + 1H), 3.48 (broad t, ³J_H-H = 7 Hz, 2 + 2H), 3.10 (t, ³J_H-H = 6 Hz, 2 + 2H), 2.74 (t, ³J_H-H = 6 Hz, 2 + 2H), 2.12 (m, 1 + 1H), 1.85 (m, 1 + 1H), 1.57 and 1.50 (d and d, ³J_P-H = 14 Hz, 3 + 3H), 1.35 and 1.34 (t, ³J_H-H = 7 Hz, 3 + 3H), 1.25-1.15 (four d, ³J_H-H = 6.5 Hz, 3 + 3 + 3 + 3H).³¹P NMR (CD₃OD): d 60.13 and 59.14. MS (CI, NH₃): 295([M + H]⁺).

Phenylphosphinic Acid Ethyl Ester 4b. 4b
was purchased from Aldrich or was synthesized in a 90% yield via a reaction similar to the preparation of 4a. (3-Amino-1-hydroxypropyl)-phenylphosphinic Acid Ethyl Ester 5b.
Phenylphosphinate 4b (440 mg, 2.6 mmol), diisopropyl ethylamine (470 µl, 2.7 mmol), and (3-oxopropyl)carbamic acid benzyl ester (7) (550 mg, 2.7 mmol) were dissolved in 10 ml of dry DME. The mixture was heated at room temperature for 4 days, DME and the amine were evaporated, and the crude mixture was directly chromatographed on silicagel (CH₂Cl₂/MeOH 95/5) yielding 780 mg (80%) of (3-benzyloxycarbonylamino-1-hydroxypropyl)phenylphosphinic acid ethyl ester as a colorless oil, ca. 50/50 mixture of the two diastereomers. ¹H NMR (CDCl₃): d 7.80 (m, 2 + 2H), 7.54 (m, 1 + 1H), 7.44 (m, 2 + 2H), 7.29 (m, 5 + 5H), 5.88 and 5.85 (t, ³J_H-H = 6 Hz, 1 + 1H), 5.05 and 5.03 (s, 1 + 1H), 4.08 (m, 2 + 2H), 3.92 (m, 1 + 1H), 3.35 (m, 2 + 2H), 1.95 (m, 1 + 1H), 1.67 (m, 1 + 1H), 1.26 and 1.25 (t, ³J_H-H = 7 Hz, 2 + 2H). ¹³C NMR (CDCl₃): d 157.2, 136.7, 132.8, 132.7, 132.5, 128.7, 128.5, 128.1, 128.0 (d, ¹J_P-C = 110 Hz), 67.6 and 67.2 (d, ¹J_P-C = 130 Hz), 66.7, 61.6 (d, ²J_P-C = 7 Hz), 37.3 (d, ³J_P-C = 14 Hz), 30.8 (d, ²J_P-C = 14 Hz), 16.6 (d, ³J_P-C = 5 Hz). ³¹P NMR (CDCl₃): d 41.44. IR (neat) 3307, 3064, 3983, 2953, 1715, 1694, 1682, 1538, 1439, 1257, 1186, 1124, 1027, 959. MS (CI, NH₃): 378 ([M + H]⁺), 395 ([M + NH₄]⁺). High-resolution mass spectroscopy (HRMS) (CI, [M + H]⁺, C₁₉H₂₅NO₅P) calc.: 378.1470, found 378.1472.

