3662
NOTES
reduced pressure and stirred into excess water. The still clear solution was acidified with dilute hydrochloric acid, and the resulting yellow precipitate was filtered, washed with water, and recrystallized t o yield fluffy yellow cr! stals melting a t 296-297". T h e product appears t o be a nitrohydroxy-methoxy-benzoic acid. Anal. Calcd. for C s H j S 0 6 : C, 45.08; 1 1 . 3.31. Fourid: C, 45.10; H, 3.32. Inasmuch as Bolliger and Reuter3 reported it ineltilit: point of 220" for 2-hydroxy-3-11iethos~--~-~iitroberlzoic acid, this compound is probably :I-h\-dros!.-2-tnetho~y-~-Iiitrobenzoic acid.
Acknowledgment.-The authors wish to thaiik A h . Harold Willemsen for the analyses and Dr. R . Donovick of the Squibb Institute for Medical Research for the values of the inhibiting concentrations for the pathogenic microiirganisms reportetl in this paper. ( 3 ) A. Bolliger a n d P.Reuter, J . Prdic. iiiiy. SOL.S.5'. i v a l e s , 72, 3'29 (1939).
THEINSTITCIX OF PAPER CHEMISTRY
APPLETOS, \VISCOXSIX
VOl. 77
found in the absorption spectrum of 3-(p-nitrophenylazo)-p-cresol,6 determined for comparison. The absorption spectrum of poly-p-(l-hydroxynaphthyl4-azo)-phenylalanine ( n average 20) in ethanolamine shows, between 2500 and 6000 A . , one characteristic peak, a t 5180 A. (E 10000). A copolymer of L-tyrosine and L-lysine, in a nio1ar ratio of 1 : 10, was coupled with p-nitrobenzene diazonium chloride. The azopolypeptide obtained resembled polylysine in its solubility, influence on blood clotting' and antibacterial activity.8 rl copolymer of p-aniinophenylalanine and L-aspartic acid, in a niolar ratio of 1:9, was diazotized and coupled with a-naphthol. The colored derivative thus obtained resembled polyaspartic acid in solubility and biological activity.'m5 These findings indicate that azo derivatives of a-amino acid copolymers containing a small percentage of aromatic amino acids might be used as tagged compounds iii certain biological investigations.
Experimental All melting points are uncorrected. The coupliiig rcacAzo Derivatives of Some Aromatic Poly-a-amino tions were carried out in 1N sodium hydroxide or sodium Acids carbonate a t 0-5" in the usual way. Absorption measurcments were made on a Beckman niodcl DU spcctrophoB Y k11CHAEL S E L A AND EPHR.4141 KATCHALSKI tometer, a t approximately 25 '. 3,3 '-Di-(p-nitropheny1azo)-DL-tyrosine anhydride was preRECEIVED DECEMBER29, 1951 pared by coupling DL-tyrosine anhydride" (1 mole) with pnitrobenzene diazonium chloride ( 2 moles), precipitated In this paper the synthesis and properties of some azo derivatives of poly-a-amino acids containing from reaction mixture with dilute hJ-drocliloric acid and rearomatic amino acid residues are described. These crystallized from t-butyl alcohol; m . p . 243 -2G5". yield 917,. Anal. Calcd. for C3&dSs0,: C , .57.i'; H , :3.9;S , 17.9. compounds may serve as models in the study of azo- Found: C, 57.3; H,3.9; X, 17.6. proteins, which are widely used in immunological The brown azo compound diisolves rcadilh- i i i LLCY toiic, studies as well as in the investigation of the chemi- pyridine, butylamine, ethanolamitie arid ethylenediainitle, i cal and biological properties of The sparingly soluble in ethanol, chloroform and dioxane, a i i r i i colored azopolypeptides obtained represent a new insoluble in water, ether, benzene and carbon tetrachloride. I t s solutions in aqueous or alcoholic iodium hydrosidc :arc group of polymeric dyes.3 Their synthesis permits red-violet. the attachment through an azo link of different T h e azo derivative of t!-rosiiie a~il~yclri,lc r!ioved a b a compounds to polyamino acids containing residues single red-violet band o:i ;LII activntcd alurnina column, of tyrosine, histidine, tryptophan or p-aminophen- when 1 v'- sodium hydroxide \va, piised t!lrough the colunin. \\71ierl the substance mi\ reduced and Iiyclrolyzcd for 12 ylalanine. hours in a boilirig solutioii of itannous ciiloride ( 1 0 % ) iii Polv-3-(p-nitroplieiiylazo)-L-tyrosiiie and poly- t i ,V Iiytlrociiloric acid, ~ i i i t l;ti1 aqueous solutioli of the c1ric.d 3,.5-di-(p-bromophenylazo)-L-tyrosinewere pre- rc:tctioii tnikture n.a\ i r u i i on a paper clii-oiti~itogr-iiii j, pared by coupling poly-L-tyrosine4 with p-nitro- drveloped with n-butJ-1 :ilcoliol ~wdtcr-aceticw i t 1 ( 4 : .i1: oiilyone i i ) u t ,witli iti 0 . 2 8 , was obt:iiiicd upoii 5pr;tyiiig ivitli benzene or p-broniobenzene diazonium salts, re- ninhydri:i. One i p o t , with K , 0.80, wa, ubtaiiicri ~ v l i e ~ i spectively. For coniparison 3,3'-di-(p-nitropheiiyl- the paper c!iromatogram \viis developed witli u-propyl azo)-DL-tyrosine anhydride was prepared. Poly-p- alcohol-Cl.BC/o aqueous :t:iiinonia (2: 1). Paper clcctro( 1-hydroxynaphthyl-4-azo) phenylalanine was phoresis of the above hydrolysate was carric. man Xo. 1 filter paper in acetate buffer of p obtained by coupling a-naphthol with diazotized strengt!i 0.2, a t a potential gradient of 1U poly-p-amino-DL-phenyla1aiiine.jThe diazonium temperature. A single spot, ' i t :L distance of 8.7 c:n. from salt derived from poly-p-aminophenylalanine also the origin toward the cathuc!e, was revealed, aftcr t \ v o hours, with ninhydrin. Tyrosi!ie did not move froin the was coupled with tyrosine and with polytyrosine. under the same conditions. T h e homogeneity of the The absorption spectrum of poly-3-(p-nitrophen- origin azo derivative of the tyrosine anliytlrid:, the lack of tJ-rosiiic y1azo)-tyrosine ( n average 30) owas determined a t in the hydrolysate of the reduction product, a b \vel1 a i tllc
FH 13 between 2500 and 6000 A. Two characteristic peaks were found, a t 3460 A. (residue rnolar extinction coeficient, E 10000) and a t 3300 A. ( E 6330). 9 n identical spectruni was found for 3,3'-di(P-nitropbeny1azo)-tyrosineanhyd;ide. Two peaks, a t 3460 A. ( E 7250) and a t 3300 A. ( E G330) were
(1: K . Landsteiner, "The Specificity of Srrulogical Keactioni." Harvard University P r e s , Cambridge. .\lass., l Y i 5 . ( 2 ) K. Herriott, i l i i v u i i ~ c siii l'ruleitz C ' h e t i i . , 3, lij!) (1947). 1 Fui- u t h c r e ~ a n i p l c auf p o l b - u e r i c d y c a . !c 'I 11 1,irwr rlii
