added to incipient cloudiness. I t was recrystallized from alcohol. The properties of these salts, obtained during the preliminary screening with other reagents, are listed in Table 111. ACKNOWLEDGMENT
The authors thank E. J. Seus for fruitful discussions, and Donald Ketchum and the Analytical Department of the Research Laboratory of the Eastman Kodak Co. for microanalyses. Gifts of t-butyl- and t-octylamines from
(8) Kidd, D. A. .4.,J . Chem. SOC.1961, p. 4678. (6) Langford, R. B., Lawsm, 11. D., J. Chem. Edzcc. 34, 510 (1957). (7) Neville, K . G., J . Org. Chem. 23, 937 (1958). (8)Willems, J., Bull. SOC. Chzm. Belg. 64,747 (1955); C.A. 50, 16669 (1956).
Rohm & Haas, and of propyl and tripropyl sultones from Shell Chemical Co. are gratefully acknowledged. LITERATURE CITED
(1) Bohme Fettchemie G.m.b.h., British patent 764,340;C.A. 51, 12956 (1957).
(2) Brown, E. L., Campbell, Neil, J . Chem. SOC.1937, p. 1699. (3) Fischer, R. F., Znd. Eng. Chem. 56, 41 (1964). ( 4 ) Helberger, J. H., blanicke, G., Hayden, R., Ann. 565, 22 (1949).
C. F. H. ALLEN C. F. ~ I U R P H Y W. E. YOERGER Rochester Institute of Technology Rochester, N. Y.
Use of Tetrachlorophthalirnide for Identification of Alkyl Halides and Alkyl Sulfonates SIR: The use of tetrachlorophthalimide for the identification of many types of aliphatic halogen compounds by heating in the absence of a solvent was described in an earlier paper ( 2 ) . The long heating period and the use of sealed tubes in some instances may have detracted to a certain extent from its many advantages. The current availability of ionizing solvents, such as dimethylformamide and dimethyl sulfoxide, and of the tetrabromoand tetraiodophthalic anhydrides led us to re-examine this useful reaction. Dimethylformamide greatly reduces the time required for a Gabriel synthesis (3-6, 8 , 1 2 ) . This advance has been confirmed in this paper; derivatives for the purposes of identification can now be prepared in 1 hour or less. Furthermore, most of the now commonly available long chain aliphatic halides have been converted to suitable reference compounds. Only one halogen of a , w-dihalides reacts under t'he conditions adopted; this is a well known characteristic of the Gabriel synthesis. Bifunctional halogen compounds such as chloroacetamide, ethyl chloroacetoacetate, and others have been successfully derivatized. When 2-chloroethyl p-toluenesulfonate was employed, only the sulfonate group reacted, resulting in a 2-chloroethyl imide. The reaction showed that the halogen was less active in the alkylation and also indicated that derivatives from esters of sulfonic acids could be readily prepared. The rate of reaction of sulfonic acid esters in dimethylformamide is surprisingly rapid; furthermore, pressure is not required because the lower members-e.g., methyl methanesulfonate-are not nearly so volatile as the corresponding halides. These observations suggest a useful laboratory synthetic route for conversion of a primary alcohol, via the est,er, to a primary amine using phthalimide itself. I h i e t h y l sulfoxide was examined 158
ANALYTICAL CHEMISTRY
for 1 hour a t each of two temperatures. At 95" C. (steam bath) the reaction took place smoothly, but a t the boiling point there was considerable decomposition with production of much color. The yields of derivatives were good in both instances. However, the possibility of side reactions with dimethyl sulfoxide (6, 11) makes dimethylformamide the preferable solvent until dimethyl sulfoxide has been shown not to interfere. The marked advantage of using dimethyl sulfoxide a t the lower temperature is the fact that such low boiling halides as methyl iodide and ethyl bromide can now be easily converted to imides without the use of a sealed tube. Because N , A"-bisphthalimidoalkanes are not produced, even as contaminants in the Gabriel reaction, and because sulfonate esters react more readily than halides with the reagent (potassium tetrachlorophthalimide), bissulfonates (1) should form such bisalkanes more easily. However, up to now, analytically pure bis compounds have not been obtained; the main contaminant, tetrachlorophthalimide, persists throughout numerous recrystallizations of the scale employed for making identifications. To learn the effect of change of halogen upon the melting point of derivatives, tetrabromo- and tetraiodophthalimides were examined in three instances. The melting point was raised 30" to 36" C. for each change (Table I). A slight spread was observed with long chain derivatives of the tetrabromoiniide. A greatly decreased solubility and apparent evidence of decomposition led to the exclusion of a further study of iodo derivatives. However, in view of the large number of tetrachloroimides already described, the general use of the chlorinated reagent has been retained; the brominated derivative might be useful in a few instances, such as with the long chain halides.
