Hapten Inhibition of Precipitation of Antisera Homologous to the o-, m-, and p-Azobenoic Acid Groups Citation Hall, Luther Axtell Richard (1946) Hapten Inhibition of Precipitation of Antisera Homologous to the o-, m-, and p-Azobenoic Acid Groups. Master's thesis, California Institute of Technology. doi:10.7907/1k41-bj64. https://resolver.caltech.edu/CaltechTHESIS:05022025-184306892 Abstract No abstract. Item Type: Thesis (Master's thesis) Subject Keywords: (Chemistry) Degree Grantor: California Institute of Technology Division: Chemistry and Chemical Engineering Major Option: Chemistry Thesis Availability: Public (worldwide access) Research Advisor(s): Pressman, David Thesis Committee: Unknown, Unknown Defense Date: 1 January 1946 Record Number: CaltechTHESIS:05022025-184306892 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:05022025-184306892 DOI: 10.7907/1k41-bj64 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 17195 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 06 May 2025 00:30 Last Modified: 06 May 2025 00:35 Thesis Files PDF - Final Version See Usage Policy. 16MB Repository Staff Only: item control page

E.A.TTElJ IlifE I BIT I Ol~ OJ? :l:'1~: CIJ:='I 'I'AT IOH 0::&1 Ah 'l'I SERA EO~JOLOGOUS TO 'lTiJ~ .9.- , rg- , AhD ]2-AZOBEHZOIC AC ID GROUPS Thesis by . Luther Axtell Richard Hall In Pe.rtial :f·ulfilment of t h e Requirements for t h e Degree of lfa,ster of Science California Insti tu.te of Technolo gy Pasadena, California 1946 i To l:I01f ACl~lJ OWLEDGlIBlTT The author wishes to express his deepest a ppreci a tion and sincerest gratitude to Dr. David Pre s sman for his understa nding patience, invaluable hel p , constructiv e criticisms, and many kindnesses without which the successful completion of t h is re s earch would not have been possible. 'l'o Er. Dan Rice are due many thanks for his 4-chloro- 2i-aminoben zoic a.cid e,nd h is i mporta nt work on t h e protein analyses. To any a nd all others who h ave in any way directly or ind i r ectly assisted in t ~e comp letion of t h is research go t h e sincere gr a titud e of t h e author. ii T.AJ3LE OF COHTE:t~TS ACKHOWLEDG:i:ZEl~T T.ABL"E OF COHTEi~TS i ii INTRODUCTION 1 EXJ?ERD!J};NT.AL 4 DISCUSSION 28 39 BIBLIOGRAPEY 41 Table T.ABLE I TABLE II 27 TABLE III 29 INTRODUCTION 1. Landsteiner and his coworkers investigated the effect of position of substituents in the benzene ring of haptens and haptenic groups on the precipitation reactions of antisera and azo:proteins and the inhibition of precipitation by haptens during their extensive studies of the properties of antisera prepared by injecting animals with artificial conjugated antigens containing groups of known chemical structure. 1 They found that combination of antiserum and antigen or hapten is usually decreased by the presence of substituent groups in the precipitating antigen or hapten in positions other than those occupied in the immunizing antigen. Similar results have been obtained in work by Pauling, Pressman and collaborators with the anti, . . 2,3,4 ( sera homologous to the 2-azophenylarson1c acid, £- £-azophenylazo)phenylarsonic acid, 3 ' 4 £-azobenzoic acid, 5 and the .2,-, ~~-, and r-azophenylarsonic acid groups. 6 In order to learn more about the intermolecular forces o~erating in these serological systems and to learn more concerning the steric configuration of the antibody-hapten complex, this work was undertaken with the quantitative hapten inhibition studies of antisera homologous to the£-,~- and ~-azobenzoic acids using haptens containing both the carboxyl and azo groupings. Previously ha:pten inhibition work was done with simple substances usually containing only one homologous charged grouping. This group is presumably held in the antibody-hapten complex by a charge on the antibody of the opposite sign, by hydrogen bonds and by van der Waals forces due to the intimacy and close proximity of this grouping to the antibody formed by an homologous immunizing antigen. 7 The remaining portions of the homologous hapten are held in the antibody-hapten complex by hydrogen bonds and by van der Waals forces of attraction. 'l'he presence of a substituent,not homologous, causes steric hindrance effects. Previously various substituent groups were placed on the arsenic acid or the benzoic acid haptens and the steric effect of substitution on the combination of the antibody and haptens was discussed from the standpoint of radial dilatation required for such a hapten to fit into the antibody molecule. 6 These results were ambiguous as to showing exactly how the hapten might fit into the antibody in the cases where there were two orientations which the hapten could assume in combining v:.'i th the antibody-(e. g . ortho substituted haptens could fit into meta antiserum in two ways, meta substituted haptens could fit into ortho antiserum in two ways, etc.). To eliminate ambiguity as to how a hapten with a substituent group in a non-homolo gous position oriented itself in the antibody, the Q-, ~-, and £-(~-hydroxyphenylazo)chlorobenzoic acids were prepared and used as haptens in inhibiting the specific precipitation of antiserum specific to the _Q-, ~-, and ~-azobenzoic acid groups - hereafter called anti-Xo,-Xm, and Xp sera respectively. Theve acids have two groups(carboxyl and azo) homologous to the immunizing antigen which determine their orientation when combining with antibody prepared from antigen having the azo and carboxyl in the s~~e positions. Hence these two groupings firmly attracted to the antibody by strong forces of a more specific nature anchor the hapten in a definite configuration in the complex precluding the possibility of the hapten orienting itself in more than one way with the antibody molecule. With the hapten oriented in the antibody in a known way, the placement of a chloro group on the benzene ring in some kno~n position and the use of the subsequent hapten in the inhibition studies would give a measure of the steric effect of the substituent on the combination of antibody ~nd hapten. All the monochloromonoazobenzoic acids except ~-chloro4-azobenzoic acid were prepared and their-relative combining powerswi th the antibodies were determined. The results of the investigation are given in this thesis. EXPE1UI0'GNTAL 4. S11'.0?LE HAPTEHS: The benzoic acid used as hapten was a Merck product of acidic equivalent weight of 123.1 and a melting point(hereafter designated by M.F.) of 119.5-120°c. Theoand ~-(.r-hydroxyphenylB,zo)benzoic acids were prepared by Dr. A. Recsei a.nd the 12-(12-hydroxyphenylazo)benzoic acid was prepared by Mr . John Bryden by the coupling of diazo- - - tized o-, m-, and .... u-aminobenzoic e.cids v:i th phenol. The preparation of the chloro substituted haptens is discussed later in this thesis. PROTEIN .AKTIGENS: The immunizing antigens used for inoculations were prepared by diazotizing 2 gre,ms of .9.-, m- and .:12-aminobenzoic a.cid a nd coupling with 200 ml. of beef serum at pE about 9. The product was purified by the method of Landsteiner and van der Scheer. 8 The azoprotein test antigens were prepared by diazotizing o. 2 gra.ms of .9.-, Q'!- &.nd 12-aminobenzoic acid and coupling the product to 3 grams of ovalbumin at pE about 9. The azoproteins were dialyzed against tap water overnight, precipitated twice at pH 3.5 and finally dissolved at pH 7 in saline. ANTISERA.: Antisera were prepared by injecting rabbits with beef serum coupled to the .Q-, m- and 12-a.minobenzoic acids. The method v:as similar to that described previously for the preparation of anti-phenylarsonic acid sera. 9 The sera were pooled according to titer. A single pool of each antiserum was used for these experiments. 