CHEMICAL AND METABOLIC STUDIES ON PHENYLALANINE*

I. THE NITRATION OF PHENYLALANINE

BY RICHARD J. BLOCK AND DIANA BOLLING

(Prom the Department of Chemistry, New York State Psychiatric Institute and Hospital, New York)

(Received for publication, March 11, 1939)

The quantitative estimation of phenylalanine depends upon the formaCon of a violet, color when the amino acid is first nitrated and then reduced with hydroxylamine and ammonia. The chemical investigations which led to this observation arc sum- marized briefly below.

Mohler (4) in 1890 observed the formation of a red color when benzoic acid was nitrated with cold fuming nitric acid and t,he resulting product was reduced with ammonium sulfide in an am- moniacal solution. This reaction was modified by Grossfeld (5) who heated benzoic acid with an excess of concentrated sulfuric acid containing 10 per cent of potassium nitrate in a boiling water bath for 20 minutes. After cooling the solution, an excess of hydroxylamine hydrochloride followed by concentrated ammo- nia was added. The solution became a cherry-red similar to the color of ferric thiocyanate.

In 1928, Kollmann (6) proposed a quantitative method for the estimation of phenylalanine in proteins which involved oxida- tion to benzoic acid and isolation of the latter by extraction with ether. Kapeller-Adler (7) hoped that by treating a protein hydrol-

* Interest in the metabolism of phenylalanine was aroused by the find- ings of Foiling (l), Penrose (2), and Jervis (3) of a new type of metabolic abnormality in man in which the organism is unable to catabolize phenyl- alanine normally. It is planned to carry out metabolic studies on patients with phenylpyruvic oligophrenia and on experimental animals. The pres- ent investigation was aided by a grant from Child Neurology Research (Friedsam Foundation) to Dr. G. A. Jervis.

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2 Phenylalanine. I

ysate with nitric acid as employed by Grossfeld, the phenyl- alanine would be first oxidized to dinitrobenzoic acid which could be estimated by reduction with hydroxylamine and ammonia. Experimental attempts to verify this idea resulted in the formation of a violet color instead of the expected cherry-red. In order to explain this discrepancy, Kapeller-Adler proposed the mechanism shown in Equation 1.

(l) (jxii~Esfi*-E::~

NOe 2

COONH, COOH COOH

R = -CH&HNH,COOH.

However, repeated attempts to isolate 3-nitroso-4-nitrobenzoic acid and 3-nitro-4-nitrosobenzoic acid from phenylalanine after nitration and reduction failed (cf. below).

The chemical reactions given in Equation 1 were based upon the following experimental evidence which was taken largely from the literature. Morner (8) isolated p-nitrobenzoic acid after treating proteins with hot concentrated nitric acid. Kapel- ler-Adler (7) observed that when p-nitrobenzoic acid was heated with 10 per cent potassium nitrate in concentrated sulfuric acid, the resulting nitration product gave a violet color on reduction with hydroxylamine and ammonia. However, the principal sup- port in favor of the reactions given in Equation 1 was based upon the studies of Meisenheimer and Patzig (9, 10).

Meisenheimer (9) in 1903 reported that when o- or p-dinitro- benzene is carefully reduced with stannous chloride or hydroxyl-

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R. J. Block and D. Bolling 3

amine in the presence of an excess of alkali, a deeply colored solution resulted (Equation 2).

(2) CsH4(NOz)z + Hz + 2KOH - CeH4(=N02K)2 + 2H20

That is, 2 hydrogen atoms entered the benzene ring in the 1,2 or 1,4 positions to form the two diacidihydrodinitrobenzenes with the structure shown in Equation 3.

(3) H =NOOK

Ii

-NOOK

or \,=NOOK

0 I I

=NOOK

Meisenheimer and Patzig (10) continued these investigations and isolated the sodium salt of diaci-p-dihydrodinitrobenzene (Equa- tion 4).

(4) p-CeHa(NOz)z + 2NHzOH + 2NaOH-p-C6H4(=NOONa)2 + 4&O + Nz

The analogous conversion of o-dinitrobenzene into diaci-o-dihy- drodinitrobenzene was not successful but the latter compound was obtained from o-nitronitrosobenzene by the addition of alkali.