(3-Benzyloxycarbonylamino-1-hydroxypropyl)phenylphosphinic acid ethyl ester was then dissolved in 20 ml of ethanol (containing ~5% water), 20 mg of Pd/C 5% was added, and the reaction mixture was left overnight under hydrogen. The palladium was filtered off and the ethanol evaporated. The resulting colorless oil was chromatographed (CH₂Cl₂/MeOH 7/3) yielding 490 mg (98%) 5b as a colorless oil and a 50/50 mixture of diastereomers. ¹H NMR (CD₃OD), carbonated amine: d 7.91 (m, 2 + 2H), 7.72 (broad t, ³J_H-H = 6.5 Hz, 1 + 1H), 7.62 (m, 2 + 2H), 4.32-4.04 (m, 3 + 3H), 3.21 (m, 2 + 2H), 2.15 (m, 1 + 1H), 2.01 (m, 1 + 1H), 1.40 and 1.38 (t, ³J_H-H = 7 Hz, 3 + 3H). ¹³C NMR (CD₃OD), carbonated amine d 134.4 (1 + 1C), 133.9, 133.8 (1 + 1C), 133.7, 130.0, 129.9 (1 + 1C), 129.8, 128.4, and 128.2 (d, ¹J_P-C = [dsim]100 Hz, 1 + 1C), 69.0 and 68.1 (d, ¹J_P-C = 120 Hz, 1 + 1C), 62.8 and 62.7 (d, ²J_P-C = 7.5 Hz, 1 + 1C) 39.1 (d, ³J_P-C = 14.5 Hz, 1 + 1C), 33.5 and 33.4 (1 + 1C), 16.9 (2C). ³¹P NMR (CDCl₃): d 43.7 and 42.85. IR (neat) 3384, 3051, 2967, 1615, 1592, 1440, 1394, 1194, 1123, 1033, 962. MS (CI, NH₃): 244 ([M + H]⁺).

(1-Hydroxy-3-isopropylamino-propyl)phenylphosphinic Acid Ethyl Ester 6b.
Phenylphosphinite 5b (450 mg, 1.85 mmol) was dissolved in 5 ml of acetone and 5 ml of MeOH. MgSO₄ (100 mg) was added. After 4 h at room temperature, MgSO₄ was filtered off, 10 mg of Pd/C5% was added, and the reaction mixture was left overnight under hydrogen. The crude reaction mixture was filtered over celite, the solvents were removed under vacuum, and 445 mg (85%) of pure 6b was recovered as a colorless oil (50/50 mixture of diastereomers). ¹H NMR (CD₃OD), carbonated amine: d 7.91 (m, 2 + 2H), 7.72 (broad t, ³J_H-H = 6.5 Hz, 1 + 1H), 7.63 (m, 2 + 2H), 4.33-4.02 (m, 3 + 3H), 3.38 (hept ³J_H-H = 6.5 Hz, 1 + 1H), 3.23 (m, 2 + 2H), 2.20 (m, 1 + 1H), 2.04 (m, 1 + 1H), 1.40 and 1.38 (t, ³J_H-H = 7 Hz, 3 + 3H), 1.37 (d, ³J_H-H = 6.5 Hz, 6 + 6H). ¹³C NMR (CD₃OD), carbonated amine d 136.4 (2C), 133.9, 133.8, 133.7, 133.6, 130.0, 129.9, 129.8, 129.6, 128.8, and 128.6 (d, ¹J_P-C = ~100 Hz, 1 + 1C), 68.4 and 67.3 (d ¹J_P-C = ~120 Hz, 1 + 1C), 63.1 and 62.9 (d, ²J_P-C = 7.5 Hz, 1 + 1C), 52.0 (1 + 1C), 43.1 and 41.4 (d, ³J_P-C = 10 Hz, 1 + 1C), 28.6 (3 + 3C), 19.2 (1 + 1C), 16.8 (1 + 1C) ³¹P NMR (CD₃OD): d 42.83 and 41.78. MS (CI, NH₃): 286 ([M + H]⁺), 298 ([M + NH₄]⁺). Hapten (Hb): {[(3-Amino-propanoyl)-isopropyl-amino]-hydroxy-propyl}-phenyl-phosphinic Acid Ethyl Ester.
