THE SPATIAL CONFIGURATION OF a-amino-p- HYDROXY -n-butyric ACID *

Transcription

1 THE SPATAL CONFGURATON OF a-amno-p- HYDROXY -n-butyrc ACD * BY CURTS E. MEYER? AND WLLAM C. ROSE (From the Laboratory of Biochemistry, University of llinois, Urbana) (Received for publication, July 20, 1936) Several months ago McCoy, Meyer, and Rose (1935) described the isolation and identification of cr-amino+-hydroxy-n-butyric acid as a cleavage product of proteins, and demonstrated that the new amino acid is an indispensable component of the diet. Since that time attention has been directed to the determination of the spatial configuration of the compound. t is the purpose of the present paper to outline these investigations and to suggest a simple name for the amino acid. EXPERMENTAL Obviously, cr-amino-/3-hydroxy-n-butyric acid may exist in four optically isomeric forms. n our previous report (McCoy, Meyer, and Rose, 1935) we pointed out that the natural acid has a specific rotation of approximately -28. Reduction with hydriodie acid and red phosphorus (cf. Abderhalden and Heyns (1934)) yielded an ar-amino-n-butyric acid having a slight but unmistakable dextrorotation. Unfortunately, it was not possible to determine accurately the specific rotation of the reduced product because of the small amount of material available at that time. We have now repeated the reduction using slightly modified conditions. For this purpose, a mixture of 1 gm. of the hydroxyamino acid, 0.3 gm. of red phosphorus, and 12 cc. of hydriodic acid (sp. gr. 1.96) was heated in a sealed tube at for 7 hours. After cooling, the contents of the tube were diluted with 300 cc. of water and evaporated in vacua to a volume of 3 cc. The * Aided by grants from the Rockefeller Foundation and the Graduate School Research Fund of the University of llinois. t Research Assistant in Biochemistry. 721

2 722 a-amino-/3-hydroxybutyric Acid latter was diluted to 250 cc. and treated with moist silver oxide for the removal of the iodide ion. The excess silver was precipitated with hydrogen sulfide, and the silver sulfide was removed by filtration. The solution was decolorized with a small amount of norit, and after filtration was concentrated in vacua to about 2 cc. Absolute alcohol was added until a faint turbidity developed, and the solution was placed in a refrigerator for crystallization. The product was recrystallized twice by dissolving in the minimum amount of water and adding absolute alcohol to incipient precipitation. The following values were obtained on analysis. GHsNOs. Calculated. C 46.57, H 8.80, N Found , 8.69, A determination of the specific rotation in aqueous solution yielded the following result blzd = 2 x (0.1495/5) = +4.w According to Oikawa (1925)) natural cy-amino-n-butyric acid has a specific rotation of Fischer and Mouneyrat (1900) report values of +S.O and -7.92, respectively, for the two enantiomorphs prepared by the resolution of synthetic or-aminobutyric acid. Evidently, almost half of our compound had undergone racemization during the reduction; but the data demonstrate conclusively that the product was the dextrorotatory form of (Yamino-n-butyric acid. The above observations suffice to establish the spatial relationship of the groups around the a-carbon atom of our hydroxyaminobutyric acid. d-cr-amino-n-butyric acid, like all other naturally occurring a-amino acids thus far investigated, belongs to the L series (cf. Clough (1918) and Levene (1926)). Thus the hydroxyaminobutyric acid present in proteins must have one of the two possible configurations indicated in Formulas and. COOH COOH HtN-C-H H,N -C-H HO-C-H H-C-OH CHs CHa

