554 FUNK : SYNTHESIS OF DL-3 : 4-DIHYDROXYPHENYLALANINE~

LXV.-Syn thesis of dl-3 : 4-DihydroxyphenylaZanine,

By CASIMIR FUNK.

THE question of the formation of adrenaline in the animal body has engaged much attention. The two amineacids that suggest them- selves as the parent substances of adrenaline are obviously tyrosine and phenylalanine, but certain considerations render it probable that the body is incapable of employing these substances for the formation of adrenaline. Thus, in cases of abnormal metabolism, tyrosine is eliminated as homogentisic acid (Wolkow and Baumann, Zeitsch. physiol. Chem., 1891, 15, 228; Fdta and Langstein, ibid., 1903, 37, 513; Abderhdden, Bloch, and Rona, ibid., 1907,52, 435), whilst no c a k h o l derivatives have been obtained from any of the aromatic amino-acids.

In the experiments which have been conducted on this point, investigators have sought to bring about a transformation of tyrosine (Ewins and Laidlaw, J. Physiol., 1910, 40, 275) into adrenaline by incubating the former substance with suprarenal gland tissue. It may well be, however, that the formation of adrenaline takes place in several steps, and that different tissues of the body are necessary.

I n order to be certain that adrenaline is really formed, it is necessary to find a chemical method by which this substance can be separated from amino-acids, and such a separation is at present being attempted.

That some substance of the nature of tyrosine or phenylalanine is the precursor of adrenaline is further suggested by the fact that all three compounds belong to the same optical ( I ) series.

The synthesis of 3 : 4-dihydroxyphenylalanine has been effected with the idea that it may be the parent substance of adrenaline; experiments are also being conducted in order to ascertain whether any of this substance is contained in proteins. ‘That this may be

* Anhydrous substance.

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FUNK : SYNTHESIS OF DL-3 : 4-DIHYDROXPPRENYLALANINE. 555

the case is indicated by the occurrence of caffeic acida in plants. A dihydroxycaff eic mid,

H O H O/-\CH,* C H2* C02H,

has been found by Lippmann in turnip leaves (Ber., 1892,25, 3220). A comparison of the formuh of adrenaline and dihydroxyphenyl-

alanine indicates a, certain similarity between these substances :

\-/

O H OH 0.1) \/

OHf)

CH,* CE(NH,)*CO,H \/

CH( 0 H)* CH, NH C H, Adrenaline. Dihy hoxyphen ylalanine.

EXPERIMENTAL. Synthesis of 3 : 4 - D i ~ ~ y d r o s y p ~ , e n y l a n ~ e ~

Dihydroxyphenylalanine was obtained according to the method described by Erlenmeyer and Halsey (.4nnaZen, 1899, 307, 138; Ber., 1897, 30, 2981) for the synthpis of tyrosine.

Cond'ensat%on of 3 : 4-Carbonyldioxyb ensaldehyde and Hippuric Acid: Lactsrnide of a-Benzoylamim-3 : 4-dihydroxychtnamic Acid,

Sixty grams of 3 : 4-carbonyldioxybenzaldehyde were mixed with 66.5 grams of hippuric acid and 30 grams of anhydrous sodium acetate, and the mixture was heated for one hour on the water-bath with 112 grams of acetic anhydride. The solution became yellow, and in a short time a yellow, crystalline substance separated out. Owing to the insolubility of this substance in the ordinary solvents, it was found necessary to purify it by boiling, first with water and then with alcohol. The crystals obtained in this way consisted of small, yellow plates, which melted about 238O. Yield=83 grams, or 74 per cent. of the theoretical. After drying in a, vacuum at 70°, the substance was analysed:

0,3116 gave 0.7604 CO, and 0.0906 H,O. 0.4266, by Kjeldahl's method, required 13.5 C.C. N/lO-H,SO,.

C = 66-55 ; H = 3.23.

N = 4.43. C,,H,O,N requires C = 66.43 ; H = 2.95 ; N = 4.56 per cent.