3-Benzyloxycarbonylaminopropionic acid (111 mg, 0.50 mmol) was dissolved in dried CH₂Cl₂. Thionyl chloride (42 µl, 1.0 mmol) was added dropwise at 0°C. The reaction mixture was brought to room temperature in 30 min. The solvents and unreacted thionyl chloride were evaporated under vacuum, and the crude reaction mixture was dissolved in dry THF. Amine 6b (132 mg, 0.46 mmol) and triethylamine (1 ml) were successively added at 0°C. The reaction mixture was allowed to stand at room temperature for 2 h. The solvents were evaporated under vacuum, and the crude product was chromatographed on silicagel (CH₂Cl₂/MeOH 98/2) yielding 143 mg (63%) of the corresponding amide (two diastereomers) as a clear oil. ¹H NMR (CD₃OD): d 7.87 (m, 2 + 2H), 7.67 (broad t, ³J_H-H = 6 Hz, 1 + 1H), 7.58 (m, 2 + 2H), 7.37 (m, 5 + 5H), 5.10 (s, 2 + 2H), 4.18-3.97 (m, 4 + 4H), 4.41 (m, 4 + 4H), 2.62 (m, 2 + 2H), 2.08 (m, 1 + 1H), 1.81 (m, 1 + 1H), 1.37 and 1.35 (t, ³J_H-H = 7 Hz, 3 + 3H), 1.20-1.13 (m, 6 + 6H). ¹³C NMR (CD₃OD): d 173.2 (2C), 158.7 (2C), 138.3 (2C), 134.2 , 134.0 (2C), 133.8, 133.7 (2C), 133.6 (2C), 129.9, 129.8 (2C), 129.7 (2C), 129.4 (3C), 129.3 (d, ¹J_P-C = 110 Hz, 2C), 129.0 (2C), 128.8 (2C), 69.3 and 68.7 (d, ¹J_P-C = 120 Hz, 1 + 1 C), 67.4 (2C), 63.0 and 62.9 (d, ²J_P-C = 9 Hz, 1 + 1C), 50.0, 49.8, 41.6 and 38.9 (d, ³J_P-C = 15 Hz, 1 + 1C), 38.3 (2C), 34.8, 34.5, 33.0, 31.6, 21.3, 21.1, 20.5 (2C), 16.8. ³¹P NMR (CD₃OD): d 43.60 and 42.68. IR (neat) 3404, 3296, 3061, 2978, 1713, 1621, 1436, 1361, 1263, 1216, 1124, 1070, 1032, 957. MS (CI, NH₃): 491([M + H]⁺). HRMS for C₂₅H₃₅N₂O₆P calc.: 490.2233, found: 490.2237. C₂₅H₃₅N₂O₆P (490.22): calc. C 61.2, H 7.19; found C 60.49, H 7.15.

The amide was dissolved in technical ethanol, 5 mg of Pd/C 5% was added, and the reaction mixture was kept under hydrogen for one night. The palladium was filtered off, and hapten Hb (95 mg, 91% yield) recovered as a pale yellow oil (50/50 mixture of diastereomers). ¹H NMR (CD₃OD): d 7.88 (m, 2 + 2H), 7.69 (t, ³J_H-H = 6 Hz, 1 + 1H), 7.60 (m, 2 + 2H), 4.20-4.00 (m, 4 + 4H), 3.46 (t, J = 8 Hz, 2 + 2H), 3.24 (m, 2 + 2H), 2.87 and 2.85 (t, J = 8 Hz, 2 + 2H), 2.10 (m, 1 +1H) 1.80 (m, 1 + 1H), 1.38 and 1.36 (t, ³J_H-H = 7 Hz, 3 + 3H), 1.23 (d, ³J_H-H = 6.5 Hz, 6 + 6H). ¹³C NMR (CD₃OD): d 173.5, 173.4, 134.2, 134.1, 133.8, 133.7 (2C), 133.6, 130.0, 129.9, 129.7, 129.6, 128.8 and 128.6 (d, ¹J_P-C = 110 Hz, 1 + 1C) 69.3 and 68.6 (d, ¹J_P-C = 120 Hz, 1 + 1C) 62.9 and 62.8 (d, ²J_P-C = 7.5 Hz, 1 + 1C), 50.1, 49.9, 41.6 and 39.8 (d, ³J_P-C = 13 Hz, 1 + 1C), 38.5, 38.4, 36.2, 35.9, 31.7 (2C), 21.3, 21.2, 20.5 (2C), 16.8 (2C). ³¹P NMR (CD₃OD): d 44.40 and 43.46. IR (neat): 3379 (broad), 2977, 1619, 1441, 1193, 1123, 1032, 956. MS (CI, NH₃): 357([M + H]⁺). HRMS for C₁₇H₃₀N₂O₄P ([M + H]⁺) calc.: 357.1943, found: 357.1946.