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VOl. 76
COMMUNICATIONS T O T H E EDITOR - -~
PUROMYCIN. SYNTHETIC STUDIES. VII. PARTIAL SYNTHESIS OF AMINO ACID ANALOGS
alanine, glycine and p-methoxy-L-phenylalanylglycine, by activation of their N-carbobenzoxy Sir: derivatives as the mixed anhydride, acid chloride or Puromycin has been shown to have the structure azide, were coupled with the “aminonucleoside” G-dimethylamino-9 - (3 ‘-p -1nethoxy-L-phenylalanyl- t o give analogs of puromycin. These compounds arnino-D-ribosyl)-purine. h i interesting type of were active against Trypanosoma equiperdum7 and structural variant would be .the exchange of the the mammary adenocarcinoma* in mice. Some of amino acid moiety for a different amino acid or pep- these analogs had anti-bacterial activity, the most tide. effective being the L-phenylalanyl analog.6 Treatment of puromycin dihydrochloride2 with It is interesting to speculate t h a t in vivo the amino phenyl isothiocyanate and triethylamine in boiling acids are enzymatically removed t o give the “arnalcohol gave a nearly quantitative yield of phenyl- inonucleoside” which is the active portion of the thiocarbamyl derivative (I), m.p. 174-175’, [ a l Z 5 ~ antibiotic against Trypanosoma equiperdum and -43.6’ (acetone). Anal. Calcd. for C29H34K8- the mammary adenocarcinoma in mice. 08:C, 57.4; H , 3 . G G ; N, 18.5; S, 5.29. Found: I,EDERI,ELABORATORIES DIVISION B. R. BAKER C, 57.3; HI 5.73; N, 1S.3; S, 5.50. Cleavage of AMERICANCYANAMID COMP.4YY JOSEPH P. JOSEPH I with boiling methanolic sodium methoxide3 was PEARLRIVER,YEWPORK JAMES H. WILLIAMS complete in 1 hour. The solution deposited 65RECEIVED .L\PRIL 11,1951 70% of a crystalline “aininonucleoside,” B-dimethylamino- 9-(8’- amino - p - D - ribofuranosyl) - purinei (II), m.p. 213-’716’, [ a I 2 ’ D -24.G’ ( H 2 0 ) . Anal. TYROSINE-0-SULFATE IN A PEPTIDE FROM FIBRINOGEN Calcd. for Cl2Hl8N6O3:C, 49.0;H , G . 1 G ; N, 88.6. Sir: Found: C, 49.4; H I 6.39; S , 2S.4. One of the two peptides released from bovine The “aminonucleoside” (11) did not exhibit the bacterial spectrum6 characteristic of puromycin,’ fibrinogen when it is clotted by thrombin, peptide but the activity against Trypanosoma eqzciperdzsiiz 13, was found to give rise to tyrosine on acid hydrolwas increased 3-4 fold.’ Puromycin has a medium ysis.’ The phenolic hydroxyl group of this tyroorder of activity against the transplanted niani- sine appears not to be free in the intact peptide, mary adenocarcinoma of the C3II mouse; the however, for it does not react with fluorodinitro“aminonucleoside” is much more active, being benzene (FDNB), and the ultraviolet absorption spectrum of the peptide shows no trace of the peaks highly effective against this In order t o establish that the “aminonucleositle” a t 2775 and 293 rnp characteristic of tyrosine in neustill had the configuration and ring size of the sugar tral and alkaline solution, respectively. There is moiety as in the original antibiotic, puromycin was now good reason to believe t h a t the tyrosine is, in resynthesized from 11. Treatment of I1 in dimeth- fact, present as the 0-sulfate derivative. The eviylformamide with the mixed anhydride9 of X-car- tlence for this is as follows. 1. Mild hydrolysis with acid, but not with albobenzoxy-p-me thoxy-L-phenylalaninelo gave 64( of pure N-carbobenzoxy puromycin ( I I I ) , ni.j). kali, liberates the phenolic group, making it reac200s-210’. Anal. Calcd. for CaoH3jNiOi: C, tive to FDNB, and causing the ultraviolet absorp59.5; H, 3.S3; N, 1G.4. Found: C, 59.4; 13, tion characteristic of tyrosine to appear. The reac3.92; N , 1G.3. Hydrogenolysis of 111 in Methyl tion is complete in 4 minutes in N HC1 a t 93’. Such Cellosolve a t GO-70” in the presence of 1Oyopalla- marked lability t o acid and stability t o alkali is dium-charcoal gave puromycin base identical with characteristic of aryl sulfates. The amount of tyrosine present, estimated spectrophotometrically an authentic sample.2 A variety of amino acids such as L-phenylalanine, after acid hydrolysis, corresponds to one mole per L-tyrosine, L-lysine, L-tryptophan, L-leucine, p- 2800 g. of peptide, in good agreenicnt with the approximate minimum molecular weight of 3000 basctl (1) C. \V. Waller, P IV, F r y t h , B . I,. Hutchings a n d J . H. Williams, on the lysine content. THISJOCRNAL, 7 6 , 2025 (1953). (2) J. N. Porter, R. I. Hewitt, C. W.IIesseltine G. R r u p k a , J . A . 2. Tyrosine-0-sulfate was prepared by the acT,owery, W. S. Wallace, N. Bohouos a n d J. 11. Williams, Anfibiofic.s tion of concentrated sulfuric acid on tyrosine in the and Chemotherapy, 2 , 409 (1952). cold2 and crystallized as the K salt. Its ultraviolet (3) I n t h e usual practice of terminal cleavage of a peptide,‘ t h e action absorption spectrum differs markedly from that of of anhydrous hydrogen chloride on t h e phenylthiourea in a n inert solvent such as nitromethane is employed. These conditions weie tyrosine, showing a much weaker absorption with considered incompatible with I. inaximum near 263 nip. It is hydrolyzed by acid (4) P.E d m a n , Acta Chein. Scond.. 4 , 283 (1950). at a rate close t o that a t which the phenolic hy( 5 ) T h e details of t h e determination of t h e configuration of t h e sugar droxyl group of the tyrosine in peptide R is libermoiety as &furanose will be published b y C. W.Waller, et al. (6) Private communication from Dr. J. N. Porter of these laboraated. tories. 3. The formation of inorganic sulfate on mild (7) R. I. Hewitt, A. Gumble, W. S. Wallace a n d J. H . Williams, A m . J. T r o p . M c d . , in press. (8) J. J. Oleson, et al., t o he published. (9) R . A. Boissonaa, Helu. Chim. Acta, 84, 874 (1951). (10) R. P. Rivers a n d J. Lerman, J . Endocrinol., 6, 223 (1948).
(1) F. R . Bettelheim a n d K. Bailey, Biochim. Biofihys. A d a , 9, 578 (1952). (2) H.C. Reitz, R. E. Ferrel, H. Fraenkel-Conrat a n d €1. S. Olcott, T H I S JOURNAL, 68, 1024 (1946).
