LABELED DIISOPROPYLPIIOSPHOROFLUORIDATE 5641
Nov. 5 , 105G
the ether then removed by distillation a t atmospheric presby bubbling anhydrous ammonia through it for ten minutes. The precipitated ( NH4)2S04was filtered and the solvent re- sure. Distillation of the crude acid gave 3.8 g. of CFzBrmoved bv evaooration under reduced oressure. The solid CFBrCOZH, b.p. 72-73' a t 2.5 mm. pressure, n% 1.4458, thus obtained 'was sublimed to yield 5:s g. (90%) of CFz- dm1 2.191, neutral equivalent 287 (calculated 286). BrCFBrCONHz, m .p. 60.6-61 .O'. Anal. Calcd. for C3HBr2FaO2: C, 12.60; Br, 55.91. .4nal. Calcd. for C3H2Br2F3NO: N, 4.92. Found: N, Found: C, 13.99; Br, 54.20. 4.86. C F Z B ~ C F B ~ C O Z C Z H ~ . grams - F O ~ ~(0.15 ~ mole) of CFzCFz=CFCONHz.-Two grams (0.03 mole) of zinc dust, BrCFBrCN, 29 g. of 90% H2SOI and 35 g. of C Z H ~ O H were a trace of zinc chloride and 100 ml. of dry acetone were heated a t reflux for nlne hours. Ether extraction of the mixed, heated to reflux, and 2 g. (0.007 mole) of CFZBrCF- product followed by fractionation g y e 28 g. (60% yield) BrCONH2 dissolved in 20 ml. of acetone added slowly to of CFzBrCFBrCO2C2H5, b.p. 81-84 a t 18-19 cm., nZ5D this mixture. Reflux was maintained for two hours after 1.426. the addition was comdeted. The solid obtained bv solvent Anal. Calcd. for C5F3BrzOzHb: C, 19.1; Br, 50.95. evaporation was s u b b e d t o yield 0.55 g. (65%) bf CFZ= Found: C, 19.1; Br, 50.97. CFCONHZ, m.p. 121.4-121.9'. Preparation of CFz=CFCOzC2H5.-About 23.6 g. (0.075 Anal. Calcd. for C3H2F3NO: C, 28.81; N, 11.20. mole) of CFzBrCFBrCO2C2H5 was added to 8.0 g. (0.12 Found: C, 28.58; N, 11.18. of zinc dust and anhydrous alcohol-free ether with CFZBrCFBrC02H.-This reaction was carried out in a mole) stirring. After the initial exothermic reaction ceased, the manner similar to the reaction described by Wiley, et a1.I4; ether was refluxed for five hours. Isolation of the product 4.4 g. (0.039 mole) of 8570 sulfuric acid was heated to 150" and fractionation yielded 6.0 g. (52% yield) of CFz=CFand 10 g. (0.039 mole) of CFlBrCFBrCN added through the COZCZH~, b.p. 100.0-100.5" a t 750 mm., nZ5D 1.3615reflux condenser over a period of four hours. The tempera1.3619. ture was kept a t 150' for two hours after the addition of the Anal. Calcd. for C~HbF302: C, 39.0; F , 37.1. Found: nitrile. The bath was then allowed to cool to 90" and 10 ml. of water was added. The temperature was kept a t 90" C, 39.0; F , 37.0. for about 15 hours. After the reaction mixture had cooled Preparation of CF2BrCFBrCOzCH2CIF7.-This ester was to room temperature, it was extracted with five 10-ml. por- prepared oia the same method used to make the saturated tions of ether. The ether extracts were combined and neuethyl ester. Fractionation of the crude product gave a tralized with a solution of sodium carbonate. After sepa67% yield of C F Z B ~ C F B ~ C O Z C H ~b.p. C ~ F72-73' ~, a t 10 ration of the water layer, the ether layer was extracted with mm., n z 5 ~1.3676-1.3680. two 10-ml. portions of water. The combined water layers Anal. Calcd. for C7F,OBr2H2O2: Br, 34.2; C, 17.9; F , were evaporated, yielding 6 g. of crude CFzBrCFBrC02Na. Evaporation of the ether solution yielded 5 g. of CFZBrCF- 40.6. Found: Br, 34.3; C, 18.1; F, 40.7. Preparation of CF~=CFCOZCHZC~F~.-A 71% yield of BrCOKHz. The crude CFzBrCFBrCOZKa was finely ground and suspended in 15 ml. of dry ether. Anhydrous CF2=CFCOzCH2C3F7, b.p. 61.0-61.8' a t 50 mm., nZ5D HCl was then passed through the ether suspension of the 1.3189, was obtained from the debromination reaction. salt for six hours. The reaction mixture was filtered and Anal. Calcd. for C ~ F ~ ~ O ZC, H ~27.24; : F, 61.68. Found: C, 27.3; F , 61.0. (14) P. F. Wiley and G. A. Nesty, "Process for Producing Esters of Acrylic Acid," U. S. P a t e n t 3,520,310 (1050). ST. PAUL,MINNESOTA
[CONTRIBUTION FROM
THE
MEDICALBIOLOGICAL LABORATORY OF THE NATIONAL DEFENCERESEARCH COUNCILT.N.O.]