5. ANTIGEN-ANTIBODY REAC'l'IOli OPTiliiiuM: For these inhibition ex- periments it was desired to find the concentrations of the antiserum and antigen that would give the optimum( that is t he greatest) amount of precipitate wh en they were mixed . Tnerefore preliminary tests were set up for the precipitation of an ti-Xo, -Xm a nd -Xp sera wi th Xo-, X11- and Xp-ovalbumin antigen solution. To 1 ml. of antiserum and 1 ml . of borate buffer( giving supernates of pH 8.0) was added 1 ml. of antigen solution(in saline) of different concentrations. The mixture stood two hours at 37°c and over two ni gh ts in the refrigerator. Cross reactions were: The anti-Xo serum gave appreciable preci pitate with Xm-ovalbumin, but none with the Xp-ovalburnin. The £,,nti-Xm serum gave very slight precipitates v:i th Xo-ova,lburnin and none v>ii th the Xp-ovalbumin. The antiXp-serum gave appreciable precipitate with the Xm-ovalbwnin and none v.ri th the Xo-ovalbun1in. In table I below the optimums observed for each antiserum and antigen are listed. TABLE I Amount of anti~:en added in EB. ~tiserum 4.7 9.4 18.8 37.5 75 150 300 A11ount of precipitate in ):lga Xo-ovalbumin an ti-Xo 11 40 119 199 315 216 76 Xm-ovalbumin anti-Xm 43 102 176 199 165 87 41 Xp-ovalbumin anti-Xp 64 117 245 314 363 363 132 a. Values are averages of triplicate analyses with a mean deviation of± 4% and are corrected for blanks of serum and buffer - 12, 12 a nd 10 µg for the Xo-, Xrri- and Xp- systems respectively. 6. REACTION OF .AJ.{TIS:SRlJM ·wrTH .ANTIGJ£H A.1!D HAPTEN: The hapten, antigen and antiserum were mixed in the proper concentrations(using 1 ml. of each) and after shaking were allowed to stand at 37°C in a water bath for two hours and then at 50c over two nights in a refrigerator. The precipitates formed were centrifuged and washed three times with 10 ml. portions of saline(0.9% sodiwn chloride) solution and were then analyzed by the standard method. 10 PROTEIN ANALYSIS: From a burette 2.5 ml. of 1.00 N sodium hydroxide solution were added to the precipitate after the wash solution had been decanted off. The precipitate dissolved and the solution was transferred to a 15 ml. graduated centrifuge tube using water to wash. The total volume now was about 6 ml. The tubes were heated for 8 minutes in a boiling water bath and then cooled for 5 minutes in a cold vvater bath. The volume was then made up to 7.5 ml. using distilled water. To each tube was added rapidly 2.5 ml. of the Folin reagent(prepared as described by Falin and Ciocalt,u 11 and diluted with two volumes of water) making the total volume 10 ml. The tubes ·were shaken immediately using rubber stoppers and after allovi.ring to stand just 10 minutes(in order to secure the maximwn blue color caused by the Folin reagent and the protein antibody), the color intensity which is a measure of the amount of antibody present was read vri th a Klett-Summerson photoelectric colorimeter using a red filter transmitting 640-700 mf• The amount of protein in pg(micrograms) was obtained by converting the Klett reading by a linear relation. 7. PREP.AHATIOH OJ? SUBSTANCES: The haptens that were used in these experiments were prepared by the coupling of phenol with the diazotized chloroa,minobenzoic acids. There are ten monochloromonoaminobenzoic acids all of which were prepared except the 3-chloro-4-aminobenzoic acid. Their preparations are described below. All observed melting points are corrected for exposed stem. 2-Chloro-3-aminobenzoic acid COOE:C . 1 O :rrn: 2 The starting material obtained commercially was 2-amino3-nitrotoluene. For the diazotization 80 grams(0.51 mols) of it were suspended in 77 ml. of cone. hydrochloric acid and 157 ml. of water. 12 An additional 137 ml. of cone. hydrochloric acid were added. The mixture was cooled to 2°c in an ice bath and 32 grams(0.51 mols) of sodium nitrite dissolved in 40 ml. of water were added dropwise with vigorous stirring over a period of several hours. After 4 hours at 2°c with vigorous stirring and with a slight excess of nitrite present(tested for by the starch-iodide test), the material was filtered to remove any unreacted 2-amino-3-nitrotoluene. The filtrate was carefully added to a solution of cuprous chloride in water and cone. hydrochloric acid. (The cuprous chloride solution was made by dissolving 160 grarns(0.63 mols) of crystallized copper sulfate and 54 grams(0.93 mole) of sodium chloride in 575 ml. of water. Th.e cuprous chloride was precipitated when 26 grams 8. (0.22 mols) of sodium bisulfite dissolved in a little water were added. The cuprous chloride was centrifuged, washed and dissolved to form Cuc1 2 - in 260 ml. of water and 200 ml. of cone. hydrochloric acid. 12 ) Nitrogen was evolved and a heavy oil was formed when the diazoniwn salt decomposed in the Cuc1 2solution. The oil was steam distilled to give pure 2-chloro-3nitrotoluene. A yield of 70 grams(85%) was obtained. M.P.= 21.5°0 1 reported= 21.5°c 13 The 2-chloro-3-nitrotoluene was oxidized to 2-chloro-3-nitrobenzoic acid by refluxing with permanganate. 14 A mixture of 25 grarns(0.146 mols) of 2-chloro-3-nitrotoluene and 54 grams (0.34 mols) of potassium permanganate in 1 liter of water was refluxed for 5 hours on an oil bath with vigorous stirring till all the permanganate was converted into manganese dioxide. From the original 25 grams, 9 grams of 2-chloro-3-nitrotoluene were recovered by steam distillation. The manganese dioxide formed was filtered off a nd washed with 500 ml. of hot water. The hot combined filtrates were acidified with 6N hydrochloric acid to pR 2. The 2-chloro-3-nitrobenzoic acid crystallized out upon t h e cooling of the solution. The acid was recrystallized from water. A yield of 10 gre.ms(55% on the basis of the 2-chloro3-nitrotoluene not recovered) was obtained. M.P. = 183°C;reported = 185°c 15 acidic equivalent weight i cal:cula.ted for C7H404NGl = 201.5; found= 205.8 and 206.2. The 2-chloro-3-nitrobenzoic acid was reduced to 2-chloro3-arninobenzoic acid by a low pressure catalytic hydrogenation using platinum oxide as the catalyst. In 100 ml. of 96% ethanol 9. 26.7 grams(0.133 mols) of the acid were dissolved and 0.1 gram of the catalyst was added. A drop of 33.8 pounds pressure(0.423 mols) of hydrogen compared to the calculated 0.399 mols was observed. The alcohol solution was carefully evaporated to a smaller volume and needles of 2-chloro-3-aminobenzoic acid crystallized out of the cooled solution. The needles were recrystallized from water to give 13 grams(60% yield) .1\/r .P.= 156-158°C; reported= 158°c 16 Acidic equivalent weight calculated for C7H602NCl= 171.5; found= 170.2 and 172.5. 2-Chloro-4-aminobenzoic acid ONH2 1J02 >- OCl !T02 COOR COOR CH3 ::;,. OCl N02 :;::;-,- ocl NH2 The starting material obtained corrunercially was 2-amino- 4-ni trotoluene of which 80 gra."!Ils( O. 55 mols) were suspended in 70 ml. of cone. hydrochloric acid and 145 ml. of water. 