3 C. E. Meyer and W. C. Rose 723 Formula is analogous to the structure of the sugar (+)- erythrose, while Formula corresponds to the configuration of d( -)-threose. n order to establish which of the above represents the correct formula, advantage was taken of the observation of Dakin (1917) that oxidation of an a-amino acid (1 mole) with chloramine-t (1 mole) yields the corresponding aldehyde with 1 less carbon atom. According to this procedure, hydroxyaminobutyric acid should yield a lactic ddehyde whose spatial configuration could be identified by further oxidation to the lactic acid. For this purpose, 2 gm. of hydroxyaminobutyric acid were dissolved in 10 cc. of water and treated with a solution of 5 gm. of chloramine-t in 40 cc. of water. The temperature of the amino acid solution was about 30, while that of the reagent was approximately 80. The mixture was stirred vigorously for 15 minutes, and was then cooled in an ice bath until most of the toluene sulfonamide had crystallized. After filtration, the precipitate was washed with a small amount of ice water, and the washings were added to the main solution. The whole was diluted to 100 cc. A portion of the above solution was tested for its optical rotation. t rotated strongly to the left, indicating that the oxidation had not proceeded to pyruvic aldehyde or acid. The biuret reaction was negative, showing the absence of hydroxyaminobutyric acid. As further proof that lactic aldehyde was actually produced, a sample of synthetic ac-amino-/3-hydroxy-n-butyric acid2 was treated with chloramine-t, as outlined above, and the resulting product was transformed into the p-nitrophenylosazone according to the method of Dakin and Dudley (1913). After purification, the derivative was found to contain per cent of nitrogen as compared with the theoretical value of per cent. Obviously, this reaction is incapable of distinguishing between lactic aldehyde and pyruvic aldehyde, but that the latter was not obtained was demonstrated by the strong bvorotation of the * Divergent opinions concerning the direction and degree of rotation of d-threose are expressed in the literature, but Hackett (1935) reports that the sugar manifests an equilibrium rotation in water of approximately This racemic modification consisted of the natural product and its enantiomorph. t was synthesized in this laboratory by Dr. H. E. Carter and Mr. H. D. West, who will publish the procedure in the near future.

4 724 a-amino-/-hydroxybutyric Acid compound produced by the degradation of the natural hydroxyaminobutyric acid. No attempt was made to isolate the lactic aldehyde formed from the natural hydroxyamino acid. Rather it was oxidized to the corresponding lactic acid, which was purified as its zinc salt. For this purpose, the solution was acidified with a few drops of hydrochloric acid and treated with bromine water until an excess had been added as indicated by the presence of an orange color after the flask has been allowed to stand in the sun. After 24 hours, the solution was concentrated in vacua to 50 cc. and repeatedly extracted with ether. 500 cc. of the solvent were used. The ether was then removed in vacua, and the residue was dissolved in 100 cc. of water. Basic zinc carbonate was added in excess, and the mixture was shaken for a few minutes and filtered. The filtrate was concentrated in vacua to incipient crystallization and treated with an equal volume of alcohol. This not only promoted the crystallization of the zinc lactate, but prevented the separation of contaminating toluene sulfonamide. One recrystallization of the zinc lactate from the minimum quantity of water yielded 1.85 gm. of pure Zn(C3H60J2.2Hz0, as compared with a theoretical yield of 2.35 gm., from 2 gm. of hydroxyaminobutyric acid. Analysis of the anhydrous salt gave the following values. Zn(C&O&. Calculated. C 29.57, H 4.14, Zn Found. ( 29.90, 4.26, 26.74, By the same procedure a second sample of zinc lactate was prepared from hydroxyaminobutyric acid, and the specific rotation of each (anhydrous salt) was determined. The results were as follows: Sample 1. & = 2 x (o.1007,5j = [af: = x (0.2619/10) = $8.02 Values reported in the literature for 2.5 per cent aqueous solutions range from +8.0 to +8.2 (cf. Cori and Cori (1929)). t is well known that the direction of rotation of zinc lactate is opposite to that of the free lactic acid from which it is prepared

