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The synthesis of radioactive l -glutamic acid
The Synthesis of Radioactive L-Glutamic
Acid
Richard W. Hendler,’ Marjorie G. Horning and Christian B. Antisen From the Laboralory of Cellular Physiology, National Heart Institde, National Institutes of Health, Department of Health, Education, and Welfare, Bethesda, Maryland Received January 21,1954
nn-Glutamic acid-5-Cl4 has been prepared by Speer et al. from isotopic KCN, with an over-ah yield of 47 $$,of theoretical (1). The same authors also prepared nL-glutamic acid-l ,2-C” with an over-all yield of 20.4 % of theoretical, starting with propionic acid-l-Cl4 using a modification of a method reported by Koegl et at. (2). Since radioactive oxalic acid is now available commercially, the over-all yield in the latter synthesis should be about 25 To. The present paper describes the synthesis of n-glutamic acid-l ,2-Cl4 with an over-all isotopic yield of 50%. The unreacted ar-ketoglutarate can be easily recovered in the last step. The reactions employed were: C*OOH CKaNz C*OOCH2 C*OOH C*OOCHs -1 C*OOCHz C*OOH I I c*=o c*=o COOC2Hr CYOOCH2 I I & CH2 - KOC,Hs CH2 + AH b*oom I I CHz CH2 I 2 CHZ I I COOK COOC2H, COOCzHs C*OOH POOH I I H-C*-NH2 c*=o glutamic acid I I dehydrogenese CH2 I
NHs , DPNH
CH2 I COOH L-glutamic acid-l, 2-C14
CH2
COOH 1 Fellow of the National
CH2 I
Foundation
for Infantile 470
Paralysis.
RADIOACTIVE GLUTAMIC ACID
471
This method could be used to synthesize optically active -1,2-O*, -5-C’*, and glutamic acid-3,4-C*, or combinations of these. EXPERIMENTAL
Methyl Oxalate-I ,2-C’* Oxalic acid (180 mg., 0.002 mole; 0.5 millicurie) was converted to the dimethyl eater with diazomethane; yield, 190 mg. (1OO~c); m.p. 53-53.5”.
a-Ketoglutaric
Acid-l ,dC’*
The procedure followed was a modification of the standard method (3). In a nitrogen atmosphere, 75 mg. (10% excess) of potassium was added to 7 ml. of dry ether, followed by 0.28 ml. of absolute alcohol. When all of the potassium had been converted to the alcoholate, the isotopic methyl oxalate (190 mg., 0.0016 mole) was transferred to the reaction vessel with a minimum of dry ether, and stirring was continued for 10 min. Three hundred milligrams (10% excess) of ethyl succinate was added to the well-stirred suspension, stirring continued for 25 min., and the mixture was allowed to stand for 2% hr. The potassium salt of oxalosuccinic ester was collected on a filter and was dissolved in 1.2 ml. of water and 0.2 ml. of concentrated HCI. The oily ester which was liberated was extracted with ether, and, after removal of the ether, 1.0 ml. of concentrated HCl was added to the residue. After standing for 48 hr., the hydrochloric acid was removed by rapid distillation until the temperature reached 140-150”. The residue was cooled quickly and crystallized from benzene-ethyl acetate. The isotopic yield, 150 mg., based on oxalic acid was 64%.
Preparation of Glutamic Acid Dehydrogenase Beef liver (1 kg.) packed in ice at the slaughterhouse was treated as described by Olson and Anfinsene (on a x-scale), except that an International refrigerated centrifuge was used rather than a Sharples centrifuge (4). The preparation was taken to the stage of dissolving the 28-4OoJcNazSO4 precipitate (Dz~+) in phosphate buffer, and bringing the NaaSOc concentration back to 28%. This solution with no further purification was used after dialysis. Enzyme activity wae assayed as described by Olson and Anfinsen (4).
Synthesis of Glutamic Acid Five milligrams (0.034 mmoles) of a-ketoglutaric acid, with a slight excess of DPNH, and an amount of enzyme predetermined in a nonisotopic experiment to be sufficient, was suspended in a total volume of 6 ml. having an NH&l concentration of 0.1 M. The reaction was allowed to proceed for approximately 45 min. Under similar circumstances a nonisotopic experiment was followed by withdrawing samples and readz In the first step of this preparation, 0.05 M phosphate buffer should be used rather than 0.5 M, as erroneously appeared in the reference cited.