12 An additional 145 ml. of cone. hydrochloric acid were added with vigorous stirring. The mixture was cooled to 2°c and 33.6(0.53 mols) of sodium nitrite dissolved in a little water were added dropwise oYer a period of several hours. The material was kept cold throughout and was filtered. The filtrate was added cautiously to a solution of cuprous chloride in water and cone. hydrochloric acid. The cuprous chloride was prepared as described previously starting with 168 grams(0.591 mols) of crystallized copper sulfate and 57 grams(0.97 mols) of sodium ctloride. With the copious evolution of nitrogen, a reddish oil was formed from which 80 grams(85% yield) of semi-pure 2-chloro-4-nitro- 10. toluene were steam distilled. 1:t.:r. = 57.6°C; reported= 65°c. 17 The 2-chloro-4-nitrotoluene was oxidized to 2-chloro-4ni trobenzoic acid by refluxing i'.ri th permanganate. 14 The mixture of 25 grams(0.146 mols) of 2-chloro-4-nitrotoluene, 2 grams of potassium hydroxide and 54 grams(0.34 mols) of potassium permanganate in 1 liter of water was refluxed on an oil bath with vigorous stirring for 2 1/2 hours till all the permanganate was converted into manganese dioxide. :&,rom the 25 grar.as of starting material, 13 grams were recovered by ste_ai'n distillation. The manganese dioxide formed was filtered and washed with 500 ml. of hot water. The combined filtrates were acidified to -pH 2 by the addition of 6N hydrochloric acid and were evaporated to a small v.olume from which the 2-chloro-4-ni trobenzoic acid crystallized out. The yield was 5.5 grams(40% on the basis of the 2-chloro-4-nitrotoluene not recovered). M.P. • 140.9°C; reported= 140-142°C. 18 Acidic equivalent weight calculated for c7H4 o4NC1 = 201.5; found= 204.8 and 205.7. The yield was not spectacular so some variations in procedure were tried. The oxidation using potassiu~ permanganate was tried using 4 liters of water instead of 1 liter of water as the medium in the hope of getting more of the"toluene" in contact with the permanganate solution decreasing the time of oxidation. The yield was 7.5 grams compared to the 5.5 grams secured previously. However the increase in yield did not warrant the greater inconvenience in handling of large volumes. The use of carbon dioxide as a buffer against the hydroxide formed by adding small pieces of dry ice to the refluxing mixture was unsuccessful due to the too rapid evolution of carbon 11. dioxide in the flask. The use of magnesium sulfate as a buffer ~o.."e (0.146 mols for the above procedureJ,'\unsatisfactory results(75Jb of the starting material was recovered and a yield of 4 grams of the desired acid was obtained). .Another modification involved the oxidation of the compound with permanganate exactly as described in the first procedure with double quantities for all reactants, but at the co·nclusion of the oxidation half as much more permanganate was added without separating the product, starting material or the manganese dioxide and the oxidation 'llvas continued. The yield was 10 grams. The yields were not spectacularly changed by the different variations in procedure. The 2-chloro-4-nitrobenzoic acid was reduced to the 2-chloro4-aminobenzoic a<?id by a low pressure catalytic hydrogenation using 0.1 gr~ns of platinum oxide as catalyst. In 100 ml. of 96% ethanol were dissolved 22.7 grams(0.113 mols) of the acid. A pressure drop of 26.8 pounds(0.335 mols) of hydrogen compared to the 0.339 mols calculated was observed. The alcohol solution was evaporated to dryness on a water bath and the residue was recrystallized from water and dilute alcohol. A little material insoluble in water and difficultly soluble in alcohol was also formed. By a neutralization equivalent of 530.3 it was believed to be a trimer of the desired chloroaminobenzoic acid. A yield of 10 grams(53%) of the 2-chloro-4-aminobenzoic acid was obtained. M.P. = 212-213°C; reported= 213°C 19 Acidic equivalent weight calculated for C7H6021JC1 = 171.5; found= 171.1 and 173.2. 12. 6-Chloro-3-arainobenzoic acid The starting material was 2-amino-5-nitrotoluene obtained commercially of which 100 grams(0.66 mols) were suspended in 82 ml. of cone. hydrochloric acid and 170 ml. of water. 12 An additional 170 ml. of cone. hydrochloric acid were added with vigorous stirring. The mixture was cooled to 2°c and 45 grarns(0.66 mols) of sodiu.~ nitrite dissolved in 115 ml. of water were added dropwise over a period of several hours. The cold solution was then filtered to remove unreacted starting material and the filtrate was carefully added to a solution of cuprous chloride in cone. hydrochloric acid and water with the evolution of nitrogen and the formation of a heavy oil. The cuprous chloride solution prepared as described previously starting with 200 grams(0.79 mols) of crystallized copper sulfate and 67 grams(l.15 mols) of sodium chloride. The oil was steam distilled to give 93 grams(93% yield) of semi-pure 2-chloro-5-nitrotoluene. M.P. = 40°c; reported= 440c. 20 The 2-chloro-5-nitrotoluene was oxidized to 2-chloro-5nitrobenzoic acid by refluxing 100 grams in 25 gram portions with permanganate. 14 The mixture of 25 grams(6.146 mols) of 2-chloro-5-nitrotoluene, 20 grams(0.36 mols) of potassium hydroxide and 54 grams(0.34 mols) of potassium permanganate in 1 liter of water was refluxed for several hours till all the permanganate was converted to manganese dioxide. By steam 13. distillation 15 grams of the starting material were recovered. The manganese dioxide formed was filtered and washed; the combined filtrates were acidified to pH 2 and the 2-chloro-5nitrobenzoic acid crystallized out upon cooling of the solution. An overall yield of 19 grams(43%) of the acid was obtained. Two samples with different equivalente weights were secured. H.P.= 162-164°C(no. 1); 163-165°C(no. 2); reported= 165°c 2l Acidic equivalent weight calculated for c 7H40 4NC1 = 201.5; foundcl91.8(no. 1) and 206.9 and 204.9(no. 2). The 2-chloro-5-nitrobenzoic acid was reduced by low pressure catalytic hydrogenation to 6-chloro-3-aminobenzoic acid using 0.1 gram of platinum oxide as catalyst. In 100 ml. of 96% ethanol were dissolved 12 grams(0.0595 mols) of the 2-chloro5Qnitrobenzoic acid(largely that of the lower M.P. and lower equivalent weight). The alcohol ,f..' solution was heated prior to the absorption of the hydrogen to facilitate the reaction. An (evejl'wye .,j W-Of') absorption of 16.5 poundif(0.206 mols) of hydrogen compared to the 0.179 mols calculated was observed. The alcohol solution was partially evaporated and allowed to cool. From this solution a precipitate separated believed at first to be the desired product, but it gave a lvI.P. of above 250°c and an equivalent weight of 110. The filtrate was then carefully evaporated almost to dryness to give a residue which was recrystallized from a minimum of water to give 2 grams(20% yield) of 6-chloro-3-aminobenzoic acid. M.P. = 186-187°C~ reported= 185°c 22 Acidic equivalent weight calcula.ted for C7H 6 0i'IC;L = 171. 5; found = 167. 8 and 178. 0. (M.P. reported= 188-188.5°c 23 also). 14. 6-Chloro-2-aminobenzoic acid The 6-chloro-2-ni trotoluene o.bte,ined com..11ercially was oxidized to 6-chloro-2-nitrobenzoic acid by refluxing with permanganate.14 A mixture of 25 grams{0.146 mols) of 6-chloro2-nitrotoluene and 54 grams(0.34 mols) of potassium permanganate was refluxed on an oil bath with vigorous stirring for several hours till all the pennanganate was converted to manganese dioxide. By steam distillation 15 grams of the starting material were recovered. The manganese dioxide was filtered and washed, and the combined filtrates were acidified with 6N hydrochloric acid to pH 2. Upon cooling of the solution 5 grams(42% yield) of 6-chloro-2-nitrobenzoic acid were obtained. M.P.