(5) o-CeHa(NOz)(NO) + 2NaOH - C&H4(=NOONa)2 + Hz0

Meisenheimer and Patzig (10) also found that when p-xylene was nitrated and the resulting dinitro-p-xylenes were treated with alkaline hydroxylamine, a red-colored compound was formed. After extraction of the alkaline solution with ether, and acidifica- tion of the aqueous residue with hydrochloric acid, a compound was isolated which they believed t,o be o-nitronitroso-p-xylene, but which may have been 2-nitro-5-nitroso-p-xylene. Meisen- heimer and Patzig summarized their findings as indicating that diaci-o-dihydrodinitrobenzene derivatives are colored violet and diaci-p-dihydrodinitrobenzene derivatives are red, an observation which we have fully confirmed.

The experiments described in this paper were undertaken in order to elucidate, if possible, the reactions involved in the for- mation of the violet color from phenylalanine. The results of our experiments confirm the suggestion of Kapeller-Adler (7) that the

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4 Phenylalanine . I

chromogen formed in the determination of phenylalanine is a deriv- ative of diaci-o-dihydrodinitrobenzene. The mechanism shown in Equation 6, which is based upon daba given in this paper, is proposed.

ti NO,

Yellow

or

R

NO

R

Violet

RI may be --CH&HNH&OOH, -CH.$H&OOH, or --CH&OOH. R may be -CH&HNH&OOH, -CH&H&OOH, -CH&OOH, or

COOH.

As mentioned above, the nitration of benzoic acid under the conditions of IMohler (4) and others (5, 11) followed by reduction of the resulting dinitro compound gave a red color. It has been reported in the literature that this color is due to 3,5-dinitroben- zoic acid. The results reported in this paper indicate that the cherry-red color is probably formed from 2,5-dinitrobenzoic acid according to Equation 7.

COOH COOH COOH I I I

,HNOs ---E- \/

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R. J. Block and D. Bolling 5

COOH I

COOH COONa

/ NO2 ,I or T-L

\/\H HCI =NOONa

COOH O,N

It appears that the positions of the nitro groups which are introduced into the benzene ring of benzoic acid and its derivatives may vary with conditions. Benzoic acid and m-nitrobenzoic acid do not yield 2,bdinitrobenzoic acid under the nitrating conditions which we employ; but 2,5-dinitrobenzoic acid was formed from o-nitrobenzoic acid. On the other hand, p-nitroben- zoic acid yields a small amount of 3,4-dinitrobenzoic acid by nitration with 20 per cent potassium nitrate in concentrated sul- furic acid.

As will be seen below, the presence of two nitro groups in the 1,2 position on the benzene ring permits, in certain cases at least, reduction of one double bond with as mild a reducing reagent as hydrogen sulfide in acid solution.

EXPERIMENTAL

Synthesis-3-nitro-4-nitrosotoluene, 2-nitroso-3-nitrotoluene, 2- nitroso-5-nitrotoluene, 3,4-dinitrotoluene, 2,3-dinitrotoluene, and 2,bdinitrotoluene were synthesized from 3-nitro-4-aminotoluene, 2-amino-3-nitrotoluene, and 2-amino-5-nitrotoluene according to the procedure of Page and Heasman (12). The nitrosonitro compounds were prepared by oxidation of the aminotoluenes wit*h persulfuric acid and the dinitrotoluenes were made by oxidation of the nitronitrosotoluenes with warm fuming nitric

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6 Phenylalanine. I

acid. The properties of these compounds are in agreement with the literature. The 2,3-, 2,5-, and 3,4-dinitrotoluenes were recrystallized from alcohol.

p-Nitrophenylalanine was prepared from dl-phenylalanine by nitration with fuming nitric acid at 0” according to the directions of Erlenmeyer and Lipp (13). M.p. 244”. Erlenmeyer and Lipp give 242-243”.