472
HENDLER,
HORNING
J 5,000 c e,ooo
c” 1 i \ 3,000 z 5 8
”
’
AND I
ANFINSEN I
I
I
‘R
:eto-glutoric
1
.J
Glutomic acid ( 79%)
#cid (2/L)
2,000 -
1,000 -
O-d 0
FIG. 1. Chromatographic
separation
of glutamic Dowex 50.
acid from cu-ketoglutaric
acid by
ing the change in optical density at 340 mp. The reaction was stopped by adding 12 ml. of alcohol, and a clear fluid was obtained by centrifugation in a clinical centrifuge. Recovery of Glutamic Acid Dowex 50 (250-400 mesh, 12 % DVB*), cycled through 4 N NH40H and 6 N HCL, was poured into a 1 X 46 cm. column and washed free of acid with distilled water. Using 1.5 N HCl as the developing agent, 3-ml. fractions were collected by use of a Technicon fraction collector. Every other tube was assayed for radioactivity by counting 0.05-ml. aliquots dried on metal planchets. A plot of radioactivity vs. tube number (see Fig. 1) showed two distinct peaks. Fractions 20-27 were pooled for the glutamic acid cut. The amounts of glutamic acid (79 %) and of cu-ketoglutaric acid (21%) isolated (by radioactivity) are what would be ex* DVB
= divinyl
benzene
RADIOACTIVE
GLUTAMIC
ACID
473
petted at equilibrium as calculated by the equilibrium equation (5). Since the unreacted cu-ketoglutaric acid can be readily recovered, there is relatively little loss of material in this procedure. Purity of Glutamic Acid The specific activity of the glutamic acid was determined on a small aliquot employing a microadaptation (6) of the Moore and Stein quantitative ninhydrin procedure (7). It was found that the specific activity (on a micromolar basis) was sixfold lower than that of the cu-ketoglutaric acid. Ten per cent of the total material was chromatographed in streaks on Whatman No. 1 filter paper in 80% phenol and in 75 y0 propanol. A radioautograph of the propanol paper showed a single strong black area where known glutamic acid migrated and a barely visible zone closer to the origin. When the paper was sprayed with ninhydrin, only one purple zone appeared, which corresponded to the position for known glutamic acid and to the radioactive area. Since there was only one ninhydrin band revealed by chromatography, it was reasonable to assume that NH3 had previously been contaminating the preparation. This assumption was confirmed by determining the specific activity of the band that had been run in 75 % propanol. The glutamic acid and a-ketoglutaric acid then had the same specific activities on a micromolar basis, and therefore the fmal purification is readily accomplished by this preparative chromatographic procedure. Position of Labeled Carbons From the route of synthesis it seems apparent that only 1 ,2-labeled glutamic acid could result. This was completely confirmed by subsequent degradations with Chloramine-T, Clostridiu.m welchii,3 and the Schmidt reaction (6). Optical Purity Cl. wekhii will degrade L-glutamic acid, whereas its optical isomer is completely inert. After about a tenfold dilution of the synthetic glutamic acid with L-glutamic acid(b), it was found that the specific activity of the y-aminobutyric acid isolated subsequent to treatment with Cl. welchii was 51 y0 that of the glutamic acid before decarboxylation. Had the synthetic glutamic acid been a racemic mixture, the relative specific activity would have been 26 %. SThe authors are grateful to Dr. Alton Meister for his gift of the C. welchii.
474
HENDLER,
HORNING
AND ANFINSEN
SUMMARY
1. The synthesis of radioactive L-glutamic acid is described. 2. The optical activity and positions of labeled carbons have been verified. REFERENCES 1. SPEER, R. J., ROBERTS, A., MALONEY, M., AND MAHLER, H. R., J. Am. Chem. Sot. 74, 2444 (1952). 2. KOEGL, F., HALBERSTADT, J., AND BARENDREGT, T. J., Rec. trav. dim. 68, 387 (1949). 3. Org. Syntheses 26,42 (1946). 4. OLSON, J. A., AND ANFINSEN, C. B., J. Biol. Chem. 197, 67 (1952). 5. OLSON, J. A., AND ANFINSEN, C. B., J. Biol. Chem. 209,841 (1953). 6. HENDLER, R. W., AND ANFINSEN, C. B., J. Biol. Chem. 299, 55 (1954). 7. MOORE, S., AND STEIN, W. H., J. Biol. Chem. 176,367 (19481.