= 161.6-163.6°C. A variation in procedure was tried. In one flask was placed in addition 25 gra..11s(0.45 mols) of potassium hydroxide to facilitate the reaction. The time needed for the oxidation with the hydroxide was half that without i t{4 rLours were cut to 2 hours). The yield was improved from 46%( for the flask v:i thout the hydroxide) to 587b(for the flask vii th it). The acid obtained was treated with norite and gave a M.P. of l6l-l62°c; reported= 161°C 24 Acidic equivalent wei ght calculated for C,jH:404NCl = 201.5; found= 205.2 and 206.2. The 6-ch loro-2-nitrobenzoic acid was reduced by ca talytic lovv pressure hydrogenation to 6-chloro-2-am.inobenzoic acid 15. using 0.12 gram of platinum oxide as catalyst. In 90 ml. of 96% ethanol were dissolved 28 grams(0.14 mols) of 6-chloro-2-nitrobenzoic acid. The solution was heated prior to the absorption of tbe hydrogen to facilitate the reduction. An absorption of (f'"'eSSIAl'e.d vof') 34.5 pounds.40.43 mols) of hydrogen compared to the 0.42 mols calculated was observed. The alcohol solution ,vas evaporated almost to dryness on a water bath and the residue(M.P. = 135°c) was recrystallized from benzene to give 7 gran1s ( 29;t) yield of 6-chloro-2-a.minobenzoic acid. M.P. = l46-l47°C; reported= 146-147°C 25 Acidic equivalent weight calculated for c 7:a 6oz-·W l = 171.5; found= 168.1 and 168.4. 4-Chloro-2-aminobenzoic acid COOE ON02 "> oli02 COOH ")t o 1m2 Cl Cl Cl The 4-chloro-2-nitrotoluene obtained commercially was oxidized to 4-chloro-2-nitrobenzoic acid by refluxing with permanganate. 14 .A mixture of 25 grams(0.146 mols) of 4-chloro2-nitrotolutine and 54 grams(0.34 mols) of potassium permanganate in l liter of water 1ivas refluxed with vigorous stirring on an oil bath till all the permanganate was converted to manganese dioxide. By steam distillation 14 grams of the starting material were recovered. The manganese dioxide was filtered and washed; the combined filtrates were acidified to pH 2 with 6N hydrochloric acid. Upon cooling 6 grams(46% yield) of 4-chloro-2-nitrobenzoic acid crystallized out. M.P. = 141-143°C; reported= 140-143°C 26 16. The procedure above was carried out using 50 grams of 4-chloro-2-nitrotoluene, 108 grams of potassium permanganate and 5 grams of potassium hydroxide. 1'he yield was only 27% with 24 grams of the starting material being recovered . .Acidic equivalent weight calculated for C?1{ 40 4NC1= 201.5; found = 203.1 and 203.9. The 4-chloro-2-nitrobenzoic acid was reduced to 4-chloro2-aminobenzoic acid by low pressure catalytic hydrogenation using 0.1 gram of platinum oxide as catalyst. In 100 ml. of 96% ethanol were dissolved 26 grams(0.129 mols) of 4-chloro- 2-nitrobenzoic acid. The solution was heated prior to the absorption of the hydrogen to facilitate the reaction. An lt'"~SSc.c.'V'e d yo() absorption of 28.8 pounds10.361 mols) of hydrogen compared to the 0.387 mols calculated was observed. A precipitate separated out on cooling which was recrys;tallized from difrlute alcohol to give 12 grams(55% yield) of 4-cr:loro-2-amino- = 234.5°C; reported= 235-236°C 27 Acidic equivalent weight calculated for C7H60#Cl = 171. 5; found benzoic acid. fu.P. 169.9 and 170.l. 4-Chloro-3-aminobenzoic acid COOE COOE 0 COOR :>- ► ol:02 0~2 Cl Cl Cl The 4-chloro-3-aminobenzoic acid was synthesized by Mr. De,n Rice by the nitration of .12-chlorobenzoic acid with fuming nitric acid. 28 The 4- chlo.ro-3-ni trobenzoic acid was recrystallized from aqueous alcohol. It was then reduced by catalytic 17. low pressure hydrogenation to 4-chloro-3-aminobenzoic acid using platinum oxide as catalyst. Acidic equivalent weight calculated for c 7n6 oz:w1 ( the latter compound) = 171. 5; found= 173.2 and 175.0. 5-Chloro-3-aminobenzoic acid COOR ::;;-0 Cl! 1JH2 The starting material obtained comrnercially ·was 3, 5- dini trobenzoic acid of which 100 grams(0.47 mols) were dissolved in an excess of ammonia(giving a brick-red colored solution) and boiled. 29 To the boiling solution was added via a capilla ry tube hydrogen sulfide for 20 ~inutes. The solution was heated to expel hydrogen sulfide c.nd a.'nmonia and then evaporated almost to dryness on a hot plate and completely to dryness on a ·water bath. The residue was taken up in water and the sulfur removed by filtration. The filtrate was then acidified to pH 3 with the 5-amino-3-nitrobenzoic acid crystallizing out on cooling. Evaporation of the filtrate gave more of the product. The acid was recrystallized fro r~ hot water. A yield of 60 grams(66 1"t) was obtained. If hydrogen sulfide is pc:.ssed in for over 20 :ninutes(or less for less m.aterial), appreciable quantities of undesirable sulfur and other products accumulate wh ich complicate · the isolation of the desired acid.~.P. = 205-207°C; reported= 2os 0 c 29 The 5-a.mino-3-ni trobenzoic acid was diazotiaed by dis- 18. solving 60 gra.ms(0.33 mols) of it in 170 ml. of 21.{ sodium hydroxide(0.33 mols) for the inverted diazotization method. 12 This solution was added along with 27 grarns(0.39 mols) of sodiu.In nitrite to 125 ml. of 12N hydrochloric acid slowly keeping the temperature below 10°c. 'l'he material was kept cold and was vigorously stirred for 2 hours. Excess nitrite was present at the end of the 2 hours(tested for by the starch-iodide test). The material was filtered to remove unreacted starting material and the filtrate was carefully added to a solution of CUJ;)rous chloride in 125 rnl. of cone. hydrochloric acid and 170 ml. of water. The cuprous chloride was made as previously described starting i.vi th 105. 3 grams (0.42 mols) of crystallized copper sulfate and 34.5 gram.s (0.60 mols) of sodiurn chloride. Copious quantities of nitro- gen were evolved and a reddish material formed. The material was heated to decompose any remaining diazonium. salt( "R" acid was used to test for the presence of diazoniu..rr1 salt). The precipitate was filtered and had a ~ .P. of 140-141°C. It was recrystallized from water. A yield of 15 grams(23%) was obtained. M.P.= 145.9-146.90C; reported= 147°c 30 Acidic equivalent ·weight calculated for C7H404:NCl = 201. 5; found = 201. 1 and 203.4. The diazotization of the 5-amino-3-nitrobenzoic acid did not proceed too readily. The isolation of the resulting chloro substituted acid was rendered more difficult as it was not volatile in steam. The procedure of diazotizing the 5-am.ino3-nitrobenzoic acid by forming the diazonium salt and then boiling in cone. hydrochloric acid to secure the 5-chloro3-nitrobenzoic acid was unsuccessful. 30 19. Oils and resins were obtained preventing the isolation of the desired compound. The 5-chloro-3-nitrobenzoic acid was reduced by a low pressure catalytic hydrogenation using O.l gram of platinu."'11 oxide as catalyst. In 100 ml. of 96% ethanol were dissolved 7.5 grams(0.037 mols) of 5-chloro-3-nitrobenzoic acid. The solution was heated prior to the absorption of the hydrogen to facilitate the reaction. An absorption of 7. 5 pounds Cry~ SS&.1. v-e d('()r) (0.094 mols) of hydrogen compared to the 0.111 mols calculated was observed. The alcohol solution was partially evaporated and a precipitate was formed upon the addition of a little water. A yield of 5 grams(78%) of the 5-chloro-3-aminobenzoic acid was obtained. The M.P. of the acid obtained was over a wide range-- darkening at 215°c and decomposing at 230°c. reported M.P. = 21s 0 c 30 Acidic equivalent weight calculated for C7H6 0 21Wl = 171.5; found= 172.8. 