NOTES
Jan. 5, 1954
299
in butylamine-water (9 : 1by volume) was measured between 2600 and 3000 A. Two characteristic BY ABRAKAM PATCHORNIK, MICHAELSELAAND EPHRAIM peaks, at 2840 k . (molar extinction coefficient per KATCHALSKI tryptophan residue, E 5470) and a t 2920 A. ( E 5030) RECEIVEDJULY 6, 1953 are present, slightly lower than the peaks a t 2840 I n continuation of experiments on poly-a-amino A. ( E 5780) and a t 2920 8.(E 5280) found in the abthe synthesis of polytryptophan has been sorption spectrum of DL-tryptophan in the same carried out. Poly-DL-tryptophan with a number solvent. Experimental average degree of polymerization n = 80, as deAll melting points are uncorrected. L-Tryptophan and termined by amino-N end-group analysis,a and poly-L-tryptophan ( n = 90) were prepared by bulk DL-tryptophan from hhtritional Biochemicals Corporation used throughout. polymerization of a,N-carboxy-DL- and a,N-car- were a,N-Carboxytryptophan Anhydride.-The Dt-anhydride boxy-L-tryptophan anhydride, respectively. The was prepared by passing phosgene for 45 minutes through a synthesis of poly-DL-tryptophan through the Los- suspension of DL-tryptophan in dry dioxane maintained a t sen rearrangement of sodium a-carboxy-P-3-indo- 40°, from which oxygen had been removed by a stream of nitrogen (cf. Farthing)s; yield 84%; m.p. 142" (from ethyl lylpropiono-(benzoylhydroxamate) has been men- acetate-petroleum ether) (dec. with carbon dioxide evolutioned by Hurd, et al.,4although experimental de- tion). A n d . Calcd. for C I Z H I O N ~ C, O ~62.6; : H, 4.4; N, 12.2; tails have not yet been published. wt., 230.2. Found: C, 62.5; H, 4.5; hT, 12.0; equiv. a,N-Carboxytryptophan anhydride was obtained mol wt., 228, determined by titration with sodium methoxide in from tryptophan and phosgene according to the benzene using thymol blue as indicator .7 general procedure of Farthing.6 Its constitution a,N-Carboxy-DL-tryptophan anhydride (100 mg.) yielded was proved by the analytical results, the quantita- upon treatment with hot N hydrochloric acid 89.5 mg. of tive evolution of carbon dioxide on heating and the tryptophan (101% of the theoretical), as calculated from the extinction of the solution a t 2805 A and pH 12, using the quantitative yield of tryptophan on treatment with molar extinction coefficient of tryptophan e 5430.8 When a hydrochloric acid. Despite the known acidity of drop of the neutralized solution was run on a descending the N H group of indoles, the possibility of reaction paper chromatogram using 1-butanol-glacial acetic acidbetween this group and phosgene was ruled out by water (4:1:5) as the mobile phase, one spot was obtained after spraying with ninhydrin, with Rr 0.46, identical with the negative reaction of skatole with this reagent that of an authentic sample of DL-tryptophan run on the .. . under similar experimental conditions. same strip. a,N-Carboxv-DL-trvDtoDhan anhydride is soluble in On alkaline hydrolysis POIY-DL- and poly-L-tryptophan yield tryptophan quantitatively. No at- ethyl acetate, dioxane and-benzene. a,N-Carboxy-L-tryptophan anhydride was prepared from tempt was made to recover the optically active x,-tryptophan analogously to the DL-derivative; yield 95%; tryptophan from the hydrolysate of poly-L-trypto- m.p. 135' (from ethyl acetate-petroleum ether). phan as the hydrolytic conditions used are known to Anal. Calcd. for C12H10x203: C, 62.6; H , 4.4; N, 12.2; favor racemization.s The marked dextrorotation mol: wt.,230.2. Found: C, 62.9; H, 4.8; N, 12.2; neut. (in dimethylformamide) of the poly-L-tryptophan, equiv., 225.7 DL-Tryptophan methyl ester hydrochloride was prepared and the fact that no racemization occurs during the the N-carboxy anhydride in the usual way (cf. preparapolymerization of N-carboxy-a-amino acid anhy- from tion of E-N-carboxylysine methyl ester hydrochlorides) : drides,'V2 suggest that the polymer derived from m.p. 225" (from methanol-ethyl acetate). KO depression a,N-carboxy-L-tryptophan anhydride contains L- in melting point was obtained on admixture with an authentic sample of DL-tryptophan methyl ester hydrochloride.'O tryptophan residues exclusively. Anal. Calcd. for C1zH16Nz02C1: C, 56.6; H, 5.9; N, As expected, no cross-links between a-carboxyl C1, 13.9; CHIO, 12.2. Found: C, 56.8; H, 5.8; N , and indole-" groups occurred during polymeri- 11.0; 10.9; C1,13.9; CH30, 12.2. zation. The formation of such links would cause a Po1ytryptophan.