Table I. Melting Points in "C. of Derivatives from Different Haloimides
Substituent C1, -(CH,)&?: 194 130 -Ci,H,g -Ci6H33 ( 1 ) 129-30
Br, 230-2 167 163-4
14
264-7 ( d ) 201 ( d ) 197 ( d )
EXPERIMENTAL
Reagents. The three tetrahalophthalimides were obtained by the previously described procedure (1) or by the use of formamide (9, 10). The second was preferable for the bromo and iodo imides; iodine vapor was noticed if overheating occurred (9). The potassium derivative of the iodinated imide was best prepared by the addition of methanolic potassium hydroxide to a dirnethylformamide solution of the imide. All the bromo and iodo imides were recrystallized from dimethylformamide. The alkyl halides and sulfonates used were Eastman chemicals, or were prepared by standard procedures. With the higher alkyl ptoluene-sulfonates, the crude ester remaining after evaporation of the solvent from the ether extract ( 7 ) was used directly-undecyl p-toluenesulfonate, f.p., 30" to 32" C. A specimen of dodecenyl chloride from Rohm and Haas was used-b.p., 108" C. 3 mm., n ~ *= j 1.4617. I3ismethanesulfonates are described in a patent (1).
of Derivatives. &IT BOILINGPOIKT. The potassium
Preparation THE
tetrahaloimide, 0.009 mole; a slight molar excess of halogen compound or sulfonate; and 15 ml. of dimethylformamide or dimethyl sulfoxide were thoroughly mixed and refluxed for 1 hour. X h e n the sulfonate esters in dimethylformamide were employed, the temperature began to rise rapidly a t 80" to 90" C., and the appearance of the suspended solid changed (gelatinous) by 130" C. The mixture was then quenched with 50 to 70 nil. of water to precipitate the product, which was extracted with 50 to 75 nil. of acetone or 25 to 50 nil. of chloroform. Chloroform, however, did not dissolve the
Table II.
Properties of N-Substituted Tetrachlorophthalamides
Analyses M.P., "C.
Substituent
1 135-6'3' 135-6 * 130a 167d 201d 163-4' -
1
-CH;C€LCl -CH,CH2CHzCH,Cl -CHzCH,CH2CN -CHzCH2CH2CN -CH&ONH2
L "
Form u 1a CieHniClrXOt CisH2rCIaXOz C2oH2,CLS02 CzzHzqC14N02 C22H29Br4?i02 C22H291aX92 C.,H,,RrA O1
.
Calrulated H
C 50 51 53 55 40
9 8 0 0 2
42 0
4 5 5 6 4
9 2 7 0 4
4 9
c1 33 32 31 29 48 60 46
4 3 4 6 5. 0' 5"
Found Yc H
C 51 52 52 55 40
0 0 7 3 2
5 5 5 6 4
41 7
0 5 4 2 4
CT 33 32 31 29 48 59 46
4 7
7 4 2 6 Se
9, 3e
"
1 1 50 9 34 5 11 50 9 CioHaCl5XOz 34 6 38 4 2 2 47 3 38 2 2 3 47 0 CI2HsCIaN02 27 2 1 1 60 46 27 1 1 3 60 1" C,,HeBrrN202 ' 19 8 0 8 71 Of 0 8 70 7 1 C12HJaS292 20 1 35 2 1 2 41 4 35 4 CtnHLCLh 2 0 9 1 2 41 7 3.0 35 7 41 9 -cH;coK(GH,)~ C;;H;,ci,s2o, 42.2 3.0 3F,. 5 -CH( CIBHBB)COOCIHO C?~H,&lrN04 56.5 6 6 24.9 56.3 6.5 24 6 -CH( Ci6H3r)COOC2Hs C2sH,,Bra?iOc 4 1 . 4e 41.8" 40.4 2.3 34 1 -CH( COCH,)COOC2H, 40.7 2.2 34.4 CiaHsClrN05 ' From chloroform-methanol. c Kork of bV. E. Yoerger. From dimethglformamide. Bromine. Iodine. a From acetone. K, calcd., 3.7%; found, 3.55;. C1 deriv., m.p. 194 ( I ) . 1 N, calcd., 8.2Yc; found, 7.9%. From ethanol. From acetic acid. K,ralcd. l.8Yc; found, l . 8 7 c .
