1602
DAVIDPRESSMAN, ARTHURB. PARDEEAND LINUSPAULINC
A n d . Calcd. for ClaHsClnO: C, G2.18; H, 3.21; C1, 28.24. Found: C, 62.41; H, 3.62; C1, 28.35. The same ketone was prepared by heating a t reflux a mixture of bis-(o-chloropheny1)-carbinolarid sodium dichromate in sulfuric acid and acetic acid for three hours. The neutral fraction gave upon distillation a nearly quantitative yield of oil, b. p. 127-135” (1 nim.), which up011 crystallization from ethanol, melted a t 4 5 . 4 4 6 . 6 (cor.). 2,4-Dinitrophenylhydrazones of the Isomeric Dichloro1.0 g. (0.0073 mole) of 2,4-dinitrobenzophenones.-To phenylhydrazine dissolved in 2 ml. of concentrated sulfuric acid 15 ml. of 95% ethanol was added; this solittion was then mixed with a solution of 1.25 g. (0.005 mole) of the ketone dissolved in 25 ml. of 95y0 ethanol. In most cases crystallization was complete in one to two hours; the o,o’-dichlorobenzophenonewas allowed to stand overnight as crystallization was very slow. Yields were not calculated, but were high. The crude 2,4-ditiitrophenylhydrazones were dissolved in hot pyridine and recrystallized by the addition of warm 95% ethanol. This is similar t o the method of Brady.@ The properties of the dinitrophenylhydrazones are given in Table IV.
Summary
TABLEI V 2,4-DINlTROPHENYLHYDRAZONESO F ISOMERIC DICHLOROBENZOPHENONES
Isomer
-M.
p . of derivativeCrude, ‘C. Pure, ‘C
200-205 206-208 225-228 230-231 0,P‘ o,m’ 253-257 255-257 vz,nl’ 234-238 235-238 253-256 258-260 m,p‘ p,p‘ 195-203 238-240 Calcd. for CIgH120aNdC12: C, O,O’
[CONTRIBUTION FROM
THE
-Analyses,” C H
%-
53.3 2.8 2.8 52.8 52.8 3.0 53.2 2.8 2.8 52.9 53.3 2.8 52.9; H, 2.8; N,
CI
13.2 13.1 12.9 12.9 12.8 13.2 13.0.
Lesser amounts of twelve other organic impurities have been found, the presence of which may be explained on the basis of side reactions involving chloral, chlorobenzene, sulfuric acid, aild impurities in the starting materials. Work on the proof of structure, including synthesis, of the by-product materials, is described. The o,p’- and the nz,p’-isomersof p,p’-DDT and various derivatives of these compounds are described. The synthesis of all the isomeric dichlorobenzophenones with one chlorine atom on each ring and of the 2,4dinitrophenylhydrazones of these ketones is described.
Studies have been made of the composition of several samples of technical D D T and a sample of “by-product oil” recovered from a process of refinement of crude D D T prepared from “chlorinated alcohol” and chlorobenzene. Technical DDT has been found to contain upwards of 707, of 1trichloro-2,2-bis-(p-chlorophenyl)-ethane (p,p’DDT), the most active insecticidal ingredient. The major impurity is 1-trichloro-2-o-chlo- BELTSVILLE, MU. MASS. rophenyl-2-p-chlorophenylethane (o,p’-DDT). CAWBRIDGE, COLLEGE PARK,MD. COLTJMBUS, OHIO
(29) Brady, J . Chcm. Soc., 756 (19311.