-The DL-polymer was prepared by the significant increase in the content of a-amino groups polymerization of a twice recrystallized a,N-carboxy-DLof the polymer. The solubility of the L- and DL- tryptophan anhydride at 150" in a high vacuum (10-4 tryptophan polymers in various organic solvents mm.) .l The polymer obtained was dissolved in hot glacial acetic acid and was precipitated with water slightly acidisupports the linear structure of these preparations. fied with hydrochloric acid; quantitative yield. The infrared absorption in the region 2.6 to 14 p Anal. Calcd. for poly-DL-tryptophan ( n average 80) : of films of poly-L-tryptophan and pdy-DL-trypt0- C, 70.9; H, 5.4; N, 15.0; amino-N, 0.094. Found: C, phan cast from pyridine and acetone, respectively, 69.0; H, 5.3; N, 15.0; amino-N, 0.094.8 I n a preliminary experiment it mas found that the carbon were found identical with that reported for polyDL-tryptophan prepared by an independent dioxide evolved during polymerization amounted to 96% of theoretical. meth~d.~ The typical strong absorption a t 2.9 p thePoly-DL-tryptophan ( n average 80) is soluble in dioxane, indicates the presence of free indole N H groups in acetone, butylamine, pyridine, dimethylformamide and hot both polymers. glacial acetic acid. It is sparingly soluble in methanol, The absorption spectrum of poly-DL-tryptophan ethanol and ethyl acetate. I t is insoluble in benzene, car-
Polytryptophan
(1) E. Katchalski, I. Grossfeld and M. Frankel. THISJOURNAL, 70, 2094 (1948). (2) E. Katchalski and P. Spitnik, ibid., 73, 3992 (1951); A. Berger and E. Katchalski, ibid., 73, 4084 (1951); E. Katchalski and M. Sela, ibid., 75, 5284 (1953). (3) D.D. Van Slyke, J . Biol. Chcm., 8 3 , 425 (1929). (4) L. Bauer, Ph.D. Dissertation, Northwestern University, 1952,in C . D. Hurd, L. Bauer and I. M. Klotz, THISJOURNAL, 75, 624 (1953). (5) A. C. Farthing, J. Chem. SOC.,3213 (1950). (6) E. Abderhalden and L. Baumann, Z . physiol. Chcm., 65, 412 (1908); A. Neuherger, Advances Protein Chem., 4, 340 (1948).
bon tetrachloride and water. Poly-L-tryptophan was prepared analogously t o the DLpolymer; [ a I z 6 +147O ~ ( c 6 in dimethylformarnide). Anal. Calcd. for poly-i-tryptophan (n average 90) : C,
(7) A. Berger, M. Sela and E. Katchalski, A n a l . Chem., 26, 1554 (1953) ( 8 ) G.H. Beaven and E. R. Holiday, Advances Prolein Chcm., 7 , 319 (1952). (9) M.Bergmann, L. Zervas and W. F. Ross,J. B i d . Chcm., 111,245 (1935). (10) D. 0. Holland and J. H. Nayler, J . Chcm. SOC.,286 (1953).
N Ol’hb
300
70.9; H, 5.4; N, 15.0; amino-N, 0.083. Found: C, 69.6; H, 5 . 5 ; N, 15.3; amino-N, 0.0S3.3 Poly-L-tryptophan differs in its solubility from the DLpolymer. It is soluble in dimethylformamide and pyridine, but insoluble in dioxane, acetone, butylamine, hot glacial acetic acid, methanol, ethanol and ethyl acetate. An aqueous suspension of polytryptophan turns deep violet when treated with Hopkins-Cole reagent; it gives a positive ninhydrin reaction and a negative picric acid test .I1 Hydrolysis of Po1ytryptophan.-Poly-DL-tryptophan (22.3 mg.) was dissolved in a mixture of dioxane (1 ml.), 4 A* methanolic sodium methoxide (2 ml.) and water (0.2 ml.). Oxygen was removed by a stream of nitrogen and the hydrolysis carried out by heating in a sealed tube a t 110-120” For 72 hours. The amount of tryptophan in the hydrolysate was deterniined by its ultraviolet absorption8and colorimetrically.” -1rzal. Calcd. for a hydrolysate of 100 mg. of PO~Y-DLtryptophan ( n average 80): tryptophan, 109 mg. Found: tryptophan 105 mg. (ultraviolet absorption8); 107 mg. (colorimetric ninhydrin determinatiou12). Poly-L-tryptophan was hydrolyzed analogously to the IiL-polymer; as it is insoluble in dioxane, pyridine was used instead. A quantitative yield of tryptophan was obtained also in this case. -4chromatographic analysis of the neutralized hydrolysate of p01y-n~- and poly-L-tryptophan carried out as above yielded one spot with Rf0.46 identical with that of authentic samples of DL- and L-tryptophan.
Acknowledgment.-This investigation was supported by a research grant (PHS (2-3677) from the National Institutes of Health, Public Health Service. (11) E. Abderhalden a n d iC, Koinni, %. pliyiiol. Ci?tinas,rrelL8. Chiith. A c t t i , 33, 1973 (1:CiO).
