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I1 I I was eluted from a silicic acid column and subjected again to countercurrent and column purification; 40 pg. of I was obtained from 100 g. of lyophilized bovine pineal glands.6 I and I1 pos5essed similar ultraviolet spectra (I, Xmax 2780 b., shoulders a t 2970, 3090 A.; 11, Xmax 2760 A., shoulders a t 2960, 3080 A.); fluorescence spectra (when Xexcitation = 304 mp, I Xmax = 333 mp, I1 Xmax = 338 mp). These data and similar blue color with Ehrlich reagent suggested that 1 is also a substituted 5-hydroxyindole, 0-Methylation a t position 5 was suggested by : failure to migrate as a cation in electrophoresis a t pH 11; lack of acid-base shift of ultraviolet absorption maxima; greatly increased lightening ability of many 5-methoxyindoles over their parent 5-hydroxyindoles. The presence of an ester or amide in the side chain a t position 3 was indicated by: detection of a carbonyl group (by infrared)' not conjugated with the indole nucleus (by ultraviolet absorption and fluorescence) ; neutrality of I and negative color reactions for aldehydes and ketones. An alcohol ester was unlikely because on hydrolysis of I with acid or base I1 was not detected. From these findings, together with observations that acetylcholine but not choline has lightening ability, we guessed that I might be I;-acetyl-5-methoxytryptamine. Synthetic N-acetyl-5-methoxytryptamine (111, 40 mg.) was prepared by reducing 100 mg. of 5methoxyindole-3-acetonitrile' with 160 mg. of sodium and 2 ml. of ethanol,*then acetylating the product with 4 ml. of both glacial acetic acid and acetic anhydride a t 100" for 1 minute. Purification was achieved by countercurrent distribution and silicic acid chromatography as for I. I11 and I were found identical with respect to: countercurrent distribution (peak tube 12) ; elution curve from silicic acid column; ultraviolet and fluorescence maxima; biologic activity (minimal lightening of isolated frog skin a t gram/ml.) ; Rf in descending chromatography systems 2-propano1:concd. NH3:water 16:1:3 (Rr 0.53); l-butano1:acetic acid:water 4:1:5 (Rzf0.9); 1-butanol: acetic acid:water:pyridine 15:5:12:10 (Rf 0 9); heptane :pyridine 7 :3 (Rf 0.10) ; heptane :pyridine G.5:3.5 (Rf0.80); benzene:ethyl acetate:water 20:1:20 (Rf 0.39). The increased lightening ability of I over Nacetyl - 5 - hydroxytryptamineg suggests that 0methylation of hydroxyindoles forms substances of increased biologic activity, in contrast to 0(6) We a r e grateful t o t h e Armour Laboratories f o r supplying us with many kilograms of bovine pineal glands. (7) We wish t o t h a n k Drs. A. Krivis a n d J. Szmuszkovicz a n d hlr. W. C. Anthony of T h e Upjohn Company for carrying out microinfrared studies, preparing numerous model compounds, supplying us with 5-methoxyindole 3-acetonitrile a n d offering m a n y helpful suggestions. (8) €1. Wieland, W. Konz a n d H. Mittasch, Ann., 613, 20 (1934). (9) J . D. Case, A . Fi T.erner and h l . R. Wright, t o be piiblishrd.