5 C. E. Meyer and W. C. Rose 725 (cf. Freudenberg (1914); Cori and Cori (1929)). Thus the lactic acid obtained by the action of chloramine-t on natural hydroxyaminobutyric acid is levorotatory, and hence belongs to the D series (cf. Levene (1926)). These facts demonstrate that the p- carbon possesses the D configuration, and that our hydroxyaminobutyric acid is an L-D type corresponding in spatial structure to d( -)-threose. The accompanying diagram illustrates the reactions involved. COOH HtN-C-H H-C-H CH, L( +)-or-aminobutyric acid C& COOH H-C-OH CH D( -)-Lactic D( -)-Lactic aldehyde acid These reactions definitely establish the spatial configuration of the new amino acid. Confirmatory evidence for the threo structure was obtained by an entirely different method of attack; namely, the transformation of the hydroxyamino acid into the corresponding dihydroxybutyric acid, and the identification of the latter as its phenylhydrazide. For this purpose, 2 gm. of natural hydroxyaminobutyric acid were dissolved in 20 cc. of water and treated with 4.9 gm. of barium nitrite monohydrate dissolved in 30 cc. of water. The solution was cooled in an ice bath. A slight excess of N sulfuric acid was added dropwise over a period of 30 minutes. After quantitative removal of the barium and sulfate ions, the solution was evaporated to a syrup in vacua. Absolute alcohol was added twice and removed, and the product

6 726 a-amino-&hydroxybutyric Acid was finally dissolved in 3 cc. of the same solvent. t is well known that nitrous acid does not induce a Walden inversion, and that a hydroxy acid so formed has the configuration characteristic of the amino acid from which it is derived (cf. Levene (1926)). Attempts were made to crystallize the dihydroxy acid; but these efforts were unsuccessful because of the extreme solubility and the relatively small amount of the compound which was available. Therefore, the phenylhydrazide was prepared by adding 2.5 gm. of phenylhydrazine to the alcoholic solution of the dihydroxy acid (3 cc.) and heating the mixture for 30 minutes at 75 (cf. Glattfeld and Woodruff (1927)). Upon standing over- Sample No TABLE Phenylhydrazides oj Dihydroxybutyric Acids Phenylhydraaide Dihydroxy acid from natural amino acid synthetic Z-Threodihydroxy acid (Glattfeld) d-threodihydroxy &Threodihydroxy (recrystallized mixture of Samples 3 and 4) dl-erythrodihydroxy acid (Glattfeld) Equal quantities of Samples 1 and 3 (for mixed m. p.) of - Melting - point C t Nitrogen* 1 per cent * Theoretical N for C~~HL~N~O~.H~O is per cent. t Glattfeld and Woodruff (1927) report a melting point of for the phenylhydrazide of dl-threodihydroxybutyric acid. night in a refrigerator, crystals were obtained. These were recrystallized three times from the minimum amounts of absolute alcohol. n a similar fashion, the phenylhydrazides of d- and l-threodihydroxybutyric acids, dkerythrodihydroxybutyric acid,8 and the dl-dihydroxy acid derived from dl-hydroxyaminobutyric acid2 3 Professor J. W. E. Glattfeld of the University of Chicago kindly furnished the samples of d- and -threo- and dl-erythrodihydroxybutyric acids. t is a pleasure to acknowledge our indebtedness to Professor Glattfeld for his cordial cooperation. The prefixes d- and Z- as used with the threo acids denote the direction of rotation, and not the series to which the compounds belong.

7 C. E. Meyer and W. C. Rose 727 were prepared. n addition, equal amounts of the phenylhydrazides of d- and kthreodihydroxybutyric acids were recrystallized together, thereby providing a racemic preparation. Hence, six phenylhydrazides were available for comparison. The melting points and analytical data are presented in Table, and demonstrate that natural hydroxyaminobutyric acid yields a dihydroxy acid whose phenylhydrazide is identical with the like derivative of one of the active (d- or l-) threodihydroxybutyric acids. Furthermore, the synthetic amino acid, consisting of the natural product and its enantiomorph, yields a dihydroxy acid whose phenylhydrazide is identical with that of dl-threodihydroxybutyric acid. The phenylhydrazide of the latter is a racemic compound rather than a racemic mixture, inasmuch as its melting point is higher than that of its components. Oddly enough, the phenylhydraaides of the d- and l-threo acids and of the dl-erythro acid melt within the same range. The fact that nitrous acid transforms natural hydroxyaminobutyric acid into a threodihydroxybutyric acid establishes the spatial configuration of the amino acid; for, as stated above, it has been shown repeatedly in this laboratory that reduction of the hydroxyamino acid yields L(+)-a-aminobutyric acid. The only configuration which can account for these findings is that shown in Formula. Thus the observations upon the dihydroxy acids are in complete accord with the data obtained by the degradation of the amino acid with chloramine-t. t is proposed that henceforth natural cr-amino-/3-hydroxy-n-butyric acid be known as d(-)- threonine,j inasmuch as it possesses a spatial configuration analogous to that of d(-)-threose (Formula ). HO-C-H CHO H-C-OH CHzOH d( -)-Threose 4 The d here refers, of course, to the series just as it does in the classification of sugars. With equal propriety the amino acid could be known as D(-)-threonine.