Vol. 67
RECEIVED MAY18, 1945
GATESAND CRELLINLABORATORIES OF CHEMISTRY, CALIFORNIA INSTITUTE OF TECHNOLOGY, No. 1006]
The Reactions of Antisera Homologous to Various Azophenylarsonic Acid Groups and the p-Azophenylmethylarsinic Acid Group with Some Heterologous Haptens* B Y D A V I D PRESSMAN,
ARTHURB. PARDEE AND LINUSPAULINC
Recently we have made quantitative studies of the combining powers of substituted phenylarsonic acids and of naphthylarsonic acids with antisera homologous to the 0-,m-,and p-azophenylarsonic acid g r o ~ i p s ~(anti-R,, ~~J -JL,and -Rp sera, respectively) and to the p-(+azophenylazo)phenylarsonic acid gr0upZt4 (anti-R; serum). The quantitative studies of these antisera have now been extended to include their reactions with substances related to phenylarsonic acid but in which the aromatic nucleus attached to the arsenic atom of the arsonic acid group is replaced by an aliphatic or alkaryl residue, or one of the ;* The Serological Properties of Simple Substances. XI. For h-0. X of this series see THISJOURNAL, 67, 1219 (1945). (1) D. Pressman, D. H Brown, and I,, Pauling, {bid., 64, 3015
(1942). (2) L. Pauling, D. Pressman, and A . I.. Grossberg. i b i d . . 66, 784 (1911). ( 3 ) L. Pauling and D . Pressman, i b i d . , 67, 1003 (1945). ( 4 ) D. Pressman, J. T . Slnynard, .9. i.. Grosshern, nnd I,. I‘diilinp, *bid.,66, 72K IlUJR)
hydroxy groups of the arsonic acid is replaced by a second organic residue to form an arsinic acid, or the arsonic acid group is altered to form the dimethylarsine dihydroxide or the arsine oxide group, or the arsenic atom is replaced by another atom (phosphorus, antimony, or sulfur). We have studied also the reactions of antiserum honiologous i o the p-azophenylmethylarsinic acid group (anti-RcH, serum) with several heterologous substances. Soirie similar reactions of antisera with heterologous substances have been studied qualitatively by other investigators. Erlenmeyer and Berger6 found that an antiserum to horse serum coupled with diazotized p-aminophenylarsonic acid formed precipitates with ovalbumin coupled with diazotized p-aminophenylphosphonic acid as well as with the antigen from diazotized p-aminophenylarsonic acid, but not with the antigen from diazot ized p-arninophenylstibonic acid, Haurowit? ’ 5 ) H Frlrnmever and R Rergrr. Riochrm Z . , 266,420 (1932)
Sept., 1945
SEROLOGICAL PROPERTIES OF SIMPLE SUBSTANCES
1603
Phenyldimethylarsine dihydroxide was prepared by addand B r e d a tested the inhibition of precipitation ing silver oxide to a solution of the hydroxybromide (see of anti-Rp serum and Rp-antigen by methylar- Steinkopf and Schwen13). The resulting solution was sonic acid, dimethylarsinic acid, di-(p-amino- used directly in the hapten inhibition experiments. phenyl)-arsinic acid, p-hydroxy phenylarsine oxide, Phenylphosphonic acid was prepared y! the method of p-aminophenylarsine oxide, p-acetaminophenyl- Michaelis14: m . p., 158'; reported, 158 . Phenylstibonic acid was prepared by the method of stibonic acid, 2-methyl-4-dimethylaminophenyl- Schmidt.l6 phosphonic acid, phosphoric acid, and arsenic Discussion acid, and found that no inhibition was produced Data concerning the hapten inhibition of preexcept a slight inhibition by arsenic acid. In the work reported here we have determined cipitation of anti-R,, - R m , -R$, and -RA serum quantitatively the combining powers of the sub- with R,-, Rm-, R9-, and Ri-ovalbumin, respecstances studied with antibody by the application tively, are given in Table I, of anti-R9 and -RL of the theory of heterogeneous antisera,2 which serum with "Chrom" R;,l6 in Table 11, and of permits the evaluation of the average inhibition anti-RcH, serum with RcH,-ovalbumin in Table constant KA and of the index u of the effective 111. The serum pools of Tables I and I1 are different. Where sufficient inhibition was obheterogeneity of the antiserum. served to permit the calculation of the heteroExperimental Methods geneity constant Q and the hapten inhibition conProtein Antigens.-The RcH,-sheep serum for inocula- stant K,' by the application of the heterogeneity tion was prepared by diazotizing 0.1, 0.2, and 0.3-g. por- t h e ~ r y these , ~ values have been included in the tions of p-aminophenylmethylarsinicacid, coupling each tables. portion with a 50-1111. portion of sheep serum a t pH about 9, The Effect of Replacing the Arsenic Atom by and then mixing the three preparations. The antigen was purified by precipitating twice a t pH 4 from 150 ml. of Phosphorus, Antimony, or Sulfur.