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methylation of catechol amines. The exquisite sensitivity of frog melanocytes to I indicates a neurohormone function, since melanocytes reflect their neural origin by responding to compounds, including acetylcholine and noradrenaline, which stimulate neurons. SECTIOX OF DERMATOLOGY AARONI3 LERNER DEPARTMEXT OF MEDICISE JAMES I). CASE YALEUNIVERSITY SCHOOL OF MEDICINE KEW HAVEX 11, CONNEClICTTT RESEARCH DIVISION RICKARD 1.. HEINZELMAN THEUPJOHN COMPAXY ~ A L A X 4 Z O 0h, ~ I C H I C A N
RECEIVED SEPTEMDER 16, 1959
WATER SUBEXCITATION ELECTRONS IN AQUEOUS FORMIC ACID RADIOLYSIS* Si?:
The sole y-ray radiolysis and lSG0 A. photolysis products of dilute formic acid are equal amounts of carbon dioxide and hydrogen. Carbon monoxide, however, is produced by formic acid excitation when the light-absorbing species is changed from water to formic acid a t 1860, 2537 and 2669 A.3.4 Although electron capture mechanisms have been suggested5.6" and may account for carbon monoxide, water subexcitation electronsst9 having a kinetic energy E < E,, the lowest excitation potential of water, provide a more likely exciting species for this reaction. G ( C 0 ) for pure 26.6 M formic acid irradiated by y-rays is 1.25 and consequently direct ionization and dissociation also produce carbon monoxide.4 But direct ionization cannot account for the unexpectedly high G ( C 0 ) of 0.25 and 0.50 in 0.1 and 1.0 M formic acid respectively. Over this concentration range, G(C02) increases and G(H,) remains unchanged. Direct action G(C02) here will be below 0.005 and 0.05. I n view of carbon monoxide formation by light, its absence in dilute solution radiolysis and the expected negligible direct ionization action in 0.1 11d solutions, direct dissociation of excited formic acid by water subexcitation electrons is postulated. Although the probability of molecular excitation is proportional to the appropriate optical constant only for fast the ratio of probabilities of two different excitations is approximately equal to the ratio of oscillator strengths down to fairly low values of Therefore we assume that all electrons including the subionization electrons excite water and formic acid in the ratio of kwcw to kfcfa t any given energy transfer ( k w , kf; molar 3s'
(1) Based o n work performed under t h e auspices of t h e U.S. Atomic Energy Commission. (2) H. Fricke a n d E . J . Hart, J. Chem. Phys., 2, 824 (1934). (3) A l . S. AIatheson, Proc. of t h e 2nd I n t l . Conf., Geneva, 1958, 29. 385, 195. (4) D. Smithies a n d E. J. H a r t , TRXSJOURNAL, i n press. (5) R. R. Williams a n d W.H . Hamill, R a d i a t i o n Research, 1, 158 (1954). (6) E. J. Hart, THIS JOURNAL. 1 6 , 4312 (1954). (7) E. H a y o n a n d J. Weiss, Proc. of t h e 2nd I n t l C o n f , Geneva, 29, 80 (1958). (8) R. L. Platzman, R a d i a t i o n Research, 1, 558 (1954); 2, 1 (1955). (9) J. Weiss, N a t u r e , 114, 78 (1984); J . chiin. p h y s . , 62, 539 (1955). (IO) H. A. Bethe, A n n . P k y s i k , 6, 325 (1930). (11) E. N. Lassettre, R a d i a t i o n Research S u p f i 1 ~ 1 1 1 ~1,n iban . (1959). (12) R. T,. P l a t i m n n , private communication.
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may complicate peptide synthesis. These functions may be blocked as a separate operation or a “selective” condensing agent may be used. This communication reports attractive examples of reagents in which the “extra” group is “protected” while thc carboxyl function is “activated.”
I1
Interaction of N-(p-nitrocarbobenzy1oxy)-L-histidine’ [m.p. 202-204’, c ~ -22.6’ ~ ~ D (C, 1.6 in G N hydrochloric acid) ] and N,N‘-diisopropylcarbodiimide in dioxane solution afforded SOY0 of the crystalline cyclized product I [found: C, 53.00; H, 3.91; N, 17.411; c r Z 5 ~-14.9’ 1.36 in tetrahydrofuran). Crystallization from tetrahydrofuran-hexane gave m.p.186-1S7°; the infrared specI , I I I I trum shows a strong band a t 1775-1780 cm.-’ atIO 9 e 7 6 5.5 5 4.5 ev. tributable to an activated amide. The benzylMOLAR EXTINCTION COEFFICIENTS amide, m.p. 190-191’ (ethanol-ether), [found: C, OF WATER A N D FORMIC AGIO. Fig. 1.-A, water vaporIs; 0, liquid water (solid line): 59.65; H, 5.33; N, 16.331, forms in nearly quanti55.5 k (dotted line) 14; 0 aq. formic acid (solid line) ; 0.1 k tative yield a t room temperature on treatment with benzylamine; there is no band a t 1775-1780 cm.