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556 FUNK : SYNTHESIS OF DL-3 : 4-DIHYDROXYPHENYLALANlNE.

a-Benaoylumino-3 ; 4-dihydroxycinnarnio A cid, HO

HO/-\CH : C( NHB+C O,H. \-/ Eighby-three grams of the lactimide prepared m above were heated

on the water-bath with 500 C.C. of 10 per cent. sodium hydroxide solution. In this way, both the lactimide and carbonyldioxy-groups were hydrolysed. After cooling and neutralising with hydrochloric acid, a resinous mass separated out, which became crystalline after keeping for some time is the cold. This was twice recrystallised from alcohol, when small, yellow plates were obtained, which, on drying in a vacuum, melted a t 210-215O. A t looo it sublimed to form colourless plates. The yield was 60 grams, or 75 per cent. of the theoretical :

0.2268 gave 0.5368 CO, and 0-0902 H,O. 0-39, by Kjeldahl's method, required 12.2 C.C. N/10-H2S0,.

C= 64.55 ; H=4-31.

N=4.35. 610H13Q5N requires C = 64.18 ; H = 4-38 ; N = 4.68 per cent.

Preparation o j dl-a-Benzoylamino-3 : 4-dihydroxy- fl-ph enylpropionic H O

id, HO/\CH~*CH(NHB~).C~~H. L/ The 60 grams of the substance just described were suspended

in 600 C.C. of water, and 750 grams of sodium amalgam added in small portions during forty-five minutes. The solution was neutralised with hydrochloric mid, and concentrated under diminished pressure in a stream of coal gas. Small, yellow, four- sided plates separated out. Yield, 19.6 grams, or 32 per cent. of the theoretical. By following the directions of E. Fischer (Ber., 1899, 32, 3638), who, in his work on tyrosine, boiled the correspond- ing solution with 33 per cent. aqueous sodium hydroxide, all this substance seemed to be destroyed.

The crystals were recrystallised from water, and contained one molecule of water of crystallisation, which was expelled when heated in a vacuum a t l l O o . They are sparingly soluble in cold, and readily so in hot, water, also in alcohol, glacial acetic acid, or ethyl acetate, but not in benzene. The anhydrous substance melts at 190-195O:

1.092 lost 0.0596 H,O. H20=5*45, C,,H,,0,N,H20 requires H20 = 5-64 per cent.

0.2616 gave 0.614 COz and 0.1133 H,O. C=64*01; H=4*80.

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FUNK : SYNTHESIS OF DL-3 : 4-DlHTDROXYPHENYLALANINE. 557

0.3248, j y Kjeldahl’s method, required 10.0 C.C. N / 10-H,S04. N =4.30.

C,6H,,0,N requires C = 63.75 ; H = 5.03 ; N =4*65 per cent.

- dl-3 : 4-Dihydl.o~~~,henyZala?tine, HO’ \CH,*CH(NH,)*CO,H. \-/ 18.2 Grams of the benzoyl compound were hydrolysed by boiling

with sixty times its weight of 20 per cent. hydrochloric acid under a reflux condenser. The solution was left overnight in the cold, and the benzoic acid which separated out was collected. The filtrate was extracted with ether, and the aqueous solution con- centrated in a vacuum. The residue obtained was dissolved in a, small quantity of water, neutralised with ammonia, and con- centrated under diminished pressure in an atmosphere of coal gas. On recrystallising from water, 5.7 grams of spindle-shaped crystals were obtained, corresponding with a yield of 47 per cent. of the theoretical. After being dried in a vacuum, the crystals melted at 263-!272O:

0.21 gave 0.4218 CO, and 0.1042 H,O. 0.1838, by Ejeidahl’s method, required 9.0 N / lo-H2S04.

C = 54.75 ; H =5*51.

N = 6.85. CSH,,04N requires C = 54.79 ; H = 5-63 ; K = 7.10 per cent.

This substance is readily oxidised in neutral, and more readily in alkaline, solution, wit’h the formation of a black pigment. With nitric acid, a red coloration is obtained. Millon’s reagent in the cold gives an orange colour, but on heating, reduction takes place. With ferric chloride, a green colour is formed, which, on boiling or on the addition of ammonia, becomes dazk red. The substance reduces Fehling’s solution on heating. It dissolves in water more easily than does tyrosine, and gives a crystalline hydrochloride, but an ethyl ester hydrochloride could not be obtained.

I am at present preparing the active I-compound and some derivatives of it, and intend shortly to publish some experiments on the physiological action of these compounds. I wish to express my thanks to Dr. Harden for granting me the use of his laboratory.

LISTER INSTITUTE OF PREVENTIVE MEDICINE, CHELSEA, S. W.

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