Acid
Richard W. Hendler,’ Marjorie G. Horning and Christian B. Antisen From the Laboralory of Cellular Physiology, National Heart Institde, National Institutes of Health, Department of Health, Education, and Welfare, Bethesda, Maryland Received January 21,1954
nn-Glutamic acid-5-Cl4 has been prepared by Speer et al. from isotopic KCN, with an over-ah yield of 47 $$,of theoretical (1). The same authors also prepared nL-glutamic acid-l ,2-C” with an over-all yield of 20.4 % of theoretical, starting with propionic acid-l-Cl4 using a modification of a method reported by Koegl et at. (2). Since radioactive oxalic acid is now available commercially, the over-all yield in the latter synthesis should be about 25 To. The present paper describes the synthesis of n-glutamic acid-l ,2-Cl4 with an over-all isotopic yield of 50%. The unreacted ar-ketoglutarate can be easily recovered in the last step. The reactions employed were: C*OOH CKaNz C*OOCH2 C*OOH C*OOCHs -1 C*OOCHz C*OOH I I c*=o c*=o COOC2Hr CYOOCH2 I I & CH2 - KOC,Hs CH2 + AH b*oom I I CHz CH2 I 2 CHZ I I COOK COOC2H, COOCzHs C*OOH POOH I I H-C*-NH2 c*=o glutamic acid I I dehydrogenese CH2 I
NHs , DPNH
CH2 I COOH L-glutamic acid-l, 2-C14
CH2
COOH 1 Fellow of the National
CH2 I
Foundation
for Infantile 470
Paralysis.
RADIOACTIVE GLUTAMIC ACID
471
This method could be used to synthesize optically active -1,2-O*, -5-C’*, and glutamic acid-3,4-C*, or combinations of these. EXPERIMENTAL
Methyl Oxalate-I ,2-C’* Oxalic acid (180 mg., 0.002 mole; 0.5 millicurie) was converted to the dimethyl eater with diazomethane; yield, 190 mg. (1OO~c); m.p. 53-53.5”.
a-Ketoglutaric
Acid-l ,dC’*
The procedure followed was a modification of the standard method (3). In a nitrogen atmosphere, 75 mg. (10% excess) of potassium was added to 7 ml. of dry ether, followed by 0.28 ml. of absolute alcohol. When all of the potassium had been converted to the alcoholate, the isotopic methyl oxalate (190 mg., 0.0016 mole) was transferred to the reaction vessel with a minimum of dry ether, and stirring was continued for 10 min. Three hundred milligrams (10% excess) of ethyl succinate was added to the well-stirred suspension, stirring continued for 25 min., and the mixture was allowed to stand for 2% hr. The potassium salt of oxalosuccinic ester was collected on a filter and was dissolved in 1.2 ml. of water and 0.2 ml. of concentrated HCI. The oily ester which was liberated was extracted with ether, and, after removal of the ether, 1.0 ml. of concentrated HCl was added to the residue. After standing for 48 hr., the hydrochloric acid was removed by rapid distillation until the temperature reached 140-150”. The residue was cooled quickly and crystallized from benzene-ethyl acetate. The isotopic yield, 150 mg., based on oxalic acid was 64%.
Preparation of Glutamic Acid Dehydrogenase Beef liver (1 kg.) packed in ice at the slaughterhouse was treated as described by Olson and Anfinsene (on a x-scale), except that an International refrigerated centrifuge was used rather than a Sharples centrifuge (4). The preparation was taken to the stage of dissolving the 28-4OoJcNazSO4 precipitate (Dz~+) in phosphate buffer, and bringing the NaaSOc concentration back to 28%. This solution with no further purification was used after dialysis. Enzyme activity wae assayed as described by Olson and Anfinsen (4).
Synthesis of Glutamic Acid Five milligrams (0.034 mmoles) of a-ketoglutaric acid, with a slight excess of DPNH, and an amount of enzyme predetermined in a nonisotopic experiment to be sufficient, was suspended in a total volume of 6 ml. having an NH&l concentration of 0.1 M. The reaction was allowed to proceed for approximately 45 min. Under similar circumstances a nonisotopic experiment was followed by withdrawing samples and readz In the first step of this preparation, 0.05 M phosphate buffer should be used rather than 0.5 M, as erroneously appeared in the reference cited.