Synthesis of P32Labeled Diisopropylphosphorofluoridate BY R. A. OOSTERBAAN AND J. VAN ROTTERDAM RECEIVED APRIL25, 1956
A method is described for the preparation of labeled diisopropylphosphorofluoridate in water or oil solution starting from radioactive phosphoric acid. The specific radioactivity amounts to 200 microcuries/mg.
Introduction The synthesis of P32labeled diisopropylphosphorofluoridate (DFP) of high specific activity (200 microcuries/mg.) has proved to be valuable in medical work; it has been used for the study of the metabolic fate of D F P and for the determination of plasma protein turnover and the life span of erythrocytes and Moreover the material has been used for studies with the purpose of collecting information on the chemical groups of esterases which are responsible for the enzymatic and D F P binding properties of these pr0teins.~-6 (1) J. A. Cohen and M . G. P. J. Warringa, J. Clin. Inuest., 33, 459 (1954). (2) C. H. W. Leeksma and J. A. Cohen, N a f u v e , 175, 5 5 2 (1955). ( 3 ) C. H. W. Leeksma and J. A. Cohen, J . C l i x . Invest., 36, in press (195ti).
(4) R . A. Ousterbaan, P. Kunst a n d J. A. Cohen, Biochim et Biophys. A c t a , 16, 229 (19.55). ( 5 ) J. A. Cohen, R. A. Oosterbaan, M. G. P. J . Warringa a n d H. S. Jansz, Discussions Faraday Soc., 20, 114 (1950). (6) R . A. Ormterbaan, H. S . Jansr a n d J. A. Cohen, Riochim. el Biophys, Acta, 20, 402 (1956).
A number of methods for the synthesis of DFP7-lo and DFP32 11.12 have been published. These methods could not be used unmodified for our purpose. Either starting material of sufficient specific activity cannot be obtained readily or the methods are not designed for the desired microscale operation. The present method is based on the conversion of phosphoric acid, which is obtainable in carrier-free form, into P32and consequently into P3T13. The further synthesis is a micro-scale adaptation of Saunders' method, l1 according to the reaction scheme
+
+
3ROH PCla +(R0)zPOH RC1 (R0)zPOH Clz +(RO)zP(O)Cl (RO)%P(O)Cl KHnF +(RO)zP(O)F
+ +
+ 2HC1 + HC1 + NH4C1
(7) 11. McCombie, B. C. Saunders and G.J. Starey, J. Chem. S O L . , 380 (1945). ( 8 ) B. C . Saunders and G. 5. Stacey. ibid., 095 (1918). (9) H.Goldwhite a n d B. C. Saunders, ibid.. 2041 (1955). (10) C. Mnnard and H. Jean, Bull. soc. chim. France, 544 (1952) (11) B. C . Saunders and T. S. Worthy, J . Chem. Soc., 1320 (1950). (12) B. Witten and J . I. Miller, THISj o u R N A L , 7 0 , 38813(1948).
5042
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