N&&on’-The following compounds in 1 to 2 mg. quantities were nitrated by warming on the steam bath for 20 minutes with a 20 per cent solution of potassium nitrate in concentrated sulfuric acid,2 I-phenylalanine, phenylacetic acid, phenylpropionic acid, p-nitrobenzoic acid, dl-p-nitrophenylalanine, toluene, benzal- dehyde, benzoic acid, aniline, nitrobenzene, cinnamic acid, man- delic acid, phenylpyruvic acid, phenylserine, o-nitrobenzoic acid, m-nitrobenzoic acid, o-nitrotoluene, m-nitrotoluene, p-nitrotol- uene, a-amino-p-hydroxyphenylacetic acid, tyrosine, m-amino- phenylacetic acid, 2-amino-3-nitrotoluene, 3-nitro-4-aminotoluene, and 3-nitro-4-aminobenzoic acid.

The resulting reaction mixtures, as well as 2,3-dinitrotoluene, 2,4-dinitrotoluene, 2,5-dinitrotoluene, 2,6-dinitrophenol, 3,4- dinitrotoluene, 2,4-dinitrobenzoic acid, p-toluquinone, and 2,4,6- trinitroresorcinol, were reduced with hydroxylamine and am- monia (14).

By this procedure, only the two o-dinitrotoluenes and the nitrat,ion products of phenylalanine, p-nitrophenylalanine, phenyl- acetic, phenylpropionic, and p-nitrobenzoic acids gave the violet color typical of diaci-o-dinitrodihydrobenzene (cf. Figs. 1 and 2).3 2,5-Dinitrotoluene and o-nitrobenzoic acid gave the cherry- red color of diaci-p-dinitrodihydrobenzene (cf. Fig. 1). Some of t.hese data are summarized in Table I in which the amount of

1 Nitric oxide fumes were not observed during the nitration of phenyl- alanine, phenylacetic acid, etc.

2 The time of heating, which was taken from Grossfeld and Kapeller- Adler, does not appear to be of great significance, for equivalent amounts of color were developed by phenylalanine which had been heated 5, 10, 15, and 20 minutes respectively. However, when the nitration was carried out at room temperature for 30 minutes considerably less color was formed.

3 The absorption spectra were made in the Electrical Testing Labora- tories, New York.

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R. J. Block and D. Bolling 7

color from 3,4-dinitrotoluene has been assigned the value of 100 per cent.4

According to Meisenheimer and Patzig (10) (Equation 5), alkalization of 3-nitro-4-nitrosotoluene and 2-nitro-3-nitroso- toluene should result in the formation of the violet color; this was confirmed experimentally.

0 WAVELENGTH

FIG. 1 FIG. 2

FIG. 1. Absorption curves of o- and p-dinitrobenzene derivatives. Curve 13,4-dinitrotoluene, Curve II 2,5-dinitrotoluene, Curve III nitrated phenylalanine, Curve IV 2,3-dinitrotoluene.

FIG. 2. Absorption curves of nitration products of phenylalanine and related compounds. Curve I p-nitrophenylalanine, Curve II phenylalan- ine, Curve III phenylacetic acid, Curve IV phenylpropionic acid.

Isolation of Diaci-S,.&Dinitrodihydrophenylalanine-825 mg. of I-phenylalanine were dissolved in 10.7 cc. of concentrated sul- furic acid which contained 1.96 gm. of barium nitrate. The phenylalanine solution was warmed in an evaporating dish for 20

4 The quantity of chromogen was determined in the Goudsmit-Summer- son photoelectric photometer (Klett), the Zeiss step-photometer, or prefer- ably in the Evelyn photoelectric calorimeter.

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8 Phenylalanine. I

minutes on the steam bath. At the end of this period, the solu- t,ion was poured into an Erlenmeyer flask and diluted with 500 cc. of water. The aqueous solution was heated to boiling and an excess of basic lead carbonate was added. The precipitate was removed and thoroughly washed with hot water. The lead was removed from the warm filtrate with hydrogen sulfide. After the lead sulfide was centrifuged off, the solution was concentrated to 50 cc. in vacua. This solution gave the typical violet diaci-o- dihydrodinitrobenzene color on treatment with sodium carbonate; i.e., the benzene ring had been partly reduced by hydrogen sulfide. The chromogen was extracted with butyl alcohol and the solvent layer was dried over sodium sulfate. The sodium sulfate was

TABLE I Development of Color by Nitration of Various Compounds

Compound I

C&Jr

3,4-Dinitrotoluene.. 2,3-Dinitrotoluene................. I-Phenylalanine + glycine, etc..