8 728 a-amino-p-hydroxybutyric Acid The above deduction concerning the spatial structure of threonine, as determined by its relation to threodihydroxybutyric acid, obviously does not necessitate previous knowledge as to the configuration of the dihydroxy acid. Having first established the relative positions of the groups attached to the a-carbon atom of the amino acid, the configuration of the /3 position of the latter follows inevitably from the fact that a threodihydroxybutyric acid was obtained. On the other hand, our observations provide important information regarding the spatial structure of the isomerit threodihydroxy acids. Braun (1930) demonstrated the relation of dl-threo- and dl-erythrodihydroxybutyric acids to the tartaric acids; and Glattfeld and Chittum (1933) accomplished the resolution of the threo acids. But the series to which each of the latter belongs appears not to have been established. n order to obtain information of this sort, comparisons of the specific rotations of the threo acid phenylhydrazides were undertaken. The results demonstrate that the phenylhydrazide of the dihydroxy acid derived from threonine and the phenylhydrazine of Glattfeld s l-threodihydroxybutyric acid are identical. The specific rotations (in aqueous solution) are shown below. Phenylhydrazide of Dihydroxybutyric Acid Derived from d(-)-threonine [OLE = 2 x (0.0389/5) = Phenylhydrazide of -Threodihydroxybutyric Acid (Glattjeld)- L-4: = x (0.1015/5) = Thus the dihydroxy acid which is levorotatory, and yields a levorotatory phenylhydrazide, is d( -)-threodihydroxybutyric acid. ts enantiomorph is (+)-threodihydroxybutyric acid. SUMMARY A study has been made of the spatial configuration of natural a-amino-/?-hydroxy-n-butyric acid. The results demonstrate that this constituent of proteins is an L-D compound analogous in struc-

9 C. E. Meyer and W. C. Rose 729 ture to d(-)-threose. Because of this relationship, the amino acid has been named d(-)-threonine. t has been shown that the dextro- and levothreodihydroxybutyric acids described in the literature are ( +) and d( -) compounds, respectively. BBLOGRAPHY Abderhalden, E., and Heyns, K., Ber. them. Ges., 67, 530 (1934). Braun, G., J. Am. Chem. Sot., 62, 3176 (1936). Cori, C. F., and Cori, G. T., J. Biol. Chem., 61, 389 (1929). Clough, G. W., J. Chem. Sot., Tr., 113,526 (1918). Dakin, H. D., Biochem. J., 11, 79 (1917). Dakin, H. D., and Dudley, H. W., J. Biol. Chem., 16, 127 (1913). Fischer, E., and Mouneyrat, A., Ber. them. Ges., 33, 2383 (1909). Freudenberg, K., Ber. them. Ges., 47, 2027 (1914). Glattfeld, J. W. E., and Chittum, J. W., J. Am. Chem. Sot., 66,3663 (1933). Glattfeld, J. W. E., and Woodruff, S., J. Am. Chem. Sot., 49, 2309 (1927). Hackett, R. C., J. Am. Chem. Sot., 67, 2260, 2265 (1935). Levene, P. A., Chem. Rev., 2, 179 (1926). McCoy, R. H., Meyer, C. E., and Rose, W. C., J. Biol. Chem., 112, 283 (1935). Oikawa, S., Japan. J. Med. SC., Biochem., 1, 61 (1925).

10 THE SPATAL CONFGURATON OF α -AMNO-β-HYDROXY-n-BUTYRC ACD Curtis E. Meyer and William C. Rose J. Biol. Chem. 1936, 115: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1