3-Chloro-2-aminobenzoic acid and 5-Chloro-2-aminobenzoic acid Cl ~20 COOE COOH ~ ~-i l i o Cl Cl The starting material obtained commercially was !!];-chlorobenzoic acid of which 20 graxn.s ( 0 .127 mols) were dissolved in 100 ml. of fuming nitric aeid(sp.gr. = 1.49-1.50). 31 This was 20. iust sufficient to dissolve the -m-chlorobenzoic acid. The V solution was heated for 10 minutes and was then diluted with 2 liters of water. The solution(with a little sandy precipitate in it) was evaporated to dryness to give a residue of M.P. = 128-130°0( v.rhich indicated a mixture of the 3-chloro-2-nitro anri 5-chloro-2-nitrobenzoic acids from known melting points). The residue was taken up in a small amount of water to form an aqueous solution and an oil. Tne oil was allowed to solidify under the aqueous phase - which was then decanted off. Crystals that crystallized out from the aqueous phase on cooling had a M.P. of 132.7-134.7°0( still a mixture). The solidified oil was extracted two times with 100 ml. portions of hot water. An insoluble portion of 11.: .P. = 232. 5- 234°0 was obtained. This portion was chiefly the 3-chloro-2nitrobenzoic acid. Nitrations were carried out at ?o 0 c and at -20°c, but the proportions of the two isomers obtained were not noticeably altered. At 7o 0 c 60 gra.~s(0.381 mols) of fil-Chlorobenzoic acid were nitrated by dissolving in 300 ml. of fwning nitric acid. After 10 minutes t h e solution was poured into 2 liters of water. A precipitate(found later to be chiefly the 3-chloro-2-nitrobe nzoic acid) settled out and was filtered off. The filtrate was evaporated to dryness a nd the residue was extracted with successive portions of hot water w till all the 5-chloro-2nitrobenzoic acid dissolved. Upon cooling the acid crystallized out with a M.P. of 130°C. The acid was then recrystallized 21. from hot benzene. The yield was 50 grams. li . P . = 136-138°C; reported = 137-138°C ~1Acidic equivalent wei ght calculated for c 7E4 o4 NC1 = 201.5; found= 205.1 and 206.1. The impure 3-chloro-2-nitrobenzoic acids secured above were treated with hot benzene to remove the 5-chloro-2-nitrobenzoic acid. The 3-chloro-2-nitrobenzoic acid remaining behind was recrystallized from a minimu1n of hot water and alcohol. The yield was 8 grams. lf .P. = 236.3-238.3°C; reported = 31 235°c Acidic equivalent weight calculated for C7H404l'ifCl = 201.5; found= 202.8 and 203.3. The overall yield of the two isomers was 47% with the ratio of the 5-chloro to the 3-chloro as about 8 to 1. The 5-chloro-2-nitrobenzoic acid was reduced by low pressure cataly tic hydrogenation to 5-chloro-2-aminobenzoic acid using 0.14 gram of platinum oxide as catalyst. In 120 ml. of 96% eth anol were dissolved 40 graJns ( 0 .199 mols) of the 5- chloro2-aminobenzoic acid. The solution was heated prior to the absorption of t he hydro gen to facilitate the reaction. An ab(fYeSSu..Ye d-rop) sorption of 49 pounds~0.612 mols) of hydrogen compared to the 0.597 mols calculated was observed. The 5-chloro-2-aminobenzoic acid separated out on cooling of t h e solution. The acid was recrystallized from alcohol. The alcohol solution gave a striking violet fla.orescence to the glass walls of the flask in the sumli ght as reported by Eller and Klemm. 32 A yield of 21 grams ( 62%) was obtained. LI.P. = 204-205°C ( softening at 180-190°c); reported= 204°c 33 Acidic equivalent wei gh t calculated for C7E602NCl = 171.5; found= 165.3 and 165.5. 22. The 3-chloro-2-nitrobenzoic acid was reduced by low pressure catalytic hydro genation to 3-chloro-2-aminobenzoic acid using 0.1 gram. of pl a tinu.In oxide as catalyst. In 50 ml. of 96% ethanol ·were dissolved 6 grams(0.0298 mols) of the 3-chloro-2nitrobenzoic acid. Th e solution was heated prior to the ab- sorption of t h e hydrogen to facilitate the reaction. An ab(fvt>ssv.,re dv-oE>) so:rption of 7 pounds~0.0874 mols) of hydrogen compared to the o. 0 8 94 mols cal cu lated was observed. The alcohol solution 11vas partially eva::; orated and t h e 3-chloro-2-aminobenzoic acid crys tallized out on cooling . The acid gave a rema rkable violet fluorescence to the glass wall of the flask. A yield of 4 grams (78%) was obtained. U.P. = 189-190.5°C A mixture of this com- pound and the 5-chloro-2-nitrobenzoic acid had a mixed melting }Joint of 155-160°c. Acidic equivalent wei gh t calculated for C7E 6 0 2EC1= 171.5; found= 171.6 Hilbner and Weiss 34 prepared a mixture of t h e two isomers ab ove. A H. r ·. of 1360C ·was observed f or t h e ni tro acid mixture e.nd a E .P. of 148°C for t h e amino a.cid :nixture. They did not consider their compounds to be mixtures. Later however, Hiibner 31 a nd Ulrich se parated t he two nitro, but not t h e two amino, isomers. Attempted preparations of 3-Chloro-4-aminobenzoic acid method I (Ac = -C0CH3) COO:H ..... ON02 TunAc Th.e C1m2 NHAc => 0 0 => Cl NH.Ac ? Cl OCl lffiAc reduction of 4~acetylamino-3-ni trotoluene obtained commercially to 4-acetylamino-3-eJainotoluene in 77% yield by NH 2 23. low pressure catalytic h ydro ge na tion went easily. ~ . P . = 130-1 31°C; re ported= 13ID-131°C 35 Tbe diazotization of the 4-acetylamino-3-a,minotoluene to form 4-acetylamino-3-chlorotoluene ·was unsuccessful a s t he diazoniurn salt for~ned coupled internally to form l-acetyl-5-methyl-bentriazole of lJI.I' . = 130.2°c; reported= 130.5°c 35 This metho d t h erefore had to be abandoned. method II lJSu lJSu lTSu Th e succination of the 4-amino-3-nitrotoluene obtained co;umercially by fusing equal mols with succinic acid went r.: ,:,:; smooth ly. Th e molten mass was cooled and t h en taken up in 96% eth anol. Yellow crystals of :U-(4-methyl-2-nitrophenyl)euccinimide separated out on cooling. M.P. = 136.8-139.8°C. Th is material was reduced by ca talytic hydrogenation to H-(4-methyl-2-aminoph enyl·}-succinimide of l\'r .Po = 164-166°C easily. Treatment v: i t h mild sodium hydroxide in its purification cloire t h e succinimide ring to g ive p resumedly M-(4- methyl2-aminophenyl)-succinamic acid a s the compound obtained exh ibited acid characteristics. Th erefore t h is method, too, was dropped. method III (see next pa ge for formulae) 24. o~o~ lffiAc HHAc liliz The !!!- chloroaniline obtained com:rnercially was easily acetylated bv refluxing with glacial acetic acid and acetic anhydride to form !!!-chloroacetanilide. M.P. = 75-77°C; reported= 72.5°c 36 The nitration of the !!!-chloroacetanilide was carried out in glacial acetic and sulfuric acids with fuming nitric acid at temperatures below 15°c. The mixture stood over night and was then poured onto cracked ice and filtered. The mixture of the 4-nitro-3-chloroacetanilide and the 6-nitro-3-chloroaceta.nilide was hydrolyzed by boiling 1/2 hour with cone. hydrochloric acid. The reddish-yellow solution was neutralized irt an ice bath with am,"Tl.onia v.ri th the precipitate formed taken up in a minimum of 96% ethanol. The 4-nitro-3-chloroaniline crystallized out first leaving the other undesired isomer behind: (the two isomers may also be separated by steam distilling off the 6-nitro-3-chloroaniline). M.P. of the 4-nitro-3-chloroaniline = 153.2-154.7°C; reported= 156-15? 0 c 37 The diazotization of this 4-ni tro-3-chloroaniline arni• the addition of the diazonium salt formed to cuprous cyanide solution resulted in the formation of a resinous mass from which the 4-nitro-3-chlorobenzoic acid was not obtained after . . . 