-Their nearly solution and finally dissolving a t pH 8 in saline solution. equal values of Ko' strongly indicate that the The Rca,-ovalbumin test antigen was prepared by diazo- structures of phenylarsonic acid and phenylphostizing 0.18 g. of p-aminophenylmethylarsinic acid and phonic acid in aqueous solution a t pH 8 are coupling the product with 2 g. of crystallized ovalbumin a t PH 9. The azoprotein was precipitated three times with closely similar. Our results substantiate the obacid from 50 ml. of solution and was then brought to pH 8. servation by Erlenmeyer and Berger6 that pThe Ro-, Rm-,Rp-, and RL-antigens have been described azophenylphosphonic acid antigens form prepre~iously.~.~ cipitates with anti-R,, sera. The failure of HauroAntisera.-Anti-&a, serum was prepared by a method witz and Brein16to observe inhibition of precipitasimilar to that described previously7 for the preparation of anti-R, sera. The preparation of anti-R,, -Rm, -Rp, and tion of an anti-Rp serum with an Rp-antigen by a -Rk sera has been described e l s e ~ h e r e . ~ . ~Two ~ 7 different substituted phenylphosphonic acid can be atpools of anti-R, serum and of anti-RL serum were used. tributed to the steric effect of the ortho methylReaction of Antiserum with Antigen and Hapten.The reactants were mixed and permitted to stand for one group in the substance they used, 2-methyl-4hour a t room temperature and over two nights at 5". dimethylaminophenylphosphonicacid. The precipitates were centrifuged, washed three times with It is interesting that phenylphosphonic acid 10-ml. portions of 0.9% sodium chloride solution, and analyzed by our standard method.* All dilutions were combines with the antisera somewhat more strongly than does the homologous phenylarsonic made with borate buffer solution' of pH 8.0. acid. A reasonable explanation of this observaPreparation of Substances tion can be proposed. The phosphonic acid group The substances not described below have been described is smaller than the arsonic acid group, and accorde l ~ e w h e r e lor , ~ were commercial products. ingly can easily enter a cavity large enough to Benzylarsonic acid was prepare? by the method $f Quick and Adamso: m. p., 166-168 ; reported, 167-168 , accommodate an arsonic acid group. Due to the Phenylmethylarsinic acid was prepared by the method smaller size of the phosphonic acid group, its of Bertheimlo; m. p., 177-179.5'; reported, 179.5'. phinophenylmethylarsinic acid was prepared by th,e negative charge in the ionized state might apmethod of BertheimlO: m. p., 201.5-202.0"; reported, 201 . proach more closely to the positive charge which p h i n o p h e n y l a r s i n e oxide was prepared by the method is probably situated on the antibody molecule in of EFlich and Bertheim": m. p., 90-100"; reported, 80the neighboring region, and the increased coulomb 100 . Phenyldimethylarsine hydroxybromide was prepared by attraction might thus lead to a greater combining dissolving 7.2 g. of phenyldimethylarsine (prepared by the power with the antibody. However, in order to method of Winmil112)in 150 ml. of ligroin (SO-loo"), and bring this effect into play, the attached benzene adding 10 ml. of water and a 25y0 solution of bromine in petroleum ether until a permanent yellow color was ob- nucleus is shifted from the position in which it can exert the optimum combining force. This tained. The aqueous phase was separated and evaporated. The residue was recrystallized twice from 85% shift takes place with a decrease in the total freeacetone solution: m. p., 161-162'; reported, 162". energy of combination only with those antibody (6) F. Haurowitz and F. Breinl, 2.fihysiol. Chem., 214, 111 (1933). (7) L. Pauling, D.Pressman, D. H . Campbell, C. Ikeda, and M. Iknwa, THISJOURNAL, 64, 2994 (1942). ( 8 ) D. Pressman, I n d . Eng. Chem., A n a l . E d . , 16, 357 (1943). (9) A. J. Quick and R . Adams, THISJOURNAL, 44,811 (1922). (10) A. Bertheim, Bcr., 48, 860 (1916). (11) P.Bhrlich and A. Bertheim, ibid., 48,919 (1010). (12) T.F. Whmill, 1.Chrm. Soc., 101, 723 (1912).
(13) W.Steinkopf and G. Schwen, Ber., 64,2795 (1921). (14) A. Michaelis, ibid., 6, 810 (1873). (15) H. Schmidt, A n n . , 421, 174 (1920).
(16) "Chrom" R;r is BOIS
DAVID PRESSMAN, ARTHURB. PARDEE AND LINUSPAULING
1GO4
Vol. 67
TABLEI EFFECTOF HAPTESS ON
-Rm, -Rp, AND
TIIE PRECIPITATIUX U F .%NTI-& FILXINS,
-l