472
HENDLER,
HORNING
J 5,000 c e,ooo
c” 1 i \ 3,000 z 5 8
”
’
AND I
ANFINSEN I
I
I
‘R
:eto-glutoric
1
.J
Glutomic acid ( 79%)
#cid (2/L)
2,000 -
1,000 -
O-d 0
FIG. 1. Chromatographic
separation
of glutamic Dowex 50.
acid from cu-ketoglutaric
acid by
ing the change in optical density at 340 mp. The reaction was stopped by adding 12 ml. of alcohol, and a clear fluid was obtained by centrifugation in a clinical centrifuge. Recovery of Glutamic Acid Dowex 50 (250-400 mesh, 12 % DVB*), cycled through 4 N NH40H and 6 N HCL, was poured into a 1 X 46 cm. column and washed free of acid with distilled water. Using 1.5 N HCl as the developing agent, 3-ml. fractions were collected by use of a Technicon fraction collector. Every other tube was assayed for radioactivity by counting 0.05-ml. aliquots dried on metal planchets. A plot of radioactivity vs. tube number (see Fig. 1) showed two distinct peaks. Fractions 20-27 were pooled for the glutamic acid cut. The amounts of glutamic acid (79 %) and of cu-ketoglutaric acid (21%) isolated (by radioactivity) are what would be ex* DVB
= divinyl
benzene
RADIOACTIVE
GLUTAMIC
ACID
473
petted at equilibrium as calculated by the equilibrium equation (5). Since the unreacted cu-ketoglutaric acid can be readily recovered, there is relatively little loss of material in this procedure. Purity of Glutamic Acid The specific activity of the glutamic acid was determined on a small aliquot employing a microadaptation (6) of the Moore and Stein quantitative ninhydrin procedure (7). It was found that the specific activity (on a micromolar basis) was sixfold lower than that of the cu-ketoglutaric acid. Ten per cent of the total material was chromatographed in streaks on Whatman No. 1 filter paper in 80% phenol and in 75 y0 propanol. A radioautograph of the propanol paper showed a single strong black area where known glutamic acid migrated and a barely visible zone closer to the origin. When the paper was sprayed with ninhydrin, only one purple zone appeared, which corresponded to the position for known glutamic acid and to the radioactive area. Since there was only one ninhydrin band revealed by chromatography, it was reasonable to assume that NH3 had previously been contaminating the preparation. This assumption was confirmed by determining the specific activity of the band that had been run in 75 % propanol. The glutamic acid and a-ketoglutaric acid then had the same specific activities on a micromolar basis, and therefore the fmal purification is readily accomplished by this preparative chromatographic procedure. Position of Labeled Carbons From the route of synthesis it seems apparent that only 1 ,2-labeled glutamic acid could result. This was completely confirmed by subsequent degradations with Chloramine-T, Clostridiu.m welchii,3 and the Schmidt reaction (6). Optical Purity Cl. wekhii will degrade L-glutamic acid, whereas its optical isomer is completely inert. After about a tenfold dilution of the synthetic glutamic acid with L-glutamic acid(b), it was found that the specific activity of the y-aminobutyric acid isolated subsequent to treatment with Cl. welchii was 51 y0 that of the glutamic acid before decarboxylation. Had the synthetic glutamic acid been a racemic mixture, the relative specific activity would have been 26 %. SThe authors are grateful to Dr. Alton Meister for his gift of the C. welchii.
474
HENDLER,
HORNING
AND ANFINSEN
SUMMARY
1. The synthesis of radioactive L-glutamic acid is described. 2. The optical activity and positions of labeled carbons have been verified. REFERENCES 1. SPEER, R. J., ROBERTS, A., MALONEY, M., AND MAHLER, H. R., J. Am. Chem. Sot. 74, 2444 (1952). 2. KOEGL, F., HALBERSTADT, J., AND BARENDREGT, T. J., Rec. trav. dim. 68, 387 (1949). 3. Org. Syntheses 26,42 (1946). 4. OLSON, J. A., AND ANFINSEN, C. B., J. Biol. Chem. 197, 67 (1952). 5. OLSON, J. A., AND ANFINSEN, C. B., J. Biol. Chem. 209,841 (1953). 6. HENDLER, R. W., AND ANFINSEN, C. B., J. Biol. Chem. 299, 55 (1954). 7. MOORE, S., AND STEIN, W. H., J. Biol. Chem. 176,367 (19481.