38 the hydrolysis of the intermediary nitrile with sulfuric acid. :More exact V?ork with much larger quantities of the 4-ni tro-3- 25. chloroaniline might result in the securing of the desired product. method IV COOE 0 Partial success vras a·ttained by this method, but the 4- amino-3-chlorobenzoic acid was not isolated. The ~-acetylaminobenzoic acid obtained commercially was dissolved in glacial acetic acid and a large excess of HTH(high test hypochloritecalcium hypochlorite) was added slowly to tt.e solution. 1'he solution was allowed to stand overnight with vigorous stirring. Crystals of :,1.p.· = 243. 5-245°c crystallized out and gave a positive halogen test upon elementary analysis(no chloride ion as salt was present in the unknown as the solution gave no precipitate with silver nitrate). The equivalent wei ght of t1:,e compound ii'\·as 186 indicating about 20~t halo genation of the ,£e,cetvlain.inobenzoic acid. The chlorination was repeated followed by hydrolysis T,vi th cone. hydrochloric acid. 'l'he amino acids :pre- sent i.vere not separated. J?urther work on this :,.ixture may result in the isolation of the desired 4-amino-3-chlorobenzoic acid. Azo:2.b:enol chlorobenzoic acids example: OE 0 COOH -➔ o - N = . : U - o EO Cl 26. The chloroa:.'11inobenzoic acids were dia,zotized and coupled to phenol to produce the azophenols. In the prer,aration of the 8 ,zophenols the chloro~..rninobenzo ic 1:.:,cids were dissolved in so- di um hydroxide solution. To this solution were added hydrochloric acid and a little ice to form a finely divided :precipitate and to keep the temperature lov: . Slovdy sodium nitrite dissolved in a little vmter was a:.dded to diazotize the amino group. Tr.e excess nitrite at the end oft he diazotization(tested for by the starch-iodide test) vms eliminated by the addition of 10% sulfamic acid. 'l'he diazonium salt v;_;as filtered to remove unreacted chloroaminobenzoic acid. A solution of ten times the theoretica,l amount of phenol and sufficient sodiwn carbonate dissolved in water was cooled with ice. The carbonate neutralized the hydrochloric acid present in the diazonium salt solution and brought the pH to 9 when t h e diazonium salt solution T-1:as added to the phenol-carbonate solution to form a reddish-oran£e dye. The reaction was c r,. rried out at pH 9 so a.s to keep the phenol dissolved as the ..-ohenoxide ion. After all t.r)e diazonium salt w&.s added, the solution was acidified to pH 7 ,,.,i t:h 6:i:J hydrochloric acid. The free phenol formed vras extracted wi tr~ ether. The ac;ueous phase was boiled to eliminate any residual ether and was then acidified to pH 2 v,i th the precipitation of the colored dye. The dye was filtered off and recrystallize d. from aqueous alcohol. The quantities used for the different amino acids and the yields obtained a re v iven in table II below. 2 2 4-Chloro-3-amino 5-Chloro-3-amino 6-Chloro-3-amino 2-Chloro-4-amino 9 10 4-01-3-azo 13 10 5-Chloro-3-amino general footnotes 2 20 11 16.5 1.2 0.81 11 22 11 0.81 1.62 0.81 22 16.5 1.2 1.62 22 11 16.5 22 33 22 22 44 44 33 22 33 44 grams Na2C°3 6 gms-93 % 4 gms-82% 3 gms-47 % 2 gms-62% 3 gms- 6~ ------- Yield O. 5 gms-16 % 3 gms-62% 2 gms-62% 3 gms-47 % 0.5 gms-16 % ------- - ·--- -- grams phenol 1.62 0. 81 1.2 ------ grams NaN02 A diaz otization using butyl nitrite in 96 % ethanol was tried on 10. In 20 ml. of 96% ethanol were dissolved 2 grams(0.0117 mols) of 5-chloro-3-eminobenzoic acid. The solution was cooled tot 500 after the addition of 3 grams of cone. sulfuric acid. To this solution was a.d.ded dropwise 1.4 grams(0.014 mols) of butyl nitrite. The nitrite was taken up as shown by its disappearance when tested for by the starch-iodide test. With an excess of nitrite present at the conclusion of the diazotiza.tion, the solution was poured into 250 ml. of w~ter and ice. The precipitate which formed was filtered off(the precipitate didmi 1 t appear to be diazonium salt by testing with 1iH 11 acid ) . Sulf m;-iic acid was added to eliminate the excess nitrite and the diazoniu.'Il salt solution was poured into the pheno],;:carbonate solution as above. The dye(0.5 grams= 16%yield) we$ treated with norite. All went smoothly with the exception of 4 which was slower to dia!?iotize and 10 whichi formed a gel upon the addition of the cone. hydrochloric acid (6N). Even with the inverted method the diazotization was q_uite po or for 10. 5-Cl-3-azo a. inverted method was used with hapten 10 6-01-3-azo 2-01-4-azo 5 15 15 4 2-Chloro-3-arnino 8 2-Cl-3-azo 3 10 10 4 6-Chloro-2-amino 11 20 10 20 10 3 5-Chloro-2-amino 5 6 5-01-2-azo 6-Cl-2-azo 15 20 15 20 2 4-Chloro-2-amino 5-Cl-3-azoa of 15 2 0 10 4 15 20 10 3 4 -------- ml. 6N HCl ------------- Table II ml. of gr ams of acid used 2N NaOH 3-Chloro-2-amino ---- ---- .Amino acid used 3 -------- Hapten Number 3.=Cl-~az o 4-Cl-2-azo -------- Ifa,p ten Prepared • ...J M DISCUSSION 28. The hapten-inhibi tion data in 'l'able III ,·., ere interpreted 4 according to the heterogenity theory based on the assumption that the heterogenity of antisera can be described by an erro~ function distribution in free energy of interaction of antibody and hapten in comp etition with a ntigen . .Application of this theory leads to t'he evalu;::;, tion of tvvo constants for each 9ntiboo.y-hapten-antigen system: one of these constants Kb is e.n a verag e hapten inl:.ibi tion constant representin g the a verage bond strengt~ of antibody a nd hapten relative to that of antibody .s,nd an ti g en; the other constant a- ( sigma) is the index of the effective heterogenity of the antiserum. Tte reference p oint for K~ has been ascribed t~,s K 0 = 1.00 for benzoic acid as hapten. The values of Kb range from 5.80 to less than 0.04 for the haptens studied. 'l'r,e ve.lues of 1: 0 for the unsubstituted azoha.pten ho: nologous to t he i mmunizing antigen are the l e, r g est by fe,r as is to be expe cted due to the v ery intimate manner in 1vhich the unsubstituted rom.ologous h ap ten can fit into tbe 2.ntibod.y molecule. The a ddition of a ch loro group into t he benzene ring of t h e hapten bring s &bout E'teric 11indrcince resul tin£ in the decreasing of t h e s t 2.bility of t~e antibody-hapten complex as evidenced by the lower values of K~. The dependence of the inhibition constant vii th t'he anti-Xo se ru:n on t}-,e pos iticn ,yf. t~1e c'cloTo gr oup i n the benze n e ring fo:r the 2-azo h ?.ptens is 6 > 3 = 5 > 4. T:::e val u e for benzoic acid is inte rnediary betv.·een t1·.e 6 and :.: ; VE.,lues of KQ. These values s h ow t ha t the placement of a cb loro group in position 6 has the least effect on decreasing the stability of t h e antibody- 29 . Tabl e I I I E f fe c t o f Eapt e ns on tl::e rrec i p i t a t i on o f An t i- Xo, Anti- Xra , and An t i- Xp Seru.m with Xo -, Xm- and Xp - ov a J: bumi n Resp e c tiv e ly . . =1.t1ti g en s ol ut i on i n borat e buff e r c,t pH 8 . 0, 1 ml. ( 90 u g of 1 Xo - and Xp-oval bmni n, 3 0 u g of X.'11-oval bumi n) ; a n t is erum, l rnl ; hapten so l ut ion in sali n e, 1 ml; pH of superne,t e s = 8. 0 - 8. 1 . Mol e s of Hap ten add e d 3. 9 7. 8 1 5. 6 31. 3 62 . 5 1 25 X 10 8 250 50 0 1 00 0 Ant i- Xo s erum and Xo-ova l bumi n Ea.p t ena ---0 K' crb Amoun t of p r e c ip i ta te c ----- -- 1 1 . 00 t:. ., . "'K., 5. 2 2. 5 ..,'z. 0.98 2 4 0 . 46 2 i::; \ ,l 0 . 96 2 910 630 290 6 1. 75 2 ( 780 ) 490 180 7 0 . 36 2 790 520 220 8 0 . 30 2. 5 860 560 3 00 9 0 .1 2 2. 5 950 74 0 480 lC 0 .24 ') ..., 900 6 30 290 11 0.44: 2 12 0 . 