XVEIZMANN IXSTITUTE OF SCIEXCE REHOVOTH, ISRAEL
Vol. 7ti auol and beiizeiie (1:2). The yield of the purified product was between 2.4 and 2.6 g . Equally satisfactory results were obtained by using an alternate procedure which consisted of heating equivalent amounts of the reactants in pyridine and removing the solvent by steam distillation with the addition of sodium acetate. Pyridoxal isonicotinyl hydrazone forms pale-yellow, small prisms either from dilute ethanol or from a mixture of methanol and benzene, m.p. 261-262’ dec. (cor.). Anal. Calcd. for ClrH14OsN4: C, 58.72; H , 4.94; S , 19.57. Found: C, 58.55; H, 5.03; N, 19.77. Pyridoxal nicotinyl hydrazone forms practically colorless, fine needles from dilute ethanol or thick platelets from a mixture of methanol and benzene, m.p. 235-236’ dec. (cor.). Anal. Calcd. for ClaH14Osn’a: C, 58.72; H , 4.04; S, 19.57. Found: C, 5S.68; H , 4.87; X, 19.63. These two compounds are very slightly soluble in cold water, slightly soluble in cold methanol or ethanol hut soluble in hot; soluble i l l cold 10% sodium hydroxide; very soluble in dilute niiileral acids; but insoluble in benzenc or petroleum ether. A mixture of methanol and petroleum ether may also be used for recrystallization. Quantitatively, pyridoxal nicotinyl hydrazone is considerably more soluble than pyridoxal isonicotinyl hydrazone in most of the solvents tested.
Acknowledgment.-The author is very grateful to Dr. Troy C. Daniels, Dean of U. C. College of Pharmacy, San Francisco and Dr. S.A. Peoples, Professor of Pharmacology, U. C. School of Veterinary Medicine, Davis, California, for their constant advice and encouragement. UXIT‘ERSITY OF CALIFORNIA COLLEGE OF PHARMACY, SANFRANCISCO AND SCHOOL OF VETERIKARY MEDICINE DAVIS,CALIFORXIA
A New Synthesis of Perfluoroaldehydes Nicotinyl and Isonicotinyl Hydrazones of Pyridoxal BY PETER P . T. SAH JULY 27, 1953 RECEIVED
Ur. OGDESR. PIERCE AND THOMAS G. KANE RECEIVED AUGUST10, 1953
The preparation of perfluoroaldehydes has been described by reduction of the corresponding acid‘ Nicotinyl and isonicotinyl hydrazones of pyri- or nitrile2 with lithium aluminum hydride, by doxal, two new compounds that show interesting oxidative nitration of 1,1,1-trifl~oropropane,~ and biological properties,l may be prepared easily by by the Rosemund reduction of the corresponding the following procedure. acid chloride.+ I n this Laboratory it has been Pyridoxal hydrochloride (product of Xutritional Bio- found that perfluoroaldehydes can be prepared by ‘chemicals Corporation, 2 8.) in mater (20 ml.) was treated reduction of the corresponding perfluoroacid esters with isonicotinic acid hydrazide2 or nicotinic acid hydrazide3 with lithium aluminum hydride a t - i o ” in good (1.4g . ) in 50% ethanol (20 i d . ) . Sodium acetate (1 g.) in The method employs a reverse wvater (10 nil.) was then added and the reactants were j-icld ( iO-S070). heated on the steam-bath for 10 minutes and allowed to addition technique and only small amounts of the stand for 24 hours a t room temperature. The crude crys- by-product fluorine-containing alcohol are formed. talline product &-as recrystallized from a mixture of methAn explanation of the ready formation of aldehydes, in contrast to the usual alcohol formation (1) Pyridoxal isouicotinyl hydrazone was fouiid by W. B. Sutton of from ester^,^ is not apparent a t this time and is , t h e Lilly Research Laboratories, Indianapolis, Indiana, t o possess significant antitubercular activity i n vitro a s well as i n vivo: its isomer, under investigation. Supporting work in this pyridoxal nicotinyl hydrazone, however, is much less active in oilro Laboratory has indicated that esters containing and inactive in z’ipo. Following this observation, both derivatives halogen atoms in the a- and P-positions will yield were found by D;. Louis Greenberg of t h e University of California This would indiSchool of Medicine t o be equal t o pyridoxine in their vitamin BB aldehydes on similar reductions. mtivity. Recently, both were found by Dr. R. Freedlander of Mount cate that a strong inductive effect is a determining Zion Hospital, S a n Francisco and Dr. A . Furst of Stanford Univerfactor in the reaction mechanism. sity School of lledicine to show distinct activity against mammary cancer in mice and certain leukemia in mice. The details of these biological results will be reijorted by these investigators elsewhere. ( 2 ) H . Meyer and J. LIally, .\loit&/>., 33, 303 (1912). ( 3 ) Prepared by Dr. C. T. I’eng of University of California College zlf Pharmacy according t o the method described in the literature; “Beilsteit?s IIandbuch der orpanixhen Chcrnie,” Bd. X X I I , 41 (1935), T h . Curtius :tnd E. I I o h r , E ? , , 31, 2.103 (1898). I t formed white, s t o u t neeilles o r rcxls froin