‘I dl-p-Nitrophenylalanine. Phenylacetic acid. _. . p-Phenylpropionic acid.. p-Nitrobenzoic acid.. Benzoic acid.. .

.

. . .

. . .

per cent . 100

26 . 27

. . . 16 8 7 3

. . 5

. 0

removed by filtration and thoroughly washed with butyl alcohol. The yellowish red solution was concentrated in vacua until crystals appeared in the flask. These were redissolved by warming on t,he steam bath and crystallization was allowed to take place first at room temperature and then in the refrigerator. The first crop weighed 132 mg. R/l.p. 180~-181” (uncorrected). A second crop, 430 mg., m.p. 180-181” (uncorrected), was precipitated from the mother liquors of Crop 1 by the careful addition of ether.

Recryst,allization of Crop 2 from alcohol and ether gave a light yellow product which melt,ed very sharply with decomposi- tion at 182-183’ (uncorrected), and which darkened somewhat on drying in the oven at 110” for 24 hours. The compound gives a

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R. J. Block and D. Bolling 9

negative ninhydrin reaction.5 Analysis of the oven-dried material showed the following composition C 43.6 per cent, H 4.1 per cent, X 16.2 per cent; calculated for diaci-3,4-dihydrodinit,rophenyl- alanine, C 42.0 per cent, H 4.28 per cent, N 16.3 per cent. This compound, presumably diaci3,4-dinitrodihydrophenylalanine, gives the typical violet, color with ammonia, sodium hydroxide, and sodium carbonate. The sodium salt gave the absorption curve shown in Fig. 3. The red filtrate from Crops 1 and 2 was evaporated to dryness over PzO5. The residue, a red amorphous

FIG. 3. Absorption curves of diaci-3,4-dinitrodihydrophenylalanine. Curve I diaci-3,4-dinitrodihydrophenylalanine (filtrate), Curve II nitrated phenylalanine, Curve III diaci-3,4-dinitrodihydrophenylalanine (m.p. 182”).

solid, soft’ened at approximately 65-70” and decomposed at 220- 250” (uncorrected). This substance, which is somewhat impure, when dissolved in dilute alkali, gave t’he same absorption spectrum as nitratcd and reduced phenylalanine (cf. Fig. 3)3 and it may be 3,4-dinitrodihydrobenzoic acid.

Diaci-3,4-dinit,rodihydrophenylalanine is soluble in water and warm 95 per cent alcohol but it is insoluble in absolute alcohol,

5 p-Nitrophenylalanine gives a typical ninhydrin reaction.

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10 Phenylalanine. I

ether, and benzene. It forms a yellow aqueous solution which is converted into the violet chromogen at approximately pH 9.5 (pink to phenolphthalein). Addition of acid (hydrochloric, sulfuric, and acetic) regenerates the yellow color; alkalization again gives the violet salt. Diaci3,4-dinitrodihydrophenylal- anine yields approximately 140 per cent of the color obtained from an equimolar quantity of phenylalanine.

Isolation of 3, .J-Dinitrophenylalanine-825 mg. of Z-phenylala- nine were added to 10.7 cc. of cold concentrated sulfuric acid containing 1.96 gm. of barium nitrate in the same manner as employed in the isolation of diaci-3,4-dinitrodihydrophenyl- alanine. The excess sulfuric acid was removed with calcium carbonate and the solution was extracted with butyl alcohol and then with ether. The butyl alcohol solution was saved and it was concentrated in vaczLo to dryness; the residue was dissolved in warm absolute alcohol and precipitated by the addition of ether. A crystalline precipitate appeared. This precipitate, after several recrystallizations, melted at 155” and decomposed at 182”. It gave over 160 per cent of the color obtained from an equimolar quantity of phenylalanine. The ninhydrin test was negative. The substance had the following composition, C 43.0 per cent, H 3.72 per cent, N 15.1 per cent, calculated for C9H90JV3.- $C2H,0H, C 43.1 per cent, H 4.3 per cent, N 15.1 per cent. This compound is probably 3,4-dinitrophenylalanine.