03 ( 2) 13 ') i::;. V 7 90 830 60 0 560 2 60 86 0 6 30 970 900 320 29 0 650 910 3 70 66 0 920 970 940 860 870 760 30. Table III(cont) .A nt i - X,"Yl serum and Xm-o v a l buxni n - ,. t Ea:otena __£;;o _g:__b ,.., a Vov 7. 8 15.6 31. 3 62.5 125 250 480 500 1000 1 1.00 3.5 2 0.40 3 760 490 280 3 0.48 5 630 470 330 4 0.4.1 3 720 510 5 0.14 3 710 520 460 6 0.13 3 720 580 4 60 7 2 .9 2. 5 8 0.90 4 670 520 2DO 9 0 . 63 3 770 570 290 10 0 . 68 2. 5 870 ( 540 ) 270 11 0 .4.6 2. 5 950 660 350 12 0 . 093 2 . !5 13 0.13 ( 2 . 5) 1 1.00 670 930 660 400 280 50 760 1030 690 500 _ ( 9.50) :; ?oO ; --------------------------------------------------------------Ant i-Xp serum and Xp-ov al bu."Ylin 1. 5 770 630 410 ~ "' 920 830 700 ..,7. 810 840 860 890 830 1000 4 970 880 5 1 01 0 6 1C40 (910) 850 7 1.79 1.5 9 50 660 250 8 1 .18 2. 5 870 680 400 g 1. :::, 2 2 1 010 660 300 10 0 .73 1.5 1060 91 C 500 11 0.58 (1.5) 12 5 .8 1. 5 920 710 300 13 2.1 2 960 830 580 970 900 7 50 Footnotes for Tahle III a. Haptens UEed were: l = :Sen zoic 2-,cid 2 = - o- ( D- H·.rdro:x.-vnr ,en..,,,1azo) benzoic e,cid ..;. .., "'J. 3 = 3-Chloro-2-(r:-h ydroxyphenylazo) b enzoic acid 4 :::: 4-Chloro-2-(.r:-hyd.ro:x.ypl:enylazo )benzoic acid h = 5-Ch loro- 2- (.;2-hyclro:xypr:enylazo) benzo ic acid 6 = G-Chloro-2- (.r:-hyd roAryphenylazo) benzo ic acid 7 -· 2- (.12-Hydroxyphenylazo )benzoic acid 8 = 2-Ch loro- 3- (r-hydro:xyphenylazo )benzoic acid. ,,.' g = 4-Chl o ro- :::-- ( 2-hyd roxyphenylazo ) b enzo i c 2.cid 10 = f5-Chloro-3- (r-hydroxyphenylazo )benzo ic acid 11 -· 6-Chloro- 3- (E,-hydroxy:;:r•enylazo )benzoic acid. 12 = 2-(;2-Eydrox:nJhenylazo )benzoic acid 1;:, = 2-Ch loro-4.- (,£-hydroxypc.enylazo )b enzoic s.cid b. Values of o c. in parentr.es es are assumed. The amounts of p recipitate are in parts per mille of the a mounts in the absence of hap ten, 437, 173, and 403 pg. for Xo -, X11- &.nd Xp- systems :respecti ,rely. Blanks of serum and buffer 10, 6, and 9 pg. respectively were obtained. Values are a verages of triplicate analyses ~ith a mean • t ion • o f ~- 2✓• 5o1 d evia ~ pa r entheses. , ' 1 i• c a t e ana 1 yses in • tl1e excep t f or aup ha.r,ten complex. TI-is clearly indicotes trc:t the antibody does not approach~ e hapten as closely at rosit ion 6 as at the other ~ositions since this position is adjacent to tre carboxyl group . The cnrboxyl} group ' s lar~e size does not permit the antibody to approach the benzene rine closely at tte 6 rosition . Therefore tr.e c11loro grou can fit somewhat into the small non-specific hole in the antibody at that :os ition and thus is not as effective sterically. The placement of tl~e chloro srou1) in either the 3 or 5 ::,osition causes slightl y less inhibition than benzoic a.Cid itself. Tre antibody appears to be fonaed more closely around t. e 3 and 5 rositions ( a little less so around the 3 position as it is adjacent to the 2-azo o eninr) than around position 6 . ?he values of :i are a relative measu.re of tbe closeness of fit of tre antibody to the hanten . re placement of a chloro group in position 4 causes the greatest steric hindrance thus ind icatin~ the closest fit of the &nti- o antibody for t_e 2- azo haptens at that position . model of t.,e antibody- hap ten complex shov·in[, the closeness of fit is rostulated and ascribed belm·· fro n the experimental data obtained. (Dotte lines s o , norm&l van der rrdii) .,c'E.ls 33. Th e de 1J endence of U: .e inh ibition consta.nt with t h e anti-:Xm serum on the ros i tion of t he c1".loro g roup in tte ben zene ring for t t e 3-azo hap tens is 2 "> 5 = 4 > 6 . Tn.e be n zoic acid causes greater i nhi bition tl:an any of t he 3-azo hapt ens excep t t h e nonsub:2t it uted 3-azo ha.r:ten. The place ment of t h e ch loro g roup in p o s ition 2 bev.,,e en the azo and carbo:xyl g roup s causes t h e least lowerinc of t h e i ~ 1i b ition as there t h e ch loro g roup is relatively s:12.ll co mpaTed to th e t wo larg e g roups adjacent to it on both s i des . '.i:':he a ntibo dy itself d oes not fold d own bet,r een t h e azo and carbo.xyl €:roups to app roach the hap ten at p osition 2. Therefore t h ere is a non-s p ecific hole in t h e antibody into wh ich t h e chloro g r oup in p o s i t ion 2 can fit without causing appreciable s teric h indran ce. The sub stitution of a ch loro g roup at po s ition 6 , however, caus es g rea t s teric h indr,u1 ce. Th is indicates t h at t he antibo dy a py r oache s the benzene ring a t position 6 rath er specifically i nsp i te of the fe,ct t ha t it is a djacent to t b e large carboxyl g roup and. t h u s d iffers fr o·,,1 t h e an ti-X.o antibody in t h at res})ect. The l_J l a c e1:1ent of t be ch loro g roup in t he 4 or 5 t o s i tion cau ses le ss inhibition tr·an in p osition 2 e,nd mo re t han in p osit ion 6 . '.'/i th a ch lorine i n r) osi tion 5 the ha:p ten molecule can orient itself b y sr. ifti n g both t t .e c c:,.rboxyl and azo g roups. Th e ce,rboxyl st .ifts tow8.rd t h e non- specific h ole at p osition 2 and t h e azo g roup ~ slides furth er out of t n e 3-azo o:pening in t h e anti body . This a llows the hap ten 'Nl th tte ch loro g roup at p o s ition 5 to assume a stable p o ~;i tion( the r hole h ap ten being shifted over a small distance). Tb e re is pTob e,b ly a small non-s pecific h ole 34. at position 4 since it is adjacent to the large 3-azo group . :model of the antibody-harten complex sro'\',ing tt.e closeness of fit is postulated and ascribed from the experimental data obtained. ( ott98 lineQ Qbov no _a I v - :v1 r P v' !:' '1. s re ii) The de!)endence of the inhibition cons tent v. i th tbe anti-Xp serum on tre .,..osition of t:e chloro group in tre benzene ring for the 4-azo haptens is t.at the unsubstituted is ereater than t~e 2-chloro . Both rive ereater inhibition than benzoic acid. Unfortunately the 3-chloro-4-azohapten was not prepared . The inhibition of the 2-chloro-4-azo hapten is one t ird that of the unsubstituted 4-azobapten. This shows a rather close fit of the bapten and antibody. I;evert11 eless t .e lack of steric Pindrance on tPe part of benzoic acid was not sufficient to overcome its lack of the azo homologous group and so the 2chloro- -azo ra,pten inhibited trice as much as benzoic acid itself. A model of the antibody-ha:ten co~plex showing the closeness of fit is r ostulc:., ted and &scribed beloi.,· fro ··.1 the exp erimental data obta ined . {D tte inep P~onor:1ql vnn .er "7h•,r, 1 8 r r;, J. . \J The values of Kb for the ch loro - ~-azo and the chloro-4-azo r-a,p tens v·i th the a.nti-.. o serum , for t h e ch loro-2-azo and the c,.-: loro-4-azo raptens v· i th tre anti- m serum and for the ch l oro2-azo and c loro-3-azo haptens ~i th t h e c,nti- p serum are reasonable fro m the models of t h e antibodies described above . Us ine tre anti-Xo seru..n t h e unsubstituted m- azo hapten gave much greater inhibition than did the p-azo hapten as is to be expected for steric reasons . •.n th the 3- azo haptens the dependence oft he inhibition constant on t h e r osition of t h e cbloro ['Toupin the benzene ring is 6 ";> unsubstituted ;::, 2 ;> 5 .> 4 . TI e benzoic acid is twice as E, ood an inhibitor as t ~ e 6-ch loro . There is some tendencv· for t r e 3-azo unsubstituted hapten to sv·ing t h e azo group into tr_e opening at tl:e 2 : osi tion in the c:1,nti-Xo antibody. In tree anti-Xo serum there is a non-specific hole at :c osition 6(as observed from tl~e data above v ith the 0 homolo gous t ,a :r,'tens). 