203c 153-4' h 230-20,' 264-7 300-2' 1 240-lk 609 68-71',' 173-4 *
'
Q
unused reagent. If the imide did not crystallize well, other solvents were employed; these are included as footnotes in Table TI, which lists properties of the neFv imides. Decolorizing carbon was used where needed. The volume of diniethylforniamide was increaied to 20 ml. for the bromo imide, and to 25 ml. for the iodo analog. IK THE STEAMBATH. The potassium salt, 1 gram, and 10 nil. of pure dimethyl sulfoxide were thoroughly mixed, 2.5 ml. of the halide were added, and the mixture heated was for 1 hour under a reflux condenser. After the mixture cooled, 10 ml. of water were added to the paste and the product was collected on a filter and rinsed (if colored) with 10 nil. of saturated aqueous sodium bisulfite. This product was then recryitallized as usual. (The smallest amount of halogen compound that was converted to a satisfactory derivative is 0.9 gram.) The use of the new solvents did not result in the formation of derivatives that were unattainable by the earlier
procedure. Other solvents that did not react or gave noncrystalline products were ethyl a-bromomyristate, ethyl dichloroacetate, bromochloromethane, and dichloroniethane. Chloroacetaldehyde diethylacetal gave a black solution ACKNOWLEDGMENT
We thank W.E. Yoerger for much of the work with the alkanesulfonates, and for some of the reactions a t 95' C.; and Donald Ketchum and the Analytical Department of the Research Laboratory of the Eastman Kodak Co. for the microanalyses. We also thank Rohm & Haas for a specimen of dodecenyl chloride.
(3) Billman, J. H., Cash, K. V.,Zbid., 75, 2499 (1953). ( 4 ) Zbid., 76, 1944 (1954). ( 5 ) Huebner, C. F., U. S. Patent 3,025,300 (19621: C. A . 58.3398 11963). ( 6 ) Hunsberger, I: 11.) Tien, J. l l . , Chem. Ind. 1959, p. 88. ( 7 ) Marvel, C. S., Sekera, V.C., ,'Organic Syntheses," E. C. Homing, ed., Coll. Vol. 3, p. 366, Wiley, New York, 1955. 18) Xefkens. G. H. L.. Tesser. G. I . Nivard, R . F. J., Rec.'Trav. Chem. 79; 688 (1960). ( 9 ) Pratt, D. S., Perkins, G. A,, J . d m . Chem. SOC.40, 205 (1918). (10) Pratt, D. S., Young, C. O., Zbid., p. 1417. (11) Ranky, W. O., Selson, 11. C., "Organic Sulfur Compounds," N. Kharasch, ed., Vol. 1, Chap. 17, Pergamon Press, New York, 1961. (12) Sheehan, J. C., Bolhofer, W.A , , J . Am. Chem. SOC.72, 2786 (1950). ~I
~
C. F. H. ALLEX W. R. ADAMS C. L. MYERS
LITERATURE CITED
(1) Allen, C. F. H., Laakso, T. T. .M., U. S. Patent 2,816,125 (1957). ( 2 ) Allen, C. F . H., Nicholls, R. V. V., J . Am. Chem. SOC.56, 1409 (1934).
Rochester Institute of Technology Rochester, N.Y .
Liquid Scintillation Counting of H3-Nucleic Acids SIR: Liquid scintillation counting of biological compounds such as sugars, amino acids, and proteins has been described by many workers ( I , 2, 4, 6, I O ) . The methods usually employed toluenehyamine or dioxane-water solvent systems to keep the compounds in solution. .i system including hyamine in the dioxane-a ater solvent which can be used for a number of low molecular organic compounds, including nucleic acid bases, nucleosides, and nucleotides, has been reported from this laboratory ( 9 ) . Vnfortunately, however, thls syhtem could hold only a minute amount of nucleic acids and was not suitable as a general method for the radioassay of H3-nucleic acids.
The present method, which employs digestion of the nucleic acid samples by hydrochloric acid prior to the addition of hyamine-dioxane solvent system appears to be a convenient method as a routine radioassay of nucleic acids. EXPERIMENTAL
The liquid scintillation spectrometer used in this experiment was a Packard Tri-Carb model 314.1X equipped with an automatic sample changer (Packard Instrument Co., Inc., La Grange, Ill.). Dioxane scintillator solvent consisted of 100 grams of naphthalene, 10 grams of PPO and 250 mg. of POPOP in 1 liter of dioxane ( 1 1 ) . Toluene scintillator solvent contained 4 grams of
PPO and 100 mg. of POPOP in 1 liter of toluene (8). Hydroxide of hyamine lox, 1 molar solution in methanol was purchased from the Packard Co. (this material will be called hyamine for convenience). H3-nucleic acids were prepared from Pseudomonas cells (adenine requiring mutant) which were grown in a medium containing H3-adenine. The nucleic acid fraction was purified by the sodium dodecyl sulfate-phenol method ( 5 ) . Vnless otherwise stated, this material with or without cold nucleic acid fraction prepared from Pseudomonss cells was used as a radioactive sample without further purification. Chromatographic analysis of this preparation with a methylated albuminkieselguhr column ( 7 ) revealed that this VOL. 37, NO. 1 , JANUARY 1965
159