-’. (dotted line) .I6 Although a large number of p-lactones are known, extinction coefficient and cw, cr ; concentrations of no authentic synthesis from the corresponding water and formic acid, respectively.) In Fig. 1, hydroxy acid has been reported2 until the past year.3 note that formic acid excitation dominates water We have cyclized N-trityl-~-serine~ to a fully excitation above 1800 A. even in 0.1 M formic acid. characterized crystalline /3-lactone (11), using Tu’,”Here kfcf exceeds hwtwby a factor of 100, which is far diisopropylcarbodiimide, in 15y0 isolated yield ; greater than the expected departure of the ratio of m.p. 193-194’, [cI]**D -62’ (c,0.5 in chloroform), corresponding excitation probabilities from theory. lo yKBr max 1820 em.-’, attributable to a ,&lactone group, The limits of this excitation effect can be esti- and no hydroxyl absorption band (chloroform) mated in 0.1 M formic acid by dividing G(C0) by [found: C, 79.S3; H, 5.78; N, 4.36; mol. wt., 309 the quantum yield, +(CO). One finds that the (Rast)]. Treatment of I1 with benzylamine gave, number of excitations/100 ev. varies from 1.7 for in 93% yield, the benzylamide, m.p. 146-147’, +(CO) equal to 0.14 a t ?669 A. to 0.40 for q5(CO) [ a ] 2 7 . 5 D -114’ (C,0.5 in chloroform) [found: equal to 0.58 a t lSG0 A. Thus this effect is ap- C, 79.62; H, 6.45; N, 6.291 identical with the preciable and supports Platzman’s prediction8 benzylamide obtained directly from the condensathat the subexcitation electron effect becomes tion of N-trityl-L-serine and benzykmiine in the significant a t molar concentrations of the order of presence of N,N’-diisopropylcarbodilmide. Treat0.1 and can attain a G value of the order of one. ment of I1 with L-alanine methyl ester hydrochloIf carbon monoxide originates from dissociation ride5 in the presence of triethylamine gave, in of formic acid by water subexcitation electrons, one G6Y0 yield, N-trityl-L-seryl-L-alanine methyl ester, may conclude that water contains no transitions m.p. 149-150’, [al3O”0 -41° 1 in ethanol) below 6.85 ev. A transition to a low-lying level, [found: C, 72.27; H, 6.58; N , 6.511. if allowed for these slow electrons (even though The possible utility of derivatives I and I1 as optically forbidden) would channel energy very monomers is being investigated. rapidly from the electron and formic acid woultf be DEPARTMENT OF CHEMISTRY J o m C. SIIEEHAN largely unaffected in solutions as dilute as 0.1 11s. ht ASSACHUSETTS INSTITUTE O F TECHNOLOGY KLAUSHASSPACFJER Acknowledgment.-The author wishes to thank \’IXG 1,IEFI YE11 39, MASSACHUSETTS M. S. Matheson, J. L. T/\:eeks and R. L. Platzman CAMBRIDGE RECEIVED OCTODER 1, 1969 for their cooperation and helpful discussions.
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(13) K. W a t a n a b e a n d M. Zelikoff. J. Q p l . SOC.A m . , 43, 753 (1953). (14) L. H . Dawson a n d E. D. Hulbert, i b i d . , 24, 175 (1934). (15) H. L e y and B. Arends, 2. p h y s i k . Chcm.. B17, 177 (1932).
CHEMISTRY DIVISION ARCONNEKATIONAL LABORATORY ILLINOIS EDWINJ. HART LEMONT, RECEIVED SEPTEMBER 3, 1959 ACTIVATED CYCLIC DERIVATIVES OF AMINO ACIDS
Sir: Many biologically important amino acids contain “extra” functional groups (e.g., hydroxyls, thiols, and potentially reactive heterocycles) which
(1) Prepared by t h e procedure described for t h e racemate, D. 7‘. Gish a n d F. H. Carpenter, THISJ O U R N A L , 76, 950 (19531. (2) H. E. Zaugg, “Organic Reactions,” Vol. V I I I , R . Adams, E d i t o r , John W l e y a n d Sons, Inc., New York, N. Y., 195.3, p. 307. (3) I n independent work which WYRS published after t h e complrtioil of our synthesis, yohimbic acid was cyclized t o a 8-lactone; 1’. .\. Diassi a n d C. hl. Dylion, i b i d . , BO, 374G (19.58). See also J C. Sheeban, Abstracts of Papers, 135th A. C. S. Meetirlg, April, 1559, 11. 3 - 0 . (4) Prepared essentially by t h e method reported f u r Wtrityl-D,I.serine, G . Amiard, R. Heymes a n d L. Velluz, Bull. Sac. C h i m . FranLc. 191 (1955). N-Trityl-L-serine was characterized as t h e crystalline diethylamine s;llt, m.p. 137-138’. [ U ] ~ ~-29’ D 1 in tnethanul) [ f o u n d : C , 7 4 . 1 9 ; H, 7 60; hT, 6.843. ( 5 ) Prepared hy Fischer esterification, m.p. 110-110.5°, [a]*>lu t 41’ (C, 1 i i i rn?thanol) [found: C , 34.17; H, 7.10; N,10.001.
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