'J.'he 6- chloro-3-azo hap ten causes greater inhibition than the 3-azo hapten itself since the chloro group in ~osition 6 stabilizes the antibody-hapten complex formed. TLe ~anten must swing a little so as to let the 3-azo group go into the 2-azo hole of the antibody. As this occurs the non-specific hole at 6 is able to accomodate a chloro group rithout steric hindrance permitting van der Waals attraction d.ue to the chlorine's juxtaposition along the antibody to become effective. 'I'he complex with the chloro group there a,ssumes creater stability e,s the ha.pten cannot now swing back for the 6-chloro holds the hapten in the new position by steric considerations. and as tbe e,ddi tional van d.er Vvaals forces increase the srecificity of t~e antibody for the hapten. The placerae ~t of a chloro group in the 2 r osition causes less inhibition than t1:e unsubstituted 3-azo since tr e ch loro c roup itself partially takes up tte 2-azo op ening of the antibody. T'ne effect of :'.)lacing a. chloro group at t:1e 4 or 5 position causes large ::_;teric effects a,s ex~o ected. Less inbibi tion is observed •:.-i th t h e c1-,loro c rour in the 4 position since we h2,ve seen tr.e,t the c=, nti body fi tB most closely around t h e 4 position. The unsubstituted 4- a zo J, a.pten causes very s mall inhibition of a chlorine even in the 2 position on the benzene ring reduces the stability of the antibody-hapten complex to a veyy small ainount indeed. 37. With the anti-X.m serum, the :... un,substi tuted o-azo hapten gave much g reater inhibition than did the p-azo hapten as is to be expe ct ed from the non-specific hole in t h e 2 p osition. '.'.-i t h t h e 2-azo hap ten s t l-,e depe nd enc e of t he inh i b ition constant on t"h e :pos i t ion of tt e c1:-:loro c roup i n t b e benze ne r i ng i s 3 > 4 = un substitu te d > 5 == 6. 'l'l-:e benzoic a ci d is t vd ce RS good c1,n inh i bi to r us t t .e 3-ch loro. Tl1e ch loro g roup i n :position 3 stab ilize s t t.e combina ti on s inc e t h ere i s a l a r g e h o le a t 1Jo s i tio n 3 wt j_ch c an a cco:.nodate /' t 1, e CT:l orine and ca n bring into pl ay it s van de r 1'iaals :for c es of a ttraction du e to t h e ch lori n e' s j uxtap osition a long the a nti body . With tl 1 e ch loro r roup a t p osition 4 t he r e i s s till a loo se fit of ant i b oc.y a.round t h e ha:p ten in t l; i s :p osition as i nd ic a te d ,:,: i t h t ':: e 4 -ch loro- 3- azo b.a_p ten allowing s pace fo r t h e cl::lorine to bring i n t o 1") l a,y i ts v a,n de r ~.-✓aa l s forces of nt tra ction. If t h e ch lo ro c roup i s ? l a ce d at ~os iti on 5 or 6, t h e co:,1b i ne,ti on i s c re s. tly de c rec:csed a s e x i: -i e c tecl fro m the clos eness of f it of t t e a n ti body to t t.e h apten in t he se p o s itions. Ben zoic a ci d i nh i b it s better t han t h e 2-ch lo ro - 4 - azo r:.ap t e n ':"1:- ich i n turn is a better i nt. i b itor t l1 an t h e unsubst itute d 4-a zo 1-: a:':l ten. Tr is i s ex:pe cte d for t h ere a re st e ric rea sons for t Ye r.:na ll i nr i bit ion of t h e un subs titute d 4-azo hapten. However as ha s been seen before, t he re i s a loo se f it of an tibo dy in t t .e 2 p o s iti on ,r-1. , icl--:" e; ives gre at er p l ay to t h e v an d e:r \iaa.ls for ce s of att r ac tion for a cYlorine i n the 2 :p o s i t i on. Th ese c; dded f orces stab,lt ze fre antibody-1:ap ten cornp lex formed 38. v.ci th the 2-chloro hapten. 'l i th tl-~e anti-Xp serum the unsubs tj_ tuted m-azo har,ten cave much greater inl':ibi tion tt,an did the unsubstituted o-azo hapten. This is ex: ected from steric considerations. ·di tb the anti-Xp serum values of Eb less than about O. 5 could not be determined because of the high concentrations of ~apten required. All of t he ch loro-2-azobenzoic acid hapten~ had values below 0.5 and below that for the unsubstituted 2-azo \'li th the 3-azo Laptens tbe dependence of the inhibition . constant on the ~osition of the ch loro croup in the benzene ~ ring is unsubstituted"'? 4 > 2 ::> 5 "> 6. 'l'he benzoic acid has a Kb of value between t hat of 2 and 5. The inhibition of the unsubstituted 3-azo and the 4-chloro-3-azo haptens were only a little le-ss tr1an t ha t of t:he 2- chloro-4-azo hap ten. If a ch loro group is at position 4 of the hapten, it shares the 4 opening in t h e antibody with the 3-azo group requiring great dila,tation of t h e antibody causing less combination of antibody a nd hapten. With a chloro c roup in position 2 there is some stabilizing influence of the chloro group on t h e orient a tion of the 3azo group to t he 4-azo position of the antibody. The effect is similar to t hat caused by the 6-chloro group on the 3-azo hapten in the anti-Xe serum. Ho~ever with the chloro £rouD"' in the _, 5 or 6 position the steric "hindrance brought about by t11e large chloro group does not allOY-' t h e 3-azo e roup to swing to the 4azo position. As these hap tens cannot form as stable antibody complexes,they cause much less inhibition. SUlvJLf.A...'J:(Y 39. Experirr1e nts r .ave been ;na.de on t l-: e precipitation of antisera homolo g ous to the 2-, m-, and E-azobenzoic acid g roups, 1wep2.red by inj ectinrs rabbi ts with beef serum coupled with die,zotized 2- ,:£- e,nd 12-aminobenzoic ac ids, with azoovalbu:nins containing tr.ese horriologous groups and on t he effect of various monochloro substituted azobenzoic a,cid haptens in inhibiting the p r e ci r itation. The monochloro substituted azobenzoic acid haptens ,vere pre pa,red fro:n t he corres p onding chloroami nobenzoic acid. These chloroa.minobenzoic a cids were synthesized fro m commercially ava.ilable starting raa teri a ls. They ·were tr:e n diazotized and coupled to lJhenol to for:n t he desired haptens used in these ext eriments. The closeness ,•; with which the &.ntibody fits its homolo g ous azohapten wa s determined by determining the effect of cr.loro groups in every position in t he benzene ring not occup ied by an h o:;1o lo g ous giroup on the inhibition of the specific p reci :i,; itation of the antisera.. This was done for t h e antibodies of tte anti-Xo, anti-Xrr and an ti-Xp sera u s ing t he appropriate haptens for each. Cross reactions using the 3-azo and 4-azo(monochloro substituted and unsubstituted) ha:p tens vd th the anti-Xo seru:-i1, the 2-azo and 4-azo(chloro substituted ~nd unsubstituted) haptens v:i th t h e 2.nt i-X11 -serum, a nd t r.e 2-azo and 3- azo { ch loro substituted and unsubstituted.) he,p tens with the a nti-Xp serum. '·· y•ere determined. Th e results obtained corres pond~'n ded to what v-:as expected as p redicted from the data secured from inhibition studies for tl-::.e closeness of fit of t he antibody and its 40. homologous haptens. Diagrams showing the closeness of fit of t he various antibodies a nd their h omolocous haptens are given. BIBLIOGI<.AJ?HY 41. 1. K. Landsteiner - "The Specificity of Serolo g ical Rea.ctions"Charles C. Thomas- Springfield, Illinoi.s 1936 2. D. Pressman, D. E . B ro wn, l.a ~ au1· ... 1.ng O 64 , - J- • .d' •.'11 • Cl-i em. ivOC., 3015( 1942) 3. :D. Pressman, J .T. :.:aynard, A.L. Grossberg, L. Pauling - ibid, .§~, 728(1 94.-3)4. L. r a uling , D. Fress:-.aan, A.L. Grossberg - _ibid, _ 6£, 784(1944) 5. D. Fressman, s. ~c. Swing le, A.L. Grossberg, L. I;auling - ib~d, 66, 1731(1944) 6. L. P2,uling, I). :!?ressman - J:.bt£.i.. 67, 1003(1945) 7. L. I'a ulj_ng , D. E. Campbell, D. I'ressman - Physiol. Rev.,23, 203( 1943) 8 . K. Landsteiner, J. van der Scheer - J. 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