Results

It was found that of the thirty-six compounds which were tested in equimolar quantities only seven gave the typical violet diaci-o-dihydrodinitrobenzene color. These alone will be dis- cussed separately.

3,4-Dinitrotoluene gave the greatest amount of color and was arbitrarily assigned the value of 100 per cent,.

2,3-Dinitrotoluene t.akes considerably longer to develop its maximum color, requiring approximately 120 minutes against 30 minutes for the 3,4-dinitro compound. Even then, the maximum amount of chromogen formed is only about one-quarter of that produced by 3,4-dinitrotoluene under the same conditions. The similarities in the absorption spectra of the chromogens obtained from arbitrary quantities of 2,3- and 3,4-dinitrotoluene are

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R. J. Block and D. Bolling 11

clearly brought out by the curves shown in Fig. 1 which were obtained in the Hardy recording color analyzer.3

Nitration of phenylalanine, especially in the presence of glycine and tyrosine, produces approximately the same amount of color as given by an equimolar quantity of 2,3-dinitrotoluene, but color formation is rapid, similar to 3,4-dinitrotoluene.

Nitration of p-nitrophenylalanine and phenylacetic acid gives only approximately 7 per cent of the color produced by the stand- ard, while phenylpropionic acid gives even less color than phenyla- cetic acid and only 3 per cent of that formed by 3,4-dinitrotoluene. As mentioned above, Kapeller-Adler stressed the fact that p- nitrobenzoic acid gives the violet color. It will be seen from Table I that only a very small amount of the color is obtained with p- nitrobenzoic acid.

SUMMARY

1. Nitration of phenylalanine, phenylacet’ic acid, and p- phenylpropionic acid with potassium nitrate in concentrated sulfuric acid at approximately 100” yields t’he p-nitro derivatives; these are t’hen further nitrated to the 3,4-dinitro compounds.

2. Derivatives of o-dinitrobenzene are relatively easily re- duced by hydroxylamine or hydrogen sulfide to the corresponding derivatives of diaci-o-dinitrodihydrobenzene which in alkaline solu- tion (about pH 9.5) form the violet o-quinone-like compounds of o-isodinitrodihydrobenzene. On the other hand, reduction of derivatives of p-dinitrobenzene yields, under t’he same conditions, cherry-red p-quinone-like compounds of p-isodinitrodihydroben- zene.

3. The possible mechanisms involved in formation of a violet> color from the nitration of phenylalanine followed by reduction in alkaline solution and the formation of a red color from benzoic acid under the same conditions of nitration and subsequent re- duction are given.

We wish to thank Professor Hans T. Clarke for helpful ad- vice during this investigation and Dr. J. A. St’ekol and Dr. G. A. Jervis for some of the compounds used in the nitration studies.

BIBLIOGRAPHY

1. FGlling, A., Z. physiol. Chem., 227, 169 (1934). 2. Penrose, L., Lancet, 1, 23 (1935).

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12 Phenylalanine. I

3. Jervis, G. A., J. Biol. Chem., 126, 305 (1938). 4. Mohler, M. E., Bull. Sot. chim., 3, 414 (1890). 5. Grossfeld, G., 2. Untersuch. Nahrungs- u. Genussmittel, 63, 467 (1927). 6. Kollmann, G., Biochem. Z., 194, 1 (1928). 7. Kapeller-Adler, R., Biochem. Z., 262, 185 (1932). 8. MGrner, C. T., 2. physiol. Chem., 96, 263 (1915). 9. Meisenheimer, J., Ber. them. Ges., 36, 4174 (1903).

10. Meisenheimer, J., and Patzig, E., Ber. them. Ges., 39, 2526 (1906). 11. Waelsch, H., and Klepetar, G., Z. physiol. Chem., 236, 92 (1935). 12. Page, H. J., and Heasman, B. R., J. Chem. Sot., 123, 3235 (1923). 13. Erlenmeyer, E., and Lipp, A., Ann. Chem., 219, 179 (1883). 14. Block, R. J., The determination of the amino acids, Minneapolis, re-

vised edition (1938).

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Richard J. Block and Diana BollingNITRATION OF PHENYLALANINE

STUDIES ON PHENYLALANINE: I. THE CHEMICAL AND METABOLIC

1939, 129:1-11